Forensic Science International: Genetics Supplement Series

Editorial Board

2011-12-01

Proceedings of the 24th International ISFG Congress

2011-12-01

DIP–STR: A new marker for resolving unbalanced DNA mixtures

D. Hall, V. Castella — 2011-10-03

Abstract: The genetic characterization of unbalanced mixed stains remains an important area where improvement is imperative. Most cases of aggression, homicide and sexual assault produce biological traces with relatively large amount of the victim's DNA and small amount of the aggressor's DNA. If this ratio is smaller than 1:10 it is currently not possible to obtain a conventional autosomal DNA profile of the minor contributor, with potential loss of crucial DNA evidence. Y-STR analysis represents a solution for some cases but has several limitations. We propose here a method based on a new compound genetic marker formed by a Deletion/Insertion Polymorphism (DIP) linked to a Short Tandem Repeat polymorphism (STR), that we name DIP–STR. By means of allele-specific amplifications of DIP–STR haplotypes, we can produce a high resolution autosomal DNA profile of a donor that contributes less than 0.1% to a DNA mixture. Based on these features DIP–STR markers may outperform conventional Y-STR markers in mixed stain analysis.

UTI preventing DNA degradation of storing urinary samples for genotyping

Suhua Zhang, Shumin Zhao, Ruxin Zhu, Gong Zhang, Chengtao Li — 2011-10-03

Abstract: In forensic practices, individual identification of urinary samples is necessary when sample switching or handling are suspected. DNA degradation with time elapsing and the low yield of extracted urinary DNA prevent its application. Storage of urine prior to analysis is increasingly advocated yet no best practice has emerged. To improve the genotyping based on storing urinary samples, we employed UTI (Urinary Trypsin Inhibitor) to prevent urinary DNA degradation. Urinary samples from 10 (5 females and 5 males) healthy volunteers from China were stored at −80°C with different concentrations (0–0.8μg/mL) of UTI. Urinary DNA was genotyped with Identifiler Kit at days of 1, 3, 5, 7, 9, 11 and 13 after storage. The average detection rate of STR loci of urinary DNA with or without UTI were statistically significant and UTI at 0.2μg/mL level is enough for urine storing. Also, significant differences in DNA yield were noted between female and male urine samples.

Collaborative EDNAP exercises on messenger RNA/DNA co-analysis for body fluid identification (blood, saliva, semen) and STR profiling

C. Haas, E. Hanson, N. Morling, J. Ballantyne — 2011-09-26

Abstract: RNA profiling is a new method for the identification of forensically relevant biological stains, such as blood, saliva, semen, vaginal secretions, menstrual blood, sweat and skin. To demonstrate the suitability of mRNA profiling for use in forensic casework, three collaborative exercises on RNA analysis or RNA/DNA co-analysis for body fluid identification (blood, saliva, semen) and STR profiling were organized within the European DNA Profiling Group (EDNAP). The results of these collaborative exercises support the potential use of an mRNA-based system for the identification of body fluids along with conventional DNA profiling.

Preliminary results of mitochondrial DNA sequence variation in Jujuy population (Argentina)

2011-09-26

Abstract: Jujuy is a province of Argentina, placed in the extreme northwest of the country, close to the borders with Chile, Bolivia and the Salta province of Argentina. Different studies on South American population have proposed that actual population is composed by Native American maternal lineages and European paternal lineages with different proportions among regions. The principal aim of this work is to study the mitochondrial genetic variability of the Jujuy population and also the different contributions of European and American-Native settlers. Results showed that the population sample was mainly composed by Native American haplogroups. Genetic contribution of European origin is very low. Haplogroups distribution in the province of Jujuy was related to other neighboring Native American populations. These preliminary data could be helpful for understanding the present distribution of the mitochondrial DNA haplogroups in this region.

When the alleged father is a close relative of the real father: The utility of insertion/deletion polymorphisms

M. Magalhaes, N. Pinto, C. Gomes, R. Pereira, A. Amorim, C. Alves, L. Gusmão — 2011-10-05

Abstract: The occurrence of Mendelian incompatibilities between true father and child is not an uncommon situation. Thus, in paternity investigations, when only a few are observed, it is possible that the alleged father is either the real father or his close relative. In this study, we intended to test how a set of autosomal indels can complement the results obtained with a routine battery of STRs in paternity investigations where the alleged father is, in fact, a close relative of the real one. We analyzed 100uncle/nephew and grandfather/grandchild unrelated duos, through the amplification of 15 STR markers and a set of 38 non-coding bi-allelic indels. For each duo, we determined the number of observed incompatibilities and calculated the LR assuming a father/son relationship, for both STRs and indels. As expected, in a significant proportion of cases, no incompatibilities were found. In these instances, indel's LR contribution was always in favour of the “wrong” hypothesis, strengthening a false paternity. Therefore, in dubious cases, an extended battery of indels can be useful to exclude false fathers but should be taken with caution when no incompatibilities are detected. We estimated that to obtain a satisfactory probability (less than 1 in 500) of finding zero incompatibilities between 2nd degree relatives, approximately 100 indels with maximum diversity would be necessary.

Evaluation of GeneMapper®ID-X Mixture Analysis tool

O. Hansson, P. Gill — 2011-10-04

Abstract: We have evaluated the ‘Mixture Analysis’ tool in GeneMapper®ID-X v1.1 (Applied Biosystems) to interpret two-person DNA mixtures. This package depends upon the evaluation of two different parameters – the mixture proportion (Mx) and the heterozygote balance (Hb). We analysed 3000 heterozygotes in order to determine the characteristics of heterozygote balance versus the average peak height ratio (PHR). This information was used to determine thresholds that were subsequently used to accept or to reject mixture genotypes. Apart from the evaluation of likelihood ratios conditioned on suspect's and victim's reference samples, the module can also be used to deconvolve mixtures. This may be important if a suspect or reference sample is not available for analysis. A case example was analysed in order to illustrate the principles. This evaluation forms the basis of a validation exercise to use the GeneMapper®ID-X Mixture Analysis tool in forensic casework at the Department of Criminal Forensic Genetics.

Database sample size effect on minimum allele frequency estimation: Database comparison analysis of samples of 4652 and 560 individuals for 22 microsatellites in Colombian population

2011-10-05

Abstract: With the aim of studying the effect of the database size on the frequency estimations of minimum and rare alleles, a comparative analysis was undertaken between two different size samples from a Colombian mestizo population living in Antioquia region. In this work, minimum allele frequencies (calculated using two different methods) were compared in samples with (a) size n=560, the total number of individuals in our database from Antioquia in 2008, and (b) size n=4652, the new size of the same database in 2010. When database size was increased, the total number of alleles increased in 17%, going from 257 up to 313 alleles. Although the number of alleles with low frequency became higher for an enlarged database, as it could be expected, the minimum allele frequency decreased and, therefore, the final number of alleles with a frequency below this estimate did not changed significantly.

Comparison of Identifiler®, Identifiler Plus® and Minifiler® performance in an initial paternity testing study on old skeletal remains at the forensic and legal medicine area of the Government of Andorra (Pyrenees)

2011-10-12

Abstract: The problem oftentimes faced by forensic scientists when working with DNA extracted from exhumations and skeletal remains is either DNA degradation or DNA contamination. Various methods have been used to improve the identification of skeletal remains using DNA technology. Most of these systems include short tandem repeat (STR) analyses. Herein, we describe our work on the identification of individuals involved in a paternity test where all parents, the mother and the two alleged fathers, had died between 10 and 58 years ago.Trace amounts of highly degraded human DNA were successfully extracted from each bone. Despite the presence of DNA inhibitors in the amplification, microsatellite alleles could be reproducibly amplified from the femur. DNA was extracted from bone which was turned into fine powder, then proteolytic digestion followed by phenol/chloroform purification and Centricon®-100 filtration took place. Our experience indicated that the AmpFlSTR® MiniFiler™ used as a complement to the AmpFlSTR® Identifiler Plus™ Kit forms an extremely sensitive multiplex STR amplification system. It has been successfully used to obtain multilocus STR profiles from bone samples with minimal amounts of human DNA. We report on the genotyping results obtained from each kit and the biomathematical results of paternity testing.

The Forensic Genetics Laboratory of the University of Barcelona: The evolution of paternity testing cases over the past 35 years

C. Barrot, C. Sánchez, S. Jiménez, M. Ortega, E. Huguet, M. Gené — 2011-10-13

Abstract: Biological paternity research in Catalonia (north-eastern Spain) began in 1976 when Dr. Castillo, Dr. Gelabert and Dr. Huguet studied the first case at the Department of Forensic and Legal Medicine of the University of Barcelona in cooperation with the Haemostasis and Hemotherapy Service and the Blood Bank of the Hospital Clínic in Barcelona. In the knowledge that each human is genetically unique as a result of genetic polymorphism, blood group antigen typing and other immunological techniques including human histocompatibility leukocyte antigen (HLA) typing became widely used for paternity testing and forensic science application until the end of the 1970s. However, biological tests were not accepted by the courts in Spain until 1981 when the Civil Code was reformed. With the support of Dr. Carracedo from the University of Santiago, in 1982 Dr. Huguet and Dr. Gené, who are still actively researching, set up the Haemogenetic Forensic Laboratory at the University of Barcelona and this area of the Legal Medicine Unit was thus consolidated. It was the first Forensic Genetics Laboratory in Catalonia. The aim of this study is to conduct a retrospective analysis of paternity testing cases over the past 35 years.We report on the social and legal development of the cases analysed, grouped into different periods of time. The results on paternity and non-paternity claims were studied included exclusion and non-exclusion rates. Biomathematical performance values were reported for each methodology stage (number of exclusions, average paternity probability values, population index, etc.).

X-chromosomal haplotype frequencies of four linkage groups in a North African population

2011-10-05

Abstract: Twelve X-STR loci in four clusters of linked markers were typed in a sample of 97 Moroccan men from different ethnic groups (Arabs, Berbers and Sahrawi) using the Investigator Argus X-12 Kit. Forensic statistical parameters for these 12 X-chromosome STR, particularly the haplotypes of the four closely linked X-STR trios, indicated that they are highly informative for anthropological and forensic purposes in Moroccan population.

A loophole in Spanish law and unsolved ethical issues in paternity testing

C. Barrot, C. Sánchez, S. Jiménez, M. Ortega, M. Gené — 2011-10-13

Abstract: Article 18 of the current Spanish Constitution (which came into force in 1978) defends all citizens’ right to privacy. Accordingly, the new Code of Ethics of the Spanish Medical Association includes several articles concerning studies conducted on genetic information and the acceptance of such studies by all individuals involved. As a result, a serious problem arises in paternity testing cases when investigating an alleged father–child relationship when the biological mother has not given consent for access to be gained to her genetic information.When it comes to the laboratory investigating these cases, the problem of lack of permission from a parent is greater when the child is a minor or has a mental or psychological disability because current Spanish legislation calls for informed consent to be given by legal representatives. The law does not specify what happens when one parent (for instance, the putative father) gives consent while the other (the mother) does not.There are currently no Spanish judicial records addressing the issue. Depending on the spirit of the law, it is logical to understand both how a team from a forensic genetics laboratory are likely to be sued for performing a biological paternity test without the mother's permission and how indeed the lawyer could win the case. The purpose of this study is to provide legal solutions in order to avoid potential problems arising with regard to legislation.

Experiences and surprises with PowerPlex ESI 17 and AmpflSTR NGM SElect in routine casework

2011-09-28

Abstract: The introduction of five “new European standard loci” (D1S1656, D2S441, D10S1248, D12S391 and D22S1045) in forensic DNA analysis called for the development of new Multiplex PCR kits. One of the aims was to combine the widespread SGM+ marker combination with the new markers to be amplified in a single kit. In addition, SE33 was also to be included in the new kits. We present practical experiences that have been made since the introduction of PowerPlex ESI 17 and AmpflSTR NGM SElect in our laboratory.

New validated analytical process for convicted offender samples submitted to the Canadian National DNA Data Bank

N. Laurin, A. De Moors, C.J. Frégeau — 2011-10-03

Abstract: Optimization and validation of direct amplification protocols for both AmpFlSTR® Identifiler® Direct (IDD) and PowerPlex® 16 HS (PP16HS) on the high-throughput 3730 DNA Analyzer (Applied Biosystems) were carried out in preparation for potential changes in Canadian legislation. Amplification conditions were optimized to perform direct amplification of single source blood, buccal and extracted hair roots collected from convicted offenders (CO) on FTA® paper without the need for sample purification prior to amplification. Validation was performed using IDD and PP16HS on a wide collection of blood and buccal samples from volunteer donors. In addition, 111 CO blood samples were selected to assess the robustness of the new analytical process (i.e. 0.53mm FTA disks amplified in 10μL PCR volume) and verify the stability of DNA stored on FTA paper at room temperature for up to 10 years. Studies such as reproducibility, robustness and concordance were carried out. The quality of the STR profiles generated using the new analytical process was assessed by examining profile intensity, heterozygote peak height ratios and interlocus balance. Reliable and high quality profiles from all CO samples and volunteer samples were produced under the new process giving support to its implementation.

Inferring ethnicity from the X-chromosome ALU insertions: Data from Western Mediterranean human groups

2011-09-26

Abstract: We investigated 9 X-chromosome ALU insertions, suitable for forensic purposes, in 645 healthy unrelated Mediterranean individuals (male and female). Forensic statistical parameters for these X-ALU insertions indicated that they are highly informative for forensic application in eleven Mediterranean populations.

Competition for DNA binding sites from denim dyes using the Promega DNA IQ™ paramagnetic beads

C.J. Frégeau, A. De Moors — 2011-09-26

Abstract: The competition between DNA and other components often present in casework specimen lysates (e.g. denim dyes) for the binding sites on the DNA IQ™ magnetic beads was examined. It was determined that dye/chemicals from black denim lysates interact directly with the DNA IQ™ beads and mask DNA binding sites. The competition from the black denim chemicals appear to be different from that previously reported for the Hemastix® reagents. This study strongly suggests that reducing the size of the specimen portion submitted for DNA IQ™ extraction will optimize DNA capture. In addition, it was found that a simple two- or three-fold dilution of the sample lysate following the overnight lysis prior to DNA extraction overcame the reduction noted in DNA yield and preserved portions of the lysates for subsequent analysis when required.

Validation of the fluorescence-based Sperm Hy-Liter™ kit as a means to standardize spermatozoa identification in sexual assault cases

A. De Moors, T. Georgalis, G. Armstrong, J. Modler, C.J. Frégeau — 2011-09-26

Abstract: To enhance searching and standardize the identification of spermatozoa in sexual assault exhibits, the Sperm Hy-Liter™ kit was evaluated and validated using a large number of sexual assault type specimens and 6mm circle slides. The practicality and sensitivity of the fluorescence-based protocol were compared to our current phase contrast microscopy protocol and examined in the context of an improved workflow for processing sexual assault cases. Experiments performed indicate that the Sperm Hy-Liter™ assay is (1) highly specific and valid (no interference from semen obtained from various animals and absence of positive fluorescence signals in all control slides prepared from swabs with only blood, yeast, urine, vaginal epithelial cells or fecal material), (2) sensitive and reliable (spermatozoa detected in vaginal swabs soaked in 1:1000 and 1:10,000 semen dilutions; spermatozoa detected more effectively in real casework samples containing few spermatozoa compared to phase contrast microscopy), (3) fast (1min versus average of 10min or more using phase contrast microscopy for <10 spermatozoa), (4) robust (spermatozoa detected from 24-year old suspected semen stains, no interference from spermicides, lubricants, fluorescent and non-fluorescent condoms and anti-fungal creams) and (5) simple to use.

Genetic polymorphisms of eight X-STR loci of Mentype Argus X-8 kit in Koreans

2011-10-31

Abstract: We have examined 45 Korean haplotype transfers in 41 families at eight X-linked STRs (DXS10135 and DXS8378 in linkage group 1, DXS7132 and DXS10074 in linkage group 2, HPRTB and DXS10101 in linkage group 3, DXS10134 and DXS7423 in linkage group 4) of Mentype Argus X-8 kit. The allele frequencies and haplotype frequencies within the four linkage groups were determined for females and males, respectively. Deviation from Hardy-Weinberg expectation could not be detected (p<0.05). No recombination was observed within each 4 linkage groups, however, six cases of mutation were founded in DXS10135, DXS10101, and DXS10134. Details of X-STR haplotype study in Koreans would be useful in kinship testing and forensic applications.

Automated scoring of Sperm Hy-Liter™-stained spermatozoa by the MetaSystems Metafer image analysis software system in sexual assault specimens

A. De Moors, C.J. Frégeau — 2011-09-28

Abstract: The MetaSystems Metafer image analysis software system was purchased three years ago in the hope of developing a routine approach in the RCMP Forensic Laboratories to automate the scoring of human spermatozoa in sexual assault exhibits. This would enhance case throughput, increase assay sensitivity and standardize the search for spermatozoa. The development of appropriate classifiers was challenging but essential to teach the software system to specifically recognize human spermatozoa fluorescently stained using the Sperm Hy-Liter™ kit (Independent Forensics). Optimized classifiers were tested/validated using a diverse set of slides prepared from mock sexual assault samples containing a limited or a large number of spermatozoa (fecal swabs, vaginal swabs, all mixed with different semen dilutions in addition to urine, blood and yeasts for a subset of those swabs). The performance of Metafer was recorded with respect to false positive counts, false negative counts and time required for the detection of spermatozoa in each sample. Automated spermatozoa counts were further compared to manual spermatozoa scoring in addition to comparing the time spent executing the identification. An excellent concordance was noted between automated and manual counts. The results of this study indicate that automated scoring of fluorescently stained spermatozoa in mock sexual assault exhibits can be carried out reliably and reproducibly using well-developed classifiers for the MetaSystems Metafer image analysis software system. The automated scoring of spermatozoa combining Sperm Hy-Liter™/MetaSystems Metafer will be tested on a large number of sexual assault cases as part of a pilot project within an operational setting.

X-chromosome in Italy: A database of 29 STR markers

2011-09-28

Abstract: We collected published and unpublished data from 17 contributing groups participating in the GeFI (Italian Forensic Geneticists). The total number of typed subjects was 1114 males and 777 females, coming from 11 regions of North, Centre, and South Italy, and Sardinia. Individual's multilocus genotypes included 4–12 loci. The total number of typed markers was 29, scattered along the X-chromosome genetic map in several clusters; the most used marker was DXS7423 (2429 gene copies); the mean number of subjects typed per marker was 336 for males and 208 for females. Data are available online.

Quality control of DNA from formalin fixed and paraffin wax embedded prostate biopsy for forensic analysis

V.D. Cantagalli, K.R.M. Leite, M. Srougi, G.J.F. Gattás — 2011-09-28

Abstract: In human identification laboratories the quality assurance in the harmonization of the thermocyclers and the standardization of the sensitivity tests, mainly for reduced and previously manipulated samples, as formalin fixed and paraffin wax embedded biopsies are necessary proceedings. In the present investigation it was used prostate biopsy from non tumor tissue of three individuals with Applied Biosystems kits: Identifiler, Minifiler and Yfiler. The amplification of 10ng of DNA from two biopsies was performed in three different thermo cyclers. Three biopsies were amplified using DNA concentrations that vary from 100 to 0.125ng of DNA. The efficiency of the thermocyclers was DNA sample and Kit dependent, although the best performance was achieved with the Applied's equipment. The minimal quantity of DNA to get results was 10ng but it was not proper for all the cases. The use of 10ng of DNA was sufficient for the Minifiler kit but not for the other kits. These results suggested the importance to previously know the laboratory and sample variables before performing forensic analysis in samples like prostate biopsies.

The potential risk of formalin fixed and paraffin wax embedded prostate biopsies for human identification

G.J.F. Gattás, V.D. Cantagalli, K.R.M. Leite, M. Srougi — 2011-09-28

Abstract: The genetic instability in malignant tumors and the possible DNA degradation in archived histological samples are potential confounder factors that can interfere in the human identification process using formalin fixed and paraffin wax embedded tissues. In the present investigation a total of 49 biopsies (normal and tumor) from 20 individuals were investigated (Identifiler Kit) and the autossomic STR profiles were compared to FTA blood samples. The identification of the entire profile was possible only in 4 cases (20%) and no amplification occurred in other 4 individuals (20%). Considering all the 49 biopsies, it was obtained 13 completed profiles (26%), 18 biopsies with partial profile (37%) and 18 biopsies without DNA amplification (37%). The DNA degradation due to the size of the prostate biopsies, the laboratory preparation and also the identification procedure could be responsible for the partial results observed. The data association with other DNA identification Kits (Minifiler and/or Yfiler) could be better to individual investigation.

Optimization and validation of the SNPforID 34-SNPplex for POP7™

Bram Bekaert, Jurgen Wens, Ronny Decorte — 2011-09-28

Abstract: The SNPforID 34-SNPplex is a multiplex of ancestry informative single nucleotide polymorphism markers and was originally designed for POP6™ polymer gels. Due to the higher viscosity of POP7™, a different design is necessary to resolve peaks smaller than 40bp when this type of polymer gel is used. The single original SNaPshot™ assay was separated and redesigned into two individual multiplex SNaPshot™ assays in which rs2304925 was moved in between two SNPs with larger amplicons. Minor adjustments to PCR parameters and primer concentrations further reduced noise background in comparison with the original protocol. Validation of the assay included reproducibility (100%), sensitivity (complete DNA-profiles up to 0.5ng) and specificity studies (no cross-species interaction). A population study (n=145), including 46 Belgian samples, further validated the assay for future forensic use.

An investigation of the presence of DNA on unused laboratory gloves

Runa Daniel, Roland A.H. van Oorschot — 2011-10-12

Abstract: The presence of background DNA in forensic laboratories is potentially a significant contamination risk to criminal investigations. As part of the laboratory's Environmental Monitoring (EM) program, a preliminary investigation of three brands of laboratory gloves was undertaken to determine the levels of human DNA present on unused gloves from closed and open boxes as well as the origin of stains observed on gloves. The study revealed the presence of DNA on a number of the gloves from closed boxes as well as on some gloves from open boxes. Some observed staining on gloves was indicative of rust. Regular assessment of the presence of human DNA on unused gloves is recommended. In addition, using only certified DNA-free gloves should be considered.

LMD-assisted single cell DNA typing of forensic biological evidence: Issues of the cell type and sample condition

Sergey Leonov, Elena Zemskova, Pavel Ivanov — 2011-09-26

Abstract: The practice of DNA typing of single cells in forensic biological evidence may have peculiarities. For instance, some laser microdissected (LMD) single cell preparations fail to give any PCR products while there is no such a problem with macropreparations from the same sample. As an answer, this study shows that not all the cells individually retrieved from the forensic sample are suitable for analysis. Hence, when going from the bulk to single cell preparations, a bottleneck occurs, which reduces chances for successful DNA typing. For a useful single cell analyses in forensics, single cell DNA profile recovery dependency on the cell type and sample condition must be considered.

Investigation of Paternity Loss: is statistical evaluation reliable? A case report

Nicoletta Cerri, Luciana Caenazzo — 2011-10-05

Abstract: We report the experience of a laboratory that was charged by a Court to conduct an analysis to establish the paternity of a woman: her mother was alive, the alleged father was deceased, but three of his brothers, were available for analysis. So the paternity testing was performed on 5 subjects by analyzing 15 different DNA markers (by using Identifiler Kit). The obtained results, showed two genetic incompatibility, and a discrepancy for a marker among the three brothers. The paternity investigation was concluded with an exclusion. However, a second laboratory was responsible for carrying out the alleged father exhumation. The obtained results showed incompatibility for all the 8 polymorphisms analyzed and confirmed what previous analysis conducted by the first laboratory had evidenced.

Mitochondrial DNA-control region sequence variation in the NE Portuguese Jewish community

J.C. Teixeira, I. Nogueiro, A. Goios, L. Gusmão, A. Amorim, L. Alvarez — 2011-09-28

Abstract: The cultural phenomenon of Crypto-Judaism, defined as the secret adherence to Judaism while publicly professing another faith, arose in Portugal in the beginning of the 16th century after the Decree of Expulsion and the establishment of the Inquisition. Surprisingly, the scientific community acknowledged the persistence of Crypto-Judaic communities at the beginning of the 20th century in central and north-eastern regions of the country (e.g. Bragança). In the present work, we have sampled 56 unrelated individuals from the Bragança Jewish community aiming to characterize their maternal lineage. A 3348bp mtDNA fragment was amplified and sequenced using mitochondrial-specific primers in order to obtain the entire control region. Haplogroup classification was performed according to current nomenclature. High frequencies were found for haplogroups H, HV0, T2, U2 and N1, indicating some degree of European admixture along with a remarkable signature of a Near East ancestry. These data confirm that the Crypto-Jews from Bragança were able to maintain not only their cultural identity but also some ancestral genetic identity, showing a significant population substructure within Portugal, with forensic relevance.

Population genetic data for 15 autosomal STR loci in four Venezuelan states (Aragua, Carabobo, Zulia and Táchira)

M. Figuera, A. Dictamen, C. Marrero, L. Borjas, R. Ferreira, M. Álvarez — 2011-10-05

Abstract: Fifteen autosomal DNA markers (D3S1358, TH01, D21S11, D18S51, PENTA E, D5S818, D13S317, D7S820, D16S539, CSF1P0, PENTA D, VWA, D8S1179, TPOX and FGA) were genotyped in a total of 1290 unrelated individuals living in Aragua (232 individuals), Carabobo (321), Táchira (431) and Zulia (306) States of Venezuela. STR amplification was carried out using GenePrint Fluorescent Systems (Promega Corp.) and the analysis of amplicons was performed on an ABI Prism™ 3130 Genetic Analyzer (Applied Biosystems). Statistical evaluations were performed using GENEPOP version 4.0 and PowerStats softwares. All markers obey Hardy–Weinberg law in the States of Aragua, Táchira, and Zulia (p>0.05). However, CSF1P0 marker deviates from Hardy–Weinberg expectations in Carabobo State (p=0.029), although equilibrium could be considered after sequential Bonferroni correction. When each pair of populations is compared at the genic and genotypic level, there are no differences among Aragua, Carabobo and Zulia, but all these populations are different to Táchira population. Allele, minimum, and null allele frequencies were estimated, as well as the main forensic parameters. These are the first population studies concerning these four States, and they will contribute to the conformation of a Venezuelan national database.

Using X-chromosomal markers in rape investigation

M. Lancia, S. Severini, A. Coletti, G. Margiotta, M. Dobosz, E. Carnevali — 2011-09-26

Abstract: The X-chromosomal markers are increasingly used in forensic genetics, particularly for relationship testing. Their use has become a valuable tool in complex cases of kinship but rather in criminal caseworks is still quite rare. In this paper the authors present a case of sexual assault in which the use of X chromosome polymorphisms has been crucial. The victim was a young immigrant woman found dead in her home. The main suspect was her husband. However, the couple lived in a community in which the particular cultural context suggested the involvement of the other males of the husband family. The suspect lived together with his brother, his father and his uncle. Generally, a useful tool to solve cases of sexual violence is undoubtedly the use of Y chromosome, but in this case this device could not discriminate between the four males involved. An additional factor has further complicated the situation: the most important biological evidence (typed with AmpFlSTR Identifiler and AmpFlSTR NGM) showed a mixed profile in which was very difficult to discriminate the suspects profiles. To solve the casework, the authors typed the victim, the suspects and the biological traces with 6 X-STRs in an homemade esaplex. The results showed the presence of victim and her husband profiles in the biological trace excluding his brother, father and uncle's profiles.

Colombian results of the interlaboratory Quality Control Exercise 2009–2010

2011-10-05

Abstract: Colombian Reference National Laboratory, GENES LTDA, have organized and coordinated for the past two years (2009 and 2010) the Quality Control Exercise for laboratories undertaking paternity, maternity and forensic tests with DNA markers. Twenty-two laboratories have participated in 2009, increasing the number to 27 in 2010. Laboratories in Colombia, Brazil, Ecuador, Peru, Dominican Republic and Panama have participated in these exercises. There have been some similarities in the two controls: A practical exercise, three blood samples on FTA cards were sent to each participating laboratory to be genotyped for DNA markers using the routine methodologies in their laboratories; theoretical exercises including optional and obligatory cases. For the theoretical exercises, the participating laboratories should calculate the partial and final PI or BRI (Biological Relationship Index or Paternity Index). Forty-nine and 52 markers were under consensus for 2009 and 2010, respectively, distributed in autosomal, Y and X chromosomes STR. With respect to 2008, 12 and 15 additional markers were under consensus for 2009 and 2010, respectively. The rate of reporting error was 2.9% in 2009 while in 2010, 4.7% error was reported. The Proficiency Test conducted through the Colombian National Reference Laboratory has become a useful tool for quality assurance of all Colombian laboratories and some of Latin America that do DNA testing to establish biological relationships and an excellent opportunity for ongoing training of experts from the region.

Genetic data of 10 X-STR in a Colombian population of Bolivar Department

2011-09-28

Abstract: X chromosome markers have been used in human identification and paternity testing since the early 1970s. In recent years it has been evaluated the informative power of X-linked STRs (X-STRs) for use in human identification and paternity testing, encouraging their study and identified over 200 trinucleotide and tetranucleotide loci from. The aim of this study was to study the polymorphism and some parameters of forensic interest of 10 X-STR loci (DXS8378, DXS9898, DXS7133, GATA31E08, GATA172D05, DXS7423, DXS6809, DXS7132, DXS9902 and DXS6789) in a population sample of the Department of Bolivar (Colombia) and they were added to the panel of markers currently used in forensic genetics and paternity testing.

Validation of BTA™ lysis buffer for DNA extraction from challenged forensic samples

A. Barbaro, P. Cormaci, G. Falcone — 2011-10-07

Abstract: Isolation of DNA from forensic samples is strongly conditioned by many factors such as wide variety of samples, degradation by environmental exposure, presence of PCR inhibitors or limited quantity of starting material.BTA performance in the extraction of genomic DNA has been evaluated from real casework powdered bones, teethes, carbonized tissues cigarettes, and tape adhesive lifts. DNA extracted from these samples was in sufficient quantities for downstream applications; the method was successfully able to remove inhibitors producing highly purified DNA; IPC values of Quantifiler® Human DNA Quantification Kit were consistent and within the normal range. The combination of BTA+AmpFℓSTR NGM PCR Amplification Kit provided a powerful tool for the typing of difficult forensic samples.

Recovery of DNA from paraffin embedded tumour samples of pediatric sarcomas

M. Marino, A. Mampel, S. Furfuro, M. Di María, J. Oliva, A.L. Vargas — 2011-10-04

Abstract: The main challenge of DNA extraction from old paraffin embedded tissue sections is to obtain analyzable DNA. The aim of this work was to recover DNA from paraffin embedded tumour samples. We analyzed 30 tumours pieces included in paraffin from 20 to 25 years old to research the existence of deletion/duplication in INI1/SMARCB1 gene. This is a retrospective study to improve clinical control in patients. Pediatric sarcomas are a heterogeneous group of tumours with monomorphic cytologic features that hamper the accurate diagnosis to apply specific tumour-depend therapies. Some of these neoplasias have copy number alterations of INI1 gene sequence, a tumour suppressor gene located in the long arm of chromosome 22 (22q11.2). These molecular alterations are associated with loss gene expression, aggressive tumour behaviour and lack of clinical response. The utilization of DNA extraction techniques usually used in forensic genetics, allowed to obtain appropriate DNA in all tumours to apply MLPA technique (Multiplex Ligation Probe Amplification). The obtained results allowed to characterize tumours depending on the existence or not of variation in copy gene number of INI1/SMARCB. The molecular characterization of tumours in retrospective studies is a useful complementary tool to make accurate tumour diagnosis and apply the better therapeutic option for current patients.

Haplotype frequencies and mutation rates for 17 Y-STRs in a sample from Mendoza province (Argentina)

Miguel Marino, Sandra Furfuro — 2011-10-04

Abstract: A sample of 566 unrelated males from Mendoza province was typed for 12 Y-STRs loci, 237 of these males were typed for 17 Y-STRs.When analyzing only the minimal haplotype of 9 Y-STRs we found a total of 398 different haplotypes, of which 317 were unique, and the haplotype diversity was 0.9931.If we analyzed the 12 Y-STRs, which make up the extended haplotype, we observed that 87% of the total of 470 different haplotypes are unique, and the haplotype diversity was 0.9989.In the 237 males typed with 17 Y-STRs included in the AmpFISTR YFiler Amplification Kit (AB Applied Biosystems), we found 226 different haplotypes of which 96% are unique, and the most frequent haplotype appears only 3 times in the study population.On the other hand, in a total of 173 father/son pairs, mutations were observed in DYS389I/II, DYS385a/b, DYS439 and DYS458 markers. In addition, there were duplications on DYS19 and DYS437 markers. However, the mutation rates are similar to those already described.The comparison between our data and previous one from Mendoza, other Argentinean provinces and different world populations, reflected a similar haplotype distribution to that depicted by European populations. The most frequent haplotype is the modal for the European haplogroup R1b, this supports its European origin.

Validation of AmpFLSTR NGM SElect™ PCR amplification kit on forensic samples

A. Barbaro, P. Cormaci, A. Agostino — 2011-10-07

Abstract: The AmpFLSTR NGM SElect™ is a next generation kit developed by Applied Biosystems that contains the 5 new loci specified in the recently expanded European Standard Set (ESS) together with the remaining markers from the SGM Plus® kit, plus the highly discriminating SE33. We performed a validation study of the NGM SElect Kit with evaluation of critical parameters as species specificity, sensitivity, degradation/inhibition study, performance on a wide variety of forensic samples, mixture sample analyses.Our feedback confirmed the multiplex shows a robust PCR chemistry and the improved performance requested by the forensic community for typing difficult samples and offers the option of a 29 or 30 cycle protocol for optimum data quality and sensitivity.

DNA extraction using the QIAsymphony: Evaluation of PCR inhibitor removal

V. Castella, D. Kummer, C. Gehrig, D. Hall — 2011-09-28

Abstract: The goal of this study was to compare the quantity and purity of DNA extracted from biological traces using the QIAsymphony robot with that of the manual QIAamp DNA mini kit currently in use in our laboratory. We found that the DNA yield of robot was 1.6–3.5 times lower than that of the manual protocol. This resulted in a loss of 8% and 29% of the alleles correctly scored when analyzing 1/400 and 1/800 diluted saliva samples, respectively. Specific tests showed that the QIAsymphony was at least 2–16 times more efficient at removing PCR inhibitors. The higher purity of the DNA may therefore partly compensate for the lower DNA yield obtained. No case of cross-contamination was observed among samples. After purification with the robot, DNA extracts can be automatically transferred in 96-wells plates, which is an ideal format for subsequent RT-qPCR quantification and DNA amplification. Less hands-on time and reduced risk of operational errors represent additional advantages of the robotic platform.

Use of universal reporter primers in multiplex PCR of autosomal loci

Elaine C. Favaro, Denilce R. Sumita, Martin R. Whittle — 2011-09-26

Abstract: At present forensic identification and parentage testing is invariably carried out using multiplex PCR amplification kits in which many loci, whether they be STRs, SNPs or DIPs are analyzed simultaneously using primers labelled with at least three, but usually four, different fluorophores. Should existing loci need to be substituted by newer, for instance, more polymorphic loci, new fluorescent primers need to be synthesized; indeed, the dye labels of several loci may need to be switched to allow a re-accommodation of the allele sizes in the single multiplex, and this can be costly. We sought to facilitate these substitutions and reduce costs by using four universal primers each labelled with a different fluorophore and employ these in a test multiplex PCR amplification of 20 DIP loci.

Use of matrix standards for new fluorophores in capillary sequencers

Martin R. Whittle, Elaine C. Favaro, Carlos W. Francischini, Denilce R. Sumita — 2011-09-26

Abstract: Most laboratories presently carrying out forensic identification and parentage testing use capillary DNA sequencers to separate and visualize the alleles that are produced by multiplex PCR amplification, whether they be of STR, SNP or DIP loci. Because at least four, but usually five, different fluorophores are used simultaneously, each instrument must undergo its own spectral calibration in which a matrix is created and used to correct the overlapping of fluorescence emission spectra of the mixture of dyes. For this users must acquire matrix standards in addition to the multiplex PCR kits and it is not clear what these standards consist of. In our efforts to develop new multiplex STR and DIP amplification kits using different fluorophores from those commonly used, we were obliged to produce new matrix standards to calibrate our multicapillary sequencers, and we describe how this was done.

SNPs in mitochondrial DNA coding region used to discriminate common sequences in HV1–HV2–HV3 region

C. Fridman, M.M.S.G. Cardena, E.A. Kanto, M.B.C. Godinho, F.T. Gonçalves — 2011-09-28

Abstract: Regions HV1, HV2 and HV3 of mitochondrial DNA (mtDNA) are routinely analyzed for forensic and evolutionary purposes due to the high polymorphic rate but sometimes show limited power of discrimination since several polymorphisms are very common in different populations. Genotyping additional SNPs in the coding region of mtDNA have been suggested to increase the power of discrimination between individuals who show common haplotypes in control region. Herein, we intended to evaluate the discrimination power of 26 SNPs from coding region, previously described, in 15 pairs of mother/child who could not be previously individualized and matched by HV1–HV2–HV3 analysis. These 15 pairs were divided into 7 groups of common sequences, of which 2 presented European haplogroup (Hg) H, 2 showed Amerindian Hg B4, 4 Amerindian Hg C1b (but with 2 different haplotypes), 3 African Hg L3e2b, 2 African Hg L3e1 and 2 African Hg L1b. Sequencing was performed using BygDye Terminator v3.1; capillary electrophoresis was performed on ABI3130. The SNPs were determined by comparison of the sequences obtained with reference sequence rCRS. After sequencing we could find 43 SNPs to analyze. Twenty-six out of 43 SNPs, in different combinations, were able to distinguish all pairs of mother/child except two from Hg L3e2b. The SNP 8047, observed here is not report in MITOMAP. This can be a preliminary result in the direction of having a SNP set that could be used to discriminate common sequences in mitochondrial control region in a highly mixed and heterogeneous population as the Brazilian one.

Evaluation of the relationship between mitochondrial haplogroup and development of heart failure in Brazilian sample

C. Fridman, M.M.S.G. Cardena, J.E. Krieger, A.C. Pereira — 2011-09-28

Abstract: Recently, some studies have been suggested an association between mitochondrial haplogroups (mtDNA Hg) and pathogenesis of cardiovascular diseases, showing an influence of ethnic origin with the development and prognosis of patients with heart failure (HF). Herein, we intended to evaluate the existence of a possible association of specific mtDNA Hg in 188 patients with HF, compared with 188 healthy individuals. Sequencing of regions HV1, HV2 and HV3 was performed using BygDye Terminator v3.1 and capillary electrophoresis was performed on ABI3130. Polymorphisms were determined comparing the sequences obtained with the Cambridge Reference Sequence (rCRS). In patients group, 92 individuals showed sequences of African Hg (49%) (L0 5%; L1 25%; L2 27%; L3 43%), 54 showed Ameridian Hg (29%) (A 39%; B 24%; C 24%; D 13%) and 42 individuals showed European Hg (22%) (H 10%; U 12%; K 10%; J 7%; T 7%; HV 5%; R 47%; W 2%). For control group, 68 individuals presented Ameridian Hg (36%) (A 31%; B 32%; C 24%; D 13%), 67 individuals showed African Hg (36%) (L0 11%; L1 25%; L2 22%; L3 40%; L4 2%) and 53 individuals showed European Hg (28%) (H 9%; HV 9%; T 9%; U 9%; I 8%; J 8%; K 6%; R 36%; X 4%; W 2%). Although African Hg was more prevalent among patients, we found no statistically significant difference between groups (P=0.10). The sample size is being increased in order to confirm these preliminary results.

Analysis of complex DNA mixtures using the Forensim package

Hinda Haned, Peter Gill — 2011-09-28

Abstract: We describe a new user-friendly module of the Forensim package of the R software, LRmix, which enables the interpretation of complex STR profiles involving multiple replicates, multiple contributors and low-template phenomena of dropout and drop-in.

Allele frequencies of 15 STRs in a representative sample of Entre Ríos province of Argentina population

G.G. Martinez, L.C. Schaller, A. Brondani, M. Bolea, B. Martinez-Jarreta — 2011-10-13

Abstract: Allele frequencies for 15 short tandem repeat (STR) loci present in AmpFℓSTR® Identifiler® PCR Amplification Kit (Applied Biosystems) were obtained from a sample of 839 unrelated individuals undergoing paternity testing. This sample includes individuals from all regions in Entre Rios province of Argentina. The most polymorphic loci were FGA, D18S51 and D2S1338. All the analyzed loci meet Hardy–Weinberg equilibrium after Bonferroni correction. Total non-discrimination probability and combined power of exclusion for the 15 tested STR loci were 1.91×10−18 and 0.9999995, respectively.

New alternative for human identification. Investigator IDplex Kit – QIAGEN® reproducibility: Latin American interlaboratory study

2011-10-07

Abstract: In 2010 QIAGEN® launched to eight kits of different combinations of STRs, including the Investigator IDplex Kit. This kit allows amplification in one PCR 16 markers. The aim of this study was to evaluate the reproducibility of Investigator IDplex Kit among Latin America laboratories. In the framework of the ‘III International Theoretical–Practice Course in Populations Genetic and Biologicals Filiations’ in Medellín–Colombia, all participants were invited to evaluate the reproducibility of this kit, they were provided of the necessary materials for the study. The results reported by participating were tabulated for the study the reproducibility. Results and comments were received on the agreed date of 12 of the 22 laboratories registered, one participant submits comments only. Some laboratories reported greater sensitivity Investigator IDplex Kit compared with other kits containing similar markers, also highlight the easy adaptability to existing conditions in laboratories, without involving major changes to its implementation. This paper shows the high reproducibility of Investigator IDplex Kit, a new tool offered by QIAGEN® for all laboratories that perform human identification testing and biological relationship testing using DNA markers.

Concordance testing with the new STR kits and the influence on the Swiss National DNA database

D. Dion, A. Sulzer, A. Kratzer — 2011-09-26

Abstract: The European Network of Forensic Science Institutes (ENFSI) and the European DNA Profiling Group (EDNAP) recommended the extension of the European Standard Set by 5 additional loci regarding international DNA data exchange based on the treaty of Prüm. This recommendation launched the development of new STR kits by-amongst others-Promega and Applied Biosystems (AB). Despite not being part of the EU nor the treaty of Prüm, Switzerland has decided to implement the new STR kits (including SE33). We therefore decided to conduct an in-house concordance study with 1498 reference samples analyzed with AmpFlSTR® SEfiler Plus™ and AmpFlSTR® NGM SElect™ (AB) and PowerPlex® ESX 17 and PowerPlex® ESI 17 (Promega). The concordance rate of the samples between the STR kits was greater than 99.967%.

A primer binding site mutation at the D2S1338 locus resulting in a loss of amplification

E.M. Dauber, B. Glock, W.R. Mayr — 2011-10-13

Abstract: An apparent paternal genetic incompatibility at the D2S1338 locus due to false homozygosity is described. It was disclosed after amplification with the AmpFlSTR® Identifiler® PCR Amplification Kit and reproducible with the genRES®MPX-4 DNA-Typing Kit. After application of an alternative primer set an allele *25 could be amplified, which restored Mendelian inheritance between father and child. Sequencing revealed a C>T transition at the fourth position from the 3′ end of the standard forward primer, which potentially caused the allelic loss with the primer sets in the commercial multiplex PCR kits.

Internal validation of Tecan robots (Freedom EVO® 150 and 75) for PCR and capillary electrophoresis setup

Nadja V. Morf, Andrea Sulzer, Adelgunde Kratzer, Walter Bär — 2011-09-26

Abstract: We validated and established an automated method for PCR and capillary electrophoresis setup (CE setup), using the Tecan Freedom EVO® 150 and 75 automated liquid handling workstations powered by Freedom EVOware®. The PCR setup script of the Freedom EVO® 150 was based on the HID EVOlution™ System. However, substantial changes in the HID script were required before putting the system on stream in the routine. The CE setup of the Freedom EVO® 75 was based on a script made by application specialists of Tecan. For internal validation of the two robots, we investigated repeatability, reproducibility and occurrence of contaminations. The analysis of the reproducibility data demonstrated that electropherograms of samples pipetted by the robots had a lower mean size standard peak height, than the hand-pipetted samples, whereas the mean sample peak height did not differ between the two methods. This was mainly due to precision-differences between the robots and the hand-pipetting. The investigation of the occurrence of contaminations showed that with the PCR setup robot no sample got contaminated. The CE setup robot on the contrary revealed carryovers of samples from one well to the subsequent well. After intense examination, we discovered that the tip travel height was set too low, causing the tip to touch the rim of the 96 well plate which led to the carryovers. The contact between rim and tip could not be seen by naked eye, but after increasing the tip travel height, no further contamination was observed. Our validation demonstrates that robots, even when set up by application specialists, need to undergo a proper internal validation before being put on stream in the routine.

The Pinpoint DNA Isolation System as a novel DNA sampling method in forensic biology

2011-09-28

Abstract: The Pinpoint DNA Isolation System (Zymo Research) uses a dissolved polymer compound to remove and extract DNA from slide mounted pathology specimens. This polymer is applied to a non-porous substrate on which biological material is deposited, allowed to dry, and the polymer, containing cells and DNA, is peeled off. The polymer dissolves into solution during extraction, theoretically releasing more DNA into the extract than standard cotton swabs; a notion supported by initial data. We performed preliminary experiments to test its effectiveness in comparison to wet/dry swabbing methodology for forensic samples, including replicates each of 10μL of saliva on glass slides and a pitted, non-porous surface. Results from the glass substrates showed that the initial application method of spreading with pipette tips generated significantly less DNA than the swabbing method. The mode of polymer application is being investigated further with the aim of improving DNA collection. Sampling from a pitted surface with Pinpoint exhibited significantly less variability than swabbing, but the mean quantity of DNA obtained from both collection methods was comparable. The Pinpoint system, combined with an optimised application method, may be another effective way to sample DNA in forensic casework. It has the potential to collect higher quantities of DNA than traditional methods which may be especially advantageous in casework involving trace DNA.

Evaluating the efficiency of DNA extraction methods from different substrates

Timothy J. Verdon, R. John Mitchell, Roland A.H. van Oorschot — 2011-09-28

Abstract: The efficiency of extracting DNA directly from substrates varies according to a range of factors including the type of substrate and the extraction technique. Two routinely used DNA extraction methodologies (automated DNA IQ and manual Chelex) were texts on their efficiency to extract DNA from a range of blood volumes (0.1–30μL) on plastic and cotton. The efficiency of extracting DNA from plastic appeared to be lower than from cotton for both methods, but was only statistically significant for Chelex extractions. Pairwise comparisons of blood volumes extracted using DNA IQ showed statistically significant differences. Comparisons of Chelex extractions of different blood volumes also showed significant differences for cotton, but not for plastic. The threshold effect of DNA IQ was only demonstrated at blood volumes above 15μL. This preliminary research highlights discrepancies between extraction methods and demonstrates that laboratories should be aware of the limitations of their analysis techniques, as knowledge of extraction efficiencies may assist in optimisation of methodologies and procedures. Extraction efficiency analysis will also allow for more accurate assessment of the influence of used methodologies in studies relating to determination of DNA transfer rates, and, should these transfer studies be put into practice, in casework.

Reliability of results in sibship cases

A. Sulzer, D. Dion, A. Kratzer — 2011-10-03

Abstract: Genetic exclusion of siblinghood of two persons, without parental findings, is basically not possible. Both the positive and the negative proof of descent can only be provided by statistics. Our aim is to evaluate the likelihood ratio (LR) and posterior probability (W-value) sufficient for a secured statement. Further, we evaluate the influence of the number of analysed STR systems on the LR-value by analysing different numbers of loci for 1225 unrelated couples and 61 pairs of full-sibs and testing the hypotheses full sibs versus half sibs versus unrelated.

Genetic diversity of 17 Y-chromosomal short tandem repeat loci in northeast Germany

Eileen Boljahn, Sylvio Tetzlaff, Andreas Buettner, Iris Lindner — 2011-09-28

Abstract: The 17 short tandem repeat loci included in the Y-filer™ PCR amplification kit from Applied Biosystems (AB) DYS19, DYS385, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635 and GATAH4 were amplified in a population sample composed of 531 unrelated males resident in northeast Germany. A total of 531 haplotypes were identified, of which 480 were unique. Allele frequencies were estimated and the overall haplotype diversity was 0.9993. We report some non-standard characteristics, including the infrequent microvariant alleles 16.2, 17.2 and 19.2. Furthermore 257 father–son pairs, previously confirmed by autosomal STR analysis, were typed using the same amplification kit, and 16 mutations were identified, giving an average mutation rate of 3.6×10−3.

SNP typing of crime case samples with the SNPforID multiplex assay

C. Børsting, B.B. Hjort, N. Morling — 2011-09-26

Abstract: The SNPforID multiplex was tested on typical crime case samples. In a mixture study, both controlled experiments with samples mixed in known ratios and a blind study were conducted. All mixtures were identified, including samples mixed in 1:40 ratios. For the study of degraded DNA, 50 crime case samples were selected. Complete SNP profiles were obtained in 80% of the highly degraded samples and at least 41 SNPs were typed in the last 20%. Furthermore, 30% of the highly degraded samples turned out to be mixtures.

Genetic data for the locus SE33 in a Southern Italy population with AmpFlSTR NGM SElect™ PCR amplification kit

A. Barbaro, P. Cormaci, A. La Marca — 2011-10-07

Abstract: A genetic population study for SE33 locus has performed from a sample of 130 individuals coming from South of Italy (Calabria) since at least 3 generations, using the next generation multiplex AmpFlSTR NGM SElect™ PCR Amplification Kit by Applied Biosystems.Allele frequencies and statistical parameters of forensic interest (Power of Discrimination, Power of Exclusion Matching Probability, etc.) were calculated using PowerStats v1.2 software.Results demonstrate the usefulness of SE33 for forensic identification, which should be added to the set of STR loci routinely studied in caseworks and in complex paternity cases.

Direct PCR by the AmpFlSTR NGM™ kit for database purpose

A. Barbaro, P. Cormaci, S. Votano — 2011-10-07

Abstract: To fully maximize the utility of DNA databases for tracing the origins of criminal activity, the streamlining of database sample processing must also be accompanied by high performance chemistries. Blood and buccal samples are often used for convicted-felon DNA profile databases, as these samples are easy to obtain.The AmpFlSTR NGM™ (Next Generation Multiplex) kit provides enhanced performance on degraded and inhibited samples and tolerance to PCR inhibitors, so we tested the kit ability to amplify directly blood and buccal samples deposited on special collecting cards. The ability to amplify stored DNA directly would allow the lab to get results more efficiently by eliminating the DNA purification step, minimizing the possibility of contamination reducing costs and time accelerating testing by as much as 30% so that lab faster would send consistent results to national database.

The potential forensic utility of two single nucleotide polymorphisms in predicting biogeographical ancestry

Yan Gu, Libing Yun, Lushun Zhang, Fan Yang, Yiping Hou — 2011-09-28

Abstract: DNA-based prediction of an individual biogeographical ancestry would provide some investigative lead value in specific forensic cases. Recent studies have shown that single nucleotide polymorphisms (SNPs) in pigmentation-related genes such as SLC24A5 and OCA2 can be considered as ancestry informative markers (AIMs). These markers, usually expected with large differences in phenotype and allele frequency, need to be assessed and validate on the basis of comprehensive distribution data in specific populations. In the present work we typed these two SNPs (rs1800407 located in exon 12 of the OCA2 gene and rs16891982 in the SLC45A2 gene) in 413 individuals including 210 Hans and 203 Uygurs, who are closely related for geographic range in China. Our study generates new allelic data of Chinese population and reveals the significant different distribution of pigmentation-associated SNPs between Hans and Uygurs. It has been shown that the Hans has higher frequency of homozygote than the Uygurs within two SNPs; especially heterozygous genotypes were only observed in the latter group. Furthermore, our research confirms the earlier results and supports SNP rs1800407 and rs16891982 can potentially be selected for AIMs.

Analysis of tri-allelic SNPs for forensic purpose in Chinese Han population

L. Zha, L.B. Yun, H.B. Luo, J. Yan, Y.P. Hou — 2011-09-26

Abstract: Single nucleotide polymorphisms (SNPs) have been proved to be effective forensic marker for supplementary paternity testing and forensic identification due to their low mutation rate and depending on small amplicon size. However, at least a set of 50–60 binary SNP loci can reach discrimination power of 13 STR loci that were routinely used in forensic practice. Meanwhile, binary SNP loci were ineffective to detect mixed samples, which may result in incorrect genotyping. In this study, we aimed to find out the potential forensic markers, tri-allelic SNP loci in Chinese Han population. Pyrosequencing (PSQ) with pooling samples was used to analyze candidate SNP loci and estimate allele frequencies of them. To confirm the existence of three alleles, individual samples were sequenced with PSQ method. Our results revealed that there were 10 tri-allelic SNP loci resided on 10 autosomal chromosomes in Chinese Han population. These data demonstrated that PSQ was an effective method to detect tri-allelic SNPs and estimate allele frequencies of them.

Statistical evaluation of pre-laboratory and laboratory factors that influence DNA recovery from archaeological material

Cristina Gamba, Eva Fernández, Ana María López-Parra, Eduardo Arroyo-Pardo — 2011-09-28

Abstract: We evaluated the influence of different factors over the efficiency of DNA amplification and PCR inhibition from 89 archaeological samples. We studied the effect of sample age and location together with some macroscopic features, like sample colour and fragmentation. Four different extraction methods were also tested and mtDNA HVRI was amplified and sequenced. Amplification and sequencing efficiencies were evaluated considering all the variables mentioned by a bivariate statistical analysis using SPSS 15.0 software. Results suggested that neither age nor macroscopic preservation had a direct effect on the recovery of endogenous DNA, while sample origin showed to influence significantly extraction and amplification successes. Moreover, the extraction method employed seemed to be a determining factor, influencing both inhibition and efficiency. An overall analysis of the results identified a silica-based protocol as the most efficient method for extracting DNA from archaeological material.

Oral swab, as simple as that? A case report

A. Verzeletti, N. Cerri, V. Cortellini, F. De Ferrari — 2011-09-28

Abstract: Although performing oral swabs is a very simple, fast and cheap procedure, it should always be kept in mind the importance of this step in DNA typing. We report a case of voluntary contamination of oral swabs to alter a paternity testing. Therefore, carefully evaluate the oral cavity before collecting the swab, personally collect the sample, perform at least two oral swabs at two different sites of the oral cavity.

Evaluation of PowerPlex ESI 17® amplification kit in an admixed Hispano-Amerindian population sample of Valparaíso, Chile

Manríquez Jose, Rojas Sergio, Yáñez Maria Oriana, Molina Graciela — 2011-09-28

Abstract: The PowerPlex ESI 17® (PP17) amplification kit was designed for European population (ENFSI and EDNAP recommendations). Routinely, DNA testing in our population was performed using the AmpFℓSTR® Identifiler® PCR Amplification kit (ID). Our population is an admixture between Hispanic and Amerindians. To evaluate the usefulness of the loci included in PP17 we analyzed 150 unrelated individuals’ samples obtained with previous informed consent, who attend to paternity testing. The DNA extraction was automated performed and the STR loci was PCR amplified using PP17 kit. Allelic, genotypic frequencies, Power of exclusion (PE), Match Probability (MP) and polymorphism information content (PIC) were calculated using PowerStat v12 software (Promega). This study showed a high percentage of heterozigosity in PP17 loci in the population studied, despite being designed for European population, showing that PP17 system has better informativeness than ID. The combined use of these two systems could resolve complex DNA testing, where ID alone is not sufficient to get a strong conclusion.

Analysis of severely compromised skeletal remains by the use of the latest generation kits

S. Severini, M. Lancia, S. Massetti, A. Coletti, L. Carlini, E. Carnevali — 2011-09-28

Abstract: In recent years, European working groups (ENFSI-EDNAP) have strongly encouraged the development of new amplification kits that allow to obtain DNA profiles even on difficult samples (degraded samples, presence of inhibitory substances, LCN, mixtures, etc.). To enable the amplification of difficult biological samples the new loci were designed to achieve greater resistance to inhibitors and most robust and uniform amplification. With the aim of evaluating the performance of latest generation kits in real forensic caseworks the authors retested some bone samples previously analyzed in their laboratory over the past five years with the old commercial kits. The authors compared the performance of AmpFℓSTR® NGM™ PCR Amplification kit (Applied Biosystems) with PowerPlex® ESX 17 systems (Promega). The twelve analyzed samples came from eight exhumed corpses and four bodies found outdoor in the advanced stage of putrefaction. For the genetic investigation the authors used a piece of bone taken from the femur. In conclusion, the authors can claim that the latest generation kits have proved to be decisive in all cases, including those where the previous use of traditional kits did not produce reliable and uniquely interpretable results.

Laser capture microdissection for forensic DNA analysis

Mado Vandewoestyne, Dieter Deforce — 2011-09-26

Abstract: Laser capture microdissection (LCM) is a unique tool for precise separation of target cells from forensic mixtures. Cells isolated by LCM can subsequently be used for the generation of pure DNA profiles. Although mainly used in sexual assault cases, LCM offers tremendous advantages for many different forensic applications.

Bonaparte: Application of new software for missing persons program

C.J. van Dongen, K. Slooten, M. Slagter, W. Burgers, W. Wiegerinck — 2011-09-28

Abstract: The Netherlands Forensic Institute (NFI), together with SNN at Radboud University Nijmegen, have developed new software for pedigree matching which can handle autosomal, Y chromosomal and mitochondrial DNA profiles. Initially this software, called Bonaparte, has been developed for DNA DVI. Bonaparte has been successfully applied in a real DVI case: the Afriqiyah Airways crash in Tripoli, Libya on 12 May 2010 in which 103 persons perished. The software performed excellently in terms of computational performance, stability and user-friendliness. This showed that Bonaparte is a reliable and time-saving tool which significantly simplifies and enhances a large-scale victim identification process.Bonaparte has been applied in NFIs missing persons program. For this, the software is connected to the NFI's missing persons database (CODIS). Since Bonaparte uses XML as import format, data from any source can be imported. In the new configuration, CODIS data is automatically imported into Bonaparte. Then the software automatically performs a set of direct searches, as well as searches against both partial and full family trees. For the autosomal DNA results, exact likelihood ratios are computed. Finally, match reports can be generated on demand by Bonaparte's customized reporting modules. In this way, an advanced search strategy combined with a modern, efficient work flow is realized in NFI's missing persons program.

NGM-SElect: An improvement to NGM kit in determining genetic profiles of low level DNA samples

2011-10-05

Abstract: AmpFlSTR® NGM™ is a 16-locus multiplex kit claiming improved robustness than earlier generation AppliedBiosystems kits; NGM-SElect™ was released recently as an equivalent to NGM loci plus SE33 so it was determined if NGM-SElect can be an asset compared to NGM in low-level DNA samples.

NGM SElect™ vs PowerPlex® ESI 17: Results with challenging DNA samples

2011-10-05

Abstract: Samples analysed in this study suffered from afflictions, such as low level DNA, degradation and PCR inhibitors: samples with induced degraded conditions and real crime scene samples, from a wide range of crimes.Results obtained with NGM SElect™ PCR amplification kit and PowerPlex® ESI 17 system were compared, to verify the behaviour of both kits in the presence of challenging samples.

Mitochondrial control region data of 3 ethnic groups from angola

2011-09-26

Abstract: Samples of 70 individuals from 3 ethnic groups from Angola (Bakongos, Kimbundu and Ovimbundu) were studied analysing the polymorphisms of the entire mitochondrial control region in order to verify the genetic diversity. A total of 68 different haplotypes were identified, 66 being unique. Sequence diversity was estimated to be 1.0000±0.0113 and nucleotide diversity for Bakongo, Kimbundu and Ovimbundu ethnic groups were 0.015556±0.007970, 0.016772±0.008517 and 0.016020±0.008411, respectively. It was possible to include the majority of the mtDNA haplotypes into a specific African mtDNA haplogroup.

Allele frequencies of all CODIS and four non-CODIS STR loci of an immigrant Brazilian population living in Lisbon – Preliminary results

2011-09-28

Abstract: Allele frequencies of CODIS STR loci CSF1PO, D13S317, D16S539, D18S51, D21S11, D3S1358, D5S818, D7S820, D8S1179, FGA, TPOX, TH01 and vWA together with STR loci D2S1339, D19S433, Penta D and Penta E were calculated in a sample of 187 healthy unrelated Brazilian immigrants, living in Lisbon. Some statistical parameters of forensic interest such as Hardy–Weinberg equilibrium, observed heterozygosity, expected heterozygosity, matching probability, power of discrimination, polymorphic information content, power of exclusion and typical paternity index are also presented. All the analysed loci meet Hardy–Weinberg equilibrium expectations. Our data is the first announcement of population genetic data of Brazilian immigrants living in Lisbon up to date.

Insertion/deletion polymorphisms at the X-STR DXS10146 and DXS10147 flanking regions

Atsushi Nagai, Yasuo Bunai — 2011-10-10

Abstract: Analyses of insertion/deletion (INDEL) polymorphisms at the flanking regions of the X-chromosomal short tandem repeats, DXS10146 and DXS10147, were performed in 146 unrelated Japanese males. Allele frequency of the DXS10146 locus for the long type allele containing the 17-bp INDEL element was 0.932, whereas that for the short type allele in which the INDEL element was deleted was 0.068. For the DXS10147 locus, the long type allele frequency was 0.822 and the short type allele frequency was 0.178. INDEL polymorphisms at the DXS10146 and DXS10147 loci are strongly suggested to be associated with the repeat number of the first repeat block (TTCC)x in the core repeat unit and the (AAAC)6 allele, respectively.

Identification of Azores islands haplogroups by mitochondrial DNA analysis

H. Afonso Costa, M. Carvalho, A.M. Bento, F. Balsa, M.J. Anjos, F. Corte-Real — 2011-10-03

Abstract: Mitochondrial DNA (mtDNA) has enormous potential in population genetics allowing the correlation and identification of the origin of individuals in a population. In forensic genetics mtDNA enables the identification of the matrilineal lineage from degraded samples. Genetic testing can be performed using mtDNA noncoding region and/or mtDNA coding region. However, to apply this inclusive approach as evidence in legal practice is necessary to determine the genetic structure of populations. Traditional mtDNA sequencing and Single Nucleotide Polymorphisms (SNP) analysis techniques were combined to establish the mtDNA variability of the Azorean population and classify the haplotypes into haplogroups. Forty-eight haplotypes were identified. All mtDNA haplotypes were included into specific haplogroups: forty-three haplotypes belonging to macrohaplogroup R; three haplotypes belonging to macrohaplogroup N; and two haplotypes belonging to macrohaplogroup M.

Genetic data of 17 autosomal STRs in Mendoza population (Argentina)

Miguel Marino, Sandra Furfuro — 2011-10-04

Abstract: The allele frequencies for 17autosomal short tandem repeats were determined in a sample of 1933 unrelated individuals from Mendoza, Argentina.The calculated parameters: polymorphism information content (PIC); discrimination power (DP); matching probability (MP); typical paternity index (TPI) and power of exclusion (PE), showed Penta E as the most valuable marker from the studied sample set. All loci met Hardy–Weinberg expectations using the Bonferroni correction for the number of loci analyzed.The mutation rate for these markers was analyzed by studying a total of 596 trio mother/son/father. We found a total of 14 mutations in different markers; all of them were single-step.

Discrepancies between forensic DNA databases

Ronny Hedell, Anders Nordgaard, Ricky Ansell — 2011-09-28

Abstract: In this study we present a comprehensive statistical comparison between a number of reference databases used in Sweden and two databases of DNA profiles from casework. Our results show no substantial differences with respect to various measurements of overall discrepancies but reveal significant differences for individual alleles at several loci.

Genetic characterization of Somali and Iraqi populations using a set of 33 X-chromosome Indels

2011-09-26

Abstract: The genotyping of insertion–deletion polymorphisms (Indels) has gained importance in population studies and human identification over the last few years and so has the study of the X-chromosome markers. In the present work, 245 samples from unrelated individuals from Somalia (148 males) and Iraq (58 males and 39 females) were analysed for 33 X-chromosome Indel markers. The aim was to characterize the diversity pattern in both populations, contributing at the same time to a better understanding of the genetic landscape of the Horn of Africa and the Middle East.Although intrapopulation diversity values for the Somali and Iraqi populations were similar, significant FST values were obtained between the groups. No departure from Hardy–Weinberg equilibrium was detected in the subset of 39 Iraqi women. Linkage disequilibrium analyses revealed significant association in some pairs of markers in Somalis (MID3690–MID2089; MID3719–MID2089; MID357–MID356) and Iraqis (MID3719–MID2089; MID357–MID356). Overall, statistical parameters for forensic efficiency presented high values. The results of the analyses of forensically relevant statistical parameters suggest that the 33plex system can be used for human identification and kinship testing in Somalis and Iraqis and most likely in other populations as well.

Study of 25 X-chromosome Single Nucleotide Polymorphisms in African and Asian populations

2011-10-03

Abstract: There is growing interest in the study of X-chromosome markers in population and forensic genetics. Since most of the studies focused on Short Tandem Repeats (STRs), data concerning Single Nucleotide Polymorphisms (SNPs) are still scarce.In the present work, male samples from Africa (Angola: n=46; Mozambique: n=42) and Asia (Taiwan: n=21; China: n=43; Bangladesh: n=58) were analysed for 25 SNPs located on the X chromosome to evaluate the diversity pattern in the populations from the two continents. Data were compared with previously published results from Somalia and Mediterranean populations.The results showed a clear genetic differentiation between Sub-Saharan populations and those from northern Africa and the Mediterranean. The differentiation between eastern and southern Africa was also recognizable, with Angola and Mozambique being statistically different from Somalia but not from each other. Likewise, the Asian samples formed a separate cluster with Bangladesh exhibiting a statistically significant difference from those of China and Taiwan.

Evaluation of Y-STR analyses of sperm cell negative vaginal samples

Jill Olofsson, Helle Smidt Mogensen, Benjamin Benn Hjort, Niels Morling — 2011-10-05

Abstract: In some cases of sexual assault, no sperm cells are microscopically visible in vaginal samples. In this study, sperm cell negative vaginal samples from 110 sexual assault cases in Denmark were analysed for Y-STRs with the AmpFlSTR® Yfiler kit (Applied Biosystems – AB). Selected samples were purified and concentrated using the MinElute (Qiagen) procedure. Y-DNA was detected in 49 (45%) cases. Complete or partial Y-STR profiles were obtained in 32 (29%) cases. Use of MinElute (Qiagen) significantly increased the success of typing of Y-STRs. In the majority of the cases, the Y-STR profiles observed were not found in three reference populations. Thus, informative Y-STR results were obtained in a significant proportion of the cases.

Analysis of ventricular myosin light chain genes in cardiomyopathy

2011-11-09

Abstract: Recently, numerous mutations in sarcomeric genes are reported as factor of cardiomyopathy. Hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) are general disease in cardiomyopathy and causes sudden death in young people. In this study, ventricular myosin regulatory light chain gene (MYL2) and ventricular myosin essential light chain gene (MYL3) were analyzed in order to elucidate the cause of cardiomyopathy. In MYL3, a significant missense mutation (Ala57Gly) was detected in HCM case. This mutation was located in the EF-hand domain calcium-binding site that regulates the cardiac contraction. No genetic mutation was found in our previous report except this gene, it indicated that the mutation of the MYL3 was the main factor for the HCM case. It is necessary to survey the other disease causing genes to clarify the relationship with etiology.

Laser microdissection: Checking that the dissected cells are recovered for DNA extraction

2011-10-13

Abstract: Tests were performed to check if the dissected cells are affectively retrieved into the collection tubes, if cross contamination could occur if cells were collected from multiple slides containing cells of differing origin, and if the work environment surfaces contained any DNA that may cause a contamination risk. A less than ideal laboratory environment, sampling different cells from multiple slides, and inadequate cleaning procedures may negatively affect cell collection and/or increase contamination risk. Recommendations are made to assist in limiting the risk of contamination while using laser microdissection to collect cells for forensic DNA profiling.

Comparison of extraction methods for spermatozoa recovered using laser microdissection

Skye M. Thorpe, Dionne V. Prince, Roland A.H. van Oorschot — 2011-10-13

Abstract: When collecting small numbers of cells using laser microdissection for the purpose of generating a DNA profile it is important to know that the DNA extraction process applied to such samples is appropriate. The DNA extraction process routinely used for common forensic samples may not be the optimal process for low numbers of LMD collected cells. We compare three DNA IQ modification methods with a distinct single-tube method. The latter outperformed the others in its ability to provide fuller profiles and higher peaks from DNA extracted from 50, 100 and 150 LMD collected spermatozoa.

Mitochondrial DNA in a population of individuals from the City of São Paulo. DNA extraction from head and pubic hair and blood

2011-10-03

Abstract: In biological samples with low copy DNA or DNA degraded samples, such as hair and pubic hair, there is a greater probability of obtaining a mitochondrial DNA profile. Hair and pubic hair are often found at crime scenes; in cases of sexual violence is common to find the suspect's pubic hair in the victim's body. This study analyzed, blood, head and pubic hair samples of 25 males to perform the analysis of mtDNA. Each three hair shafts were grouped and divided into 0.5cm sections: with the bulbs, and the other with 2cm, the proximal portion, without the bulbs. The DNA extraction of hair samples was performed using QIAamp DNA Micro Kit (QIAGEN) and the PCR amplification of HV1 and HV2 carried out. The amplified products were purified and sequenced using Big Dye™ Terminator and ABI 3130 (Applied Biosystems). To insure that hair samples were from the same individual, the AmpFLSTR Identifiler Kit® (Applied Biosystems) were used. The success rate of genotyping of hair samples with the bulb was: 56% for head hairs and in 64% for pubic hair samples, for a complete profile of the 15 STR (Identifiler). There was no statistically significant difference between the DNA concentration samples from hair with bulb and without bulb, as well as samples of pubic hair with bulb and without bulb. The overall success rate obtained with the extraction method used was approximately 60% and the success rate obtained in the sequencing was higher for pubic hair, both with and without bulb, than in head hair samples. Partial profiles of HV1 and HV2 were observed in most cases. Point heteroplasmy was detected in a sample of hair without bulb at hotspot position 16192 and another individual showed point heteroplasmy in pubic hair sample with bulb and without bulb at position 204, also observed in the blood sample of the same individual. In the preliminary results, 193 different haplotypes were detected; the most frequent haplotype was shared by 3 individuals. The most frequent lineage observed was African, showing the contribution of this haplotype group in São Paulo City population. This data were used to analyze and to compare the haplotypes of hair samples.

Eye colour and SNPs in Danes

J.D. Andersen, P. Johansen, H.S. Mogensen, C. Børsting, N. Morling — 2011-09-28

Abstract: Single nucleotide polymorphisms (SNPs) affect the pigmentation of the eyes, skin and hair in humans. Four SNPs located in the pigmentation genes HERC2 (rs12913832), OCA2 (rs1800407), SLC45A2 (rs16891982) and IRF4 (rs12203592) known to be important for eye colour were investigated in a Danish population sample. The rs12913832 homozygote genotype GG alone the highest likelihood ratio (LR)=29.4 for blue versus brown eye colour and, therefore, seems to be a major determinant of eye colour in Danes. Furthermore, it was shown, that the highest LR=37.5 for dark versus light eye colour was found when the genotypes rs12913832 AG and rs1800407 GG were observed. rs16891982 and rs12203592 had no discernable effect on the eye colour in Danes.

Genetic variants and skin colour in Danes

J.D. Andersen, P. Johansen, H.C. Wulf, B. Petersen, C. Børsting, N. Morling — 2011-10-03

Abstract: In this study, 33 single nucleotide polymorphisms (SNPs) in known pigmentation genes were tested for association to objective spectrophotometric reflectance measurements of the upper-inner arm and the buttock in 188 unrelated Danes. The 188 Danes were typed for 33 SNPs by two multiplex PCRs, two multiplex single base extension reactions and capillary electrophoresis. To further investigate the role of the SNPs, their allelic distribution was compared to those in a population of 36 Somalis. Four MC1R SNP alleles (R151C, R160W, D294H and 29insA) showed significant associations to light skin pigmentation in individuals who were either homozygous or compound heterozygous. This confirms the regulatory role of MC1R in skin pigmentation. SNP loci in SLC45A2, SLC24A5 and TYR showed large allele frequency differences between Somalis and Danes, which suggested a possible role in skin pigmentation. The average buttock pigmentation was significantly lighter than the average upper-inner arm pigmentation. Therefore, the buttock reflectance measurements better resemble the constitutive skin colour compared to upper-inner arm reflectance measurements and will be used in future studies.

Phadebas® Forensic Press test and the presence of amylases in body fluids naturally deposited on textile

Eva-Lena Olsén, Erika Edenberger, Marianne Mattsson, Ricky Ansell — 2011-09-28

Abstract: On clothing, in particular underwear, several body fluids can be present due to natural causes. Knowledge on limitations of tests used for locating stains or indication to their origin is therefore important. The Phadebas® Forensic Press test was used for amylase detection in single source samples on textile, and underwear with naturally deposited stains. Results show that other body fluids than saliva can react within the time frame stipulated on false positives, implying caution interpreting positive results.

An in-depth population genetic analysis of forensic short tandem repeat loci in Indonesia

2011-09-28

Abstract: Allele frequency data and knowledge of the population genetic features of relevant populations are required to substantiate the strength of forensic DNA evidence. It is conceivable that population substructure exists within Indonesia given that it is an archipelago with over 17,000 islands and encompasses numerous distinct ethnic and linguistic groups. However, the population genetic features of forensic short tandem repeat (STR) loci in Indonesia have not been examined thoroughly. Samples from 1500 unrelated Indonesian individuals representing 31 geographic sub-populations were analysed using the AmpFℓSTR® Identifiler® kit (Applied Biosystems). Departures from Hardy–Weinberg equilibrium (HWE) and linkage equilibrium (LE) were assessed using exact tests and results suggest that a number of the sub-populations, as well as the combined data set (N=1286), display significant departures from equilibrium. Ultimately, data from these STRs and other markers on this sample set will assist in forming genetically appropriate sub-population groupings for the purpose of constructing defensible forensic STR databases.

Mutation analysis of 24 autosomal STR loci using in paternity testing

2011-09-28

Abstract: To obtain the knowledge about the locus-specific mutation rate and characteristics of 24 autosomal STR loci, fifteen STR loci (D31358 et al.) were typed with the PowerPlex® 16 system (Promega, USA) for 11175 DNA-confirmed paternity testing cases (8059 trios, 1978 father-child duos and 1138 mother-child duos), and nine STR loci (D2S1772 et al.) were typed with the STRtyper®-10F/G system (Condon, China) for 4046 DNA-confirmed paternity testing cases (2115 trios and 1227 father-child duos and 704 mother-child duos). A total of 343,959 meiotic transfers were analyzed and 488 mutations were identified. The overall average mutation rate was estimated as 0.00142 and the estimated locus-specific mutation rate varied between 0.00010 and 0.00422. Mostly only one mutation occurred in a case, while there were 20 cases with double mutations and 2 cases with triple mutations investigated with 24 STR loci genotyping. One-step mutation was accounted for 98.16% of total mutations. The ratio of paternal versus maternal mutation was 3.66:1. Except for those two cases with triple mutations, the combined paternity index (CPI) value of 24 STR loci was greater than 10,000 when calculated including the mutation loci.

How useful is your X in discerning pedigrees?

C. Gomes, M. Magalhães, A. Amorim, C. Alves, N. Pinto, L. Gusmão — 2011-10-05

Abstract: It is known that autosomal unlinked markers are unable to distinguish some pedigrees (such as grandparent/grandchild, avuncular and half-siblings). This poses a problem especially in cases of identification in mass disasters, other human remains, or in heritage cases where it is crucial to define kinships. Theoretically, X-short tandem repeats (X-STRs) analysis should allow distinction of some of those pedigrees and so a practical approach was undertaken in order to quantify the informative power of a set of X-STRs currently available in the forensic community. Therefore, female/female, female/male and male/male individual pairs, linked by the above-mentioned kinships, were tested using Investigator Argus X-12 kit. For different combinations of 2 alternative hypotheses, Likelihood Ratios (LRs) were calculated for 89 female/female, 68 female/male and 63 male/male combinations of (autosomally) indistinguishable pedigrees. When LRs are assessed considering exactly one pedigree in which incompatibilities are possible, satisfactory values are obtained favouring one of the relationships. Apart from these cases, LRs were, in average, low and, in many cases, against the pedigree known to be under analysis.

Automated addition of Chelex solution to tubes containing trace items

Michael Stangegaard, Thomas M. Hansen, Anders J. Hansen, Niels Morling — 2011-09-28

Abstract: Extraction of DNA from trace items for forensic genetic DNA typing using a manual Chelex based extraction protocol requires addition of Chelex solution to sample tubes containing trace items. Automated of addition of Chelex solution may be hampered by high viscosity of the solution and fast sedimentation rate of the Chelex beads. Here, we present a simple method that can be used on an Eppendorf epMotion liquid handler resolving these issues.

Validation of the AmpFlSTR® Identifiler® Direct PCR Amplification kit in a laboratory accredited according to the ISO17025 standard

M. Stene, A. Buchard, C. Børsting, N. Morling — 2011-09-28

Abstract: A total of 249 FTA® card samples were investigated with the Identifiler® Direct kit. PCR amplification was performed according to the manufacturer's protocol with modifications in the reaction volume (10–25μL), the number of PCR cycles (25–27) and the number of FTA-card punches (1–2). Full concordance was observed between the results obtained with the Identifiler® Direct kit and the washed punches typed with the Identifiler® kit. The success rate with one 1.2mm punch in a 15μL PCR reaction volume and 25 PCR-cycles was 93.3%. After retyping of the failed samples, the combined success rate was 98.7%. Overall, the quality of the results was reduced when the number of punches was increased or the PCR reaction volume was decreased. The Identifiler® Direct master mix may be used in combination with other primer sets for direct amplification of other loci, e.g. the 49 SNPs using the SNPforID multiplex assay.

Statistical aspects of familial searching

K. Slooten, R. Meester — 2011-10-28

Abstract: Familial Searching is the process of looking for relatives, by comparing DNA profiles, of a fixed (typically unknown) individual C in a database of known persons. The appropriate likelihood ratios for kinship best summarize the genetic evidence in favour of relatedness for every database member separately. We present probabilistic models to interpret all these likelihood ratios together, and strategies to identify relatives with a certain probability.

Haplotype frequency data of the chromosome X centromere region

J. Edelmann, S. Hering, U. Schmidt, P. Saukko, R. Szibor, C. Augustin — 2011-09-28

Abstract: The centromere region of the human ChrX is known as an area with extremely low crossing-over-rates. Since tightly linked and non-recombining STRs are required for kinship testing, we validated a cluster of six STRs for forensic use in this region, where stable haplotypes can be expected. Our multiplex PCR setup comprised the simultaneous amplification of the six markers DXS10161, DXS10159, DXS10162, DXS10163, DXS10164 and DXS10165, which are located between 56 and 64 Mb distanced from the Xp telomere at Xp11.21-Xq11.1. Differences between several populations are expected. We present allele- and haplotype-frequency data of population samples from Germany, Estonia, Latvia, Lithuania, Wroclaw, Vladivostok, Finland, Japan, Ethiopia and Rwanda. Our ChrX centromere multiplex is a suitable supplement of the commercially available ChrX-Kit Argus X12 and can be used as an additional independent haplogroup.

Hierarchical analysis of 15 Y-chromosome SNPs and demographic history of Afro-derived isolated communities in Alagoas, Brazil

A.M.L. Assis, D.A. Azevedo, G.R.B. Souza, M.V.C. Santos-Filho, L.A.F. Silva — 2011-10-13

Abstract: Through analysis of 15 Y-SNP markers by applying the SNaPshot (Applied Biosystems) method, this study revealed the genetic composition and origin of paternal lineages of 209 male individuals belonging to nine Afro-derived isolated communities in Alagoas, Brazil.

Forensic entomology: Nuclear and mitochondrial markers for Diptera and Coleoptera identification

S. Ferreira, A.R. Oliveira, A. Farinha, M.T. Rebelo, D. Dias — 2011-10-10

Abstract: Accurate identification of insect specimens is an essential step in forensic entomology. Cytochrome c oxidase subunit 1 (COI) is the most used locus for insects molecular identification. However, other studies were done using other genetic markers, as mitochondrial cytochrome b (CytB) and ribossomal second internal transcribed spacer (ITS2). Also, it was shown that COI has some limitations in this field. In this work these three markers (COI, CytB and ITS2) were used, with the aim to infer about its suitability for insects with forensic relevance identification.DNA was extracted from insects found in carcasses of mammals with high homology with humans. COI, CytB and ITS2 regions were PCR amplified. Obtained sequences were matched in BLAST and BOLD-IDS online tools. Sequence divergences and phylogenetic analyses were performed for COI and CytB, using PAUP* v4.0b10 software. However, ITS2 analyses were not performed due to alignment problems.Maximum Parsimony and sequence divergences data for COI and CytB allowed the distinction of insect lineages, with good support. Despite analysis problems, ITS2 proved to be suitable for insects identification.

Human STR genotyping of DNA extracted from the stomach contents of a roof rat

M. Hara, T. Masuda, A. Takada, T. Miyazaki, H. Suzuki, A. Kido, K. Saito — 2011-10-13

Abstract: This study performed human STR genotyping of DNA extracted from the stomach contents of a roof rat found near a corpse. The sample obtained from the stomach of the roof rat was divided into three parts; the control was blood from the corpse. Samples 1–3 contained 273.7, 5.1, and 1.6pg DNA/μl, respectively. Human STR genotyping of sample 1 identified all 15 loci, whereas none was identified in samples 2 and 3. Sample 1 was fatty tissue, sample 2 was muscle, and sample 3 contained sweat glands, epithelial cells, and blood vessels. These results suggested that it is possible to detect human DNA in the stomach of a rat using human STR genotyping.

InDels in Y chromosome haplogroup definition

Joana Damas, António Amorim, Leonor Gusmão — 2011-10-13

Abstract: The characterization of Y chromosome haplogroups is currently done by genotyping SNPs and a few InDels. However, InDels are increasingly gaining importance, so the aim of this work was to create an InDel PCR multiplex allowing a fast, simple and straightforward characterization of the main Y-haplogroups. For this, we have selected the InDels already accepted by the Y Chromosome Consortium. However, due to their position in the Y chromosome phylogenetic tree, they only allow classifying chromosomes from 6 of the 20 main Y haplogroups. Thus, we have extended the search to already described and validated InDels in dbSNP and MGS. All 154 InDels retrieved from that search were subjected to multiple screenings and just 10 were found to be new, potentially polymorphic and Y specific. Their typing in samples for 13 distinct haplogroups confirmed only 2 polymorphisms (named M2 and M14). M2 is polymorphic in R haplogroup but it also shows a reversion within the R1b1b2 sub-haplogroup. Therefore, it is not recommended for the characterization and distinction between R and the other haplogroups. M14 shows variation in R and Q and so it can be used to identify samples belonging to the paragroup P, which was not possible before using the InDels in the phylogenetic tree of the Y Chromosome Consortium. The detailed analysis of all the available information allowed us to conclude that the creation of the aimed multiplex will only be possible with the detection and phylogenetic characterization of new InDels.

A comparative study on the techniques of SNPs analysis for degraded DNA samples

Y. Harayama, T. Oki, T. Hayashi, K. Tsukada, M. Ota, H. Asamura — 2011-12-01

Abstract: In the present study, we focused on X-chromosomal single-nucleotide polymorphisms (SNPs) (rs6641116, rs7471388, rs414960, and rs985251) analysis of 20 degraded DNA samples and assessed two techniques (TaqMan assays and single base extension reactions) by obtaining shorter PCR products. Results from highly degraded samples indicated that a shorter amplicon product is effective for each SNP assay. However, in some degraded samples, single base extension reactions presented difficulties in SNP discrimination despite successful TaqMan assay.

Characterization of U.S. population samples using a 34plex ancestry informative SNP multiplex

2011-10-13

Abstract: The number, scope, and ease of typing of single nucleotide polymorphism (SNP) markers makes them ideal supplements to existing forensic markers sets. SNP typing panels offer additional benefits such as the ability to type degraded DNA, paternity analysis, and the opportunity to infer externally visible traits. SNP also carry the potential to infer the most likely population of origin of an individual using SNPs with highly differentiated allele frequency distributions. One recent example is a 34-plex assay developed by Phillips et al. using SNaPshot primer extension reactions . A significant amount of population information has already been generated, published, and uploaded to the SPSmart open access SNP browsers . The original work by Phillips et al. analyzing the CEPH population diversity panel indicated a low error rate of ancestry prediction when confining comparisons to the three major population groups of Europe, Africa and East Asia. However, admixed populations commonly found in the U.S. represent a significant source of error or at least reduced assignment probabilities when making ancestry predictions. Thorough and wide population surveys in areas where admixture is the predominant pattern, is an important part of the process of assessing this potential source of classification error. In order to contribute to the data already available to end-users, we have generated allele frequencies for a set of U.S. African Americans, Caucasian and Hispanics for 34 ancestry informative SNPs. The data accumulated should contribute to improved characterization of admixed U.S. populations and their analysis through SNP genotyping.

NIST validation studies on the 3500 Genetic Analyzer

2011-10-12

Abstract: An internal validation of the Applied Biosystems 8-capillary 3500 Genetic Analyzer was performed using two commercial short tandem repeat (STR) multiplex kits (Identifiler and Identifiler Plus). Validation experiments to evaluate the performance of this CE consisted of a precision study, sensitivity study, genotype concordance, a two person mixture study, and signal normalization evaluation. The sensitivity study consisted of duplicate injections of amplified DNA template ranging from 1.0ng down to 0.01ng. These male samples were heterozygous at all loci. Statistical evaluation of the sensitivity data was performed to determine the statistical significance between employing a single threshold across all dye channels and opting for dye specific thresholds.

Genetic kinship analysis: A concordance study between calculations performed with the software Familias and algebraic formulas of the American Association of Blood Banks

D.A. Azevedo, G.R.B. Souza, I.H.E.F. Silva, L.A.F. Silva — 2011-10-13

Abstract: To validate the use of software Familias 1.97, a concordance study was performed between the likelihood ratio (LR) calculations carried out with Familias (LRF) and the algebraic formulas published by the American Association of Blood Banks (LRAABB). Complete agreement between LRF and LRAABB was observed in all kinship cases analysed. Results confirm the efficiency and reliability of Familias for LR calculations in the tested cases.

Concordance testing comparing STR multiplex kits with a standard data set

Carolyn R. Hill, Margaret C. Kline, David L. Duewer, John M. Butler — 2011-10-12

Abstract: Multiple concordance studies have been performed at NIST with a standard sample set (≈1450 U.S. population samples) using various STR multiplex kits from Applied Biosystems, Promega Corporation, and Qiagen, including many of the new generation European kits. Out of 948,301 allele comparisons, differences were observed 1109 times resulting in a concordance rate of 99.88%. These differences were reported to the collaborating commercial vendors, which in many cases lead to revised kits and a reduction in null alleles through the addition of degenerate primers or primer redesign.

Fast PCR amplification of AmpFlSTR Yfiler

K. Tsukada, Y. Harayama, M. Shimizu, Y. Kurasawa, K. Kasahara — 2011-10-03

Abstract: At a previous conference, we reported on fast PCR cycling for AmpFlSTR Identifiler by three methods. Allele typing proved successful with all these methods, although amplification times were reduced by 1/3 to 2/3 (to approximately one to two hours). For the current study, we performed PCR amplification with AmpFlSTR Yfiler using AmpliTaq Gold Fast PCR Master Mix, UP(x2). We succeeded in allele typing and reduced PCR running times by half (to approximately 110min).

Estimating stutter rates for Y-STR alleles

2011-10-07

Abstract: Stutter peaks are artefacts that arise during PCR amplification of short tandem repeats. Stutter peaks are especially important in forensic case work with DNA mixtures. The aim of the study was primarily to estimate the stutter rates of the AmpFlSTR Yfiler kit. We found that the stutter rates differ at the allelic level and with the parental peak height.

Comparison of four DNA extraction methods for forensic application

V. Bogas, F. Balsa, M. Carvalho, M.J. Anjos, M.F. Pinheiro, F. Corte-Real — 2011-10-07

Abstract: Challenging biological samples found in crime scenes are often brought to our lab. Several factors, such as degradation and the presence of inhibitors, can difficult the analysis of these samples. Chelating resin, silica membranes, silica-coated magnetic beads and paramagnetic resin were DNA extraction techniques used in this study. Our aim was to find out the DNA extraction method more suitable to overcome problems raised by these samples and to enable the identification of its genetic profile.

Comparison study of four different 16-locus “expanded ESS” STR kits

Stefan Kutranov — 2011-10-05

Abstract: New 16-locus multiplex STR kits incorporating the five additional loci in the expanded European Standard Set (ESS) are now available from three manufacturers. These kits offer a number of advantages including an increased number of loci and improved performance over previously available kits. An assessment of four of the kits by means of comparison to each other and to the Applied Biosystems AmpflSTR® SGM Plus® kit was carried out. The four kits assessed were the Applied Biosystems AmpflSTR® NGM™ (NGM), Promega PowerPlex® ESI16 (ESI16) and ESX16 (ESX16) and the Qiagen Investigator™ ESSplex (ESS) kits. All the kits showed an increase in sensitivity and in ability to overcome inhibition as compared to SGM Plus® and with no increase in stutter proportions or heterozygote imbalance. The next generation kits showed very similar performance to each other. The implications of the different kit configurations are also considered.

A single multiplex SNaPshot reaction of 42 SNPs to classify admixture populations into mitochondrial DNA haplogroups

G.G. Paneto, S. Köhnemann, J.A. Martins, R.M.B. Cicarelli, H. Pfeiffer — 2011-11-16

Abstract: SNaPshot minisequencing reaction is in increasing use because of its fast detection of many polymorphisms in a single assay. In this work we described a highly sensitive single nucleotide polymorphisms (SNPs) typing method with detection of 42 mitochondrial DNA (mtDNA) SNPs in a single PCR and SNaPshot multiplex reaction in order to allow haplogroup classification in Latin American admixture population. We validated the panel typing 160 Brazilian individuals. DNA was extracted from blood spotted on filter paper using Chelex protocol. Forty SNPs were selected targeting haplogroup-specific mutations in Europeans, Africans and Asians (only precursors of Native Americans haplogroups A2, B2, C1, and D1) and two non-coding SNPs were chosen to increase the power of discrimination between individuals (SNPs positions 16,519 and 16,362). It was done using a modified version of a previously published multiplex SNaPshot minisequencing reaction established to resolve European haplogroups, adding SNPs targeting Africans (L0, L1, L2, L3, and L*) and Asians (A, B, C, and D) haplogroups based on SNPs described at PhyloTree.org build 2. PCR primers were designed using PerlPrimer software and checked with the Autodimer program. Thirty-three primer-pairs were used to amplify 42 SNPs. Using this panel, we were able to successfully classify 160 individuals into their correct haplogroups. Complete SNP profiles were obtained from 10pg of total DNA. We conclude that it is possible to build and genotype more than 40 mtDNA SNPs in a single multiplex PCR and SNaPshot reaction, with sensitivity and reliability, resolving haplogroup classification in admixture populations.

Molecular genetic investigations of skeleton finds buried in the ground

K. Thiele, M. Kohl, H. Bruchhaus, J. Dreßler — 2011-10-04

Abstract: In the year 2003, several largely complete skeletons were found on various neighbouring spots during construction works in the vicinity of a Chemnitz detention centre, which had been used for decades. The Authority of the Inquiry did not assign systematic excavation, and so the skeleton material, which was sent in for investigation from the various finding places, also contained skeleton remains of several individuals per finding place. Police investigation, the burial place and some minor clothing remains resulted in the assumption that the graves had been laid out in the first years after World War II. In addition, it was suspected that one of the buried bodies might have been a male person who had been missing at that time.After appropriate mechanical preparation of selected bone materials the DNA-isolation followed, and subsequently quantification with the Quantifier™ Human DNA Quantification kit of the firm Applied Biosystems. The amplification of DNA was carried out with commercially available autosomal and gonosomal amplification kits of various firms. DNA fragment separation was done with ABI Prism™ 3130 Genetic Analyzer of the firm Applied Biosystems.Compared to the pre-investigation, much better and forensically usable results of the analysis were achieved by modified methodology, as will be presented in detail.

Evaluation of the IrisPlex eye colour prediction tool in a German population sample

J. Purps, M. Geppert, M. Nagy, L. Roewer — 2011-10-14

Abstract: The prediction of externally visible characteristics like hair, skin and eye colour from biological material of an individual becomes more and more important in forensic genetics particularly in cases in which a standard tandem repeat (STR) profiling failed. Liu et al. and Walsh et al. recently published a multiplex SNaPshot assay, named IrisPlex. The IrisPlex assay was described as a sensitive tool for accurate prediction of blue and brown eye colour. In our present study we evaluated the IrisPlex eye colour prediction tool in 102 individuals of a German population sample as well as in 81 individuals from Turkey, East Asia and Africa. The applicability of the IrisPlex was verified in a case study with highly degraded DNA of an exhumed skeleton. The IrisPlex prediction probabilities revealed a blue eye colour for the unknown person and a probable European descent. The IrisPlex works reliable and the prediction results are plausible. Due to its easy handling and robust design the IrisPlex is capable to be implemented in the forensic case work to provide first information of an unknown person for which no phenotypic information is available.

Sequences of microvariant/“off-ladder” STR alleles

2011-10-05

Abstract: We have recently developed a sequencing method using a second generation sequencing platform. We typed 111 samples for one or two of 10 short tandem repeat loci and discovered a high degree of sequence diversity. Most variation was seen in the D21S11 locus and least variation was found in the D18S51 locus. Our sequencing method gave a better resolution than traditional capillary electrophoresis methods, and also better resolution than other techniques, such as pyrosequencing and mass spectrometry. We found that poly-A/T stretches longer than six bases decreased the quality of reads, making sequence composition an important factor for the quality of sequencing results.

Interest of X chromosome (Argus X-12 kit) in complex kinship analysis

Laura Cainé, Raquel Carvalho, Sérgio Costa, Maria F. Pereira, Maria F. Pinheiro — 2011-10-04

Abstract: X-chromosomal short tandem repeats (X-STRs) have proven to be informative and useful in complex relationship testing. Paternity trio cases can most easily be solved with only autosomal STRs markers, while test of paternity duos involving more complex family relations could gain from X-chromosomal testing. The main goal of the present study was to investigate twelve X-STR markers (DXS7132, DXS7423, DXS8378, DXS10074, DXS10079, DXS10101, DXS10103, DXS10134, DXS10135, DXS10146, DXS10148, and HPRTB) based on a Portuguese population sample, and their ability to solve complex kinship cases. Evaluations of statistical parameters of interest were also considered.

Population genetic data for F13A01, FES/FPS, F13B and LPL in the south Portuguese population

C. Vieira Silva, A. Amorim, R. Espinheira, H. Afonso Costa, J. Costa Santos — 2011-10-05

Abstract: DNA parentage testing is currently performed using several highly polymorphic short tandem repeats (STRs). In our routine casework, we apply two validated STRs kits, in order to have results in the 13 codis loci plus D2S1338, D19S433, PENTA E, PENTA D and Amelogenin. In complex and deficient paternity cases it is often necessary to increment the number of studied STRs. For this reason, we introduced in our laboratory GenePrint® FFFL Multiplex kit, which can provide results in F13A1, FES/FPS, F13B and LPL using the GenePrint® FFFL System (Promega, USA) kit. In this study we analysed 150 unrelated and healthy individuals from south Portugal population. Allele frequencies and statistical parameters were estimated with Arlequin 3.5.1.2. Paternity Statistics were calculated using software package PowerStats v12. The forensic efficiency values suggested that loci F13A01, FES/FPS, F13B and LPL are discriminative and very useful to solve complex forensic casework, and should be added to the set of STRs loci routinely used in forensic laboratories. In conclusion, an additional 4 loci dataset was established for the south Portuguese population, which can be used for both forensic casework and in complex kinship testing.

Postmortem behaviour of rat microRNA (miRNA) as determined by comprehensive microarray analysis

Masaki Hashiyada, Kiyotaka Usui, Yoshie Hayashizaki, Jun Sakai, Masato Funayama — 2011-10-04

Abstract: We determined a number of miRNAs exist in rats after death using comprehensive microarray analysis with 350 rat miRNAs probes. Our analysis revealed that 237 miRNAs (67.7%) were under the threshold level and 105 (30%) were expressed until 48h after death. Some miRNAs showed the unstable levels of expression at postmortem. Further investigations using quantitative PCR analysis and animal experiments are required before forensic application of this knowledge, such as estimation of time of death or quantification of stress.

The effects of different adhesive tapes and of Hemastix test strips on DNA recovery using magnetic silica beads

Stefan Kutranov, Holly Cullis — 2011-10-05

Abstract: Adhesive tape is used in a number of forensic applications, including immobilising fragments of biological material and retrieval of cellular material from exhibits. A large range of adhesive tapes are available and used within the forensic community and we have assessed the effects of two types commonly used within our laboratories on DNA processing methods. Single-sided 3M Scotch™ tape and double-sided Selotape tape are used to retrieve cellular material from exhibits for subsequent DNA analysis. The adhesive from the single-sided Scotch™ tape was seen to interact with the silica magnetic beads during the DNA extraction process (Qiagen DNA Investigator chemistry on the QIAsymphony® SP instrument) and to cause a reduction in the quality of the results obtained while the double-sided tape showed little effect. This effect is most marked with low levels of cellular material and is evident from even relatively small amounts of tape. Optimisation of the lysis procedure was carried out to reduce the adhesive effect but it was not possible to entirely overcome it.In addition, we have also investigated the previously reported effects of reagents from the Hemastix® test strips (Bayer), commonly used for presumptive identification of blood at crime scenes. We demonstrate that these too can reduce DNA recovery using the Qiagen DNA Investigator magnetic bead chemistry and offer recommendations for their use to prevent this.

Individual identification of fox (Vulpes vulpes) in forensic wildlife investigations

Monique Wesselink, Irene Kuiper — 2011-10-07

Abstract: In recent casework the relatedness of a group of foxes (Vulpes vulpes) was questioned, as the manner of their relatedness might indicate the intentional release of several individuals. To this extent a fox-genotyping technique was pursued. 21 microsatellite markers (STRs) designed for individual identification of dogs (Canis lupus familiaris) were tested on foxes to determine which markers could be amplified reliably and scored unambiguously. For this subset of markers the possibility of multiplex amplification was tested, as were the variability and allele range of these markers.Approximately two third of the tested STRs of which some were combined in multiplex reactions, were amplified and scored readily in foxes. Both the number of alleles and allele range were observed to differ between dogs and foxes for most markers. To understand these differences and their possible effect on the use of these markers the sequence of several alleles was determined. Changes in both flanking regions and repeat structure were identified, however these changes did not influence the use of the STRs for identification purposes.After genotyping the case samples with this subset of markers a conclusion was formulated expressing the likelihood of a familial relationship of all individual foxes. The obtained genotypes are stored to compare with biological material found if a suspect of an intentional release were to be found.

Single multiplex system of twelve SNPs: Validation and implementation for association of SNPs with human eye and hair color

V. Kastelic, K. Drobnič — 2011-10-14

Abstract: Predictions of human traits from biological stains with genetic methods have recently gained tremendous interest in criminal investigations. Various studies have revealed that single nucleotide polymorphisms (SNPs) within the HERC2, OCA2, MC1R, SLC24A5, SLC45A2 and TYR genes have been strongly associated with pigmentation trait variations in Caucasian populations. The prediction probability estimation for eye and hair color in the investigation conducted on the Slovenian population is associated mostly with two HERC2 SNPs: rs1129038 and rs12913832, with a probability value of 0.96, as has also been indicated in previous investigations. Other SNPs have a small additional affect on the prediction of eye color (OCA2 – rs1667394, rs7170989, TYR – rs1393350, and SLC45A2 – rs16891982) or hair color (OCA2 – rs1667394, rs7495174, rs7170989, MC1R – rs1805005, and TYR – rs139335). The variation in the rest of the SNPs (MC1R – rs1805008, OCA2 – rs1800407, SLC45A2 – rs26722, and SLC24A5 – rs1426654), are not as meaningful in terms of the prediction of values for eye and hair color. The SIplex assay is designed to be very sensitive, in order to indicate all twelve SNPs genotypes with only 62.5pg of DNA. Using twelve SNPs, eight of which being more or less usable for eye and hair color prediction based on the human genotype, we managed to develop a sensitive and reliable multiplex genotyping assay.

Forensic identification of 12 mammals species based on size variation of mitochondrial cytochrome b gene using multiplex PCR assay

M. Črček, K. Drobnič — 2011-10-05

Abstract: In this study, we have applied the endpoint PCR method based on size variation of mitochondrial cytochrome b gene among mammals for identification of 17 mammals. We successfully identify 12 out of 13 mammal species common in the geographic region of Slovenia (Central Europe and West Balkan) using multiplex PCR with our own designed primers for 7 species (cat, pig, lynx, roe and red deer, wolf, bear) and a conventional system, i.e. non-fluorescent primers and agarose gel electrophoresis. Only primer designed for identification of Eurasian lynx turned out to be unspecific. Probably, because it was designed on to sequence data from species close to Eurasian lynx while sequence data for Eurasian lynx was not available in databases at the time of our research. With this method we could not distinguish between subspecies (wolf from dog, and pig from wild boar). The species-specificity of PCR amplicons was further demonstrated by the ability of the assay to accurately identify species-specific DNA from mixed samples.

Molecular autopsy in traumatic death. A case report

L. Buscemi, F. Alessandrini, M. Colombi, A. Tagliabracci — 2011-10-05

Abstract: To investigate a violent death of a 16-years-old boy that occurred at a distance of less than 24h from a minor traumatic event, a medicolegal autopsy was performed. Genetic analyses were extremely useful for the definition of the cause and circumstances of death. Authors highlight the importance in establishing clear guidelines for molecular autopsies in forensic medicine.

Practical value of the marker MUC4 for identification of vaginal secretion in penile swabs

G. Hadžić, A. Lukan, K. Drobnič — 2011-10-05

Abstract: The mRNA profiling method combined with a reverse transcription endpoint PCR method has been proven to be a suitable technique for the identification of vaginal secretion. The correct identification of a biological source is of vital importance in the samples from sexual assault cases. We have confirmed the reliability of the most common vaginal secretion mRNA-marker MUC4 to detect vaginal secretion in vaginal swabs in 33/42 samples. All negative results were obtained from a woman in menopause. Cross reactivity was detected in 3 out of 52 saliva samples and no cross reactivity (0/10) was detected in samples taken from tonsils. We evaluated the practical value of specificity of MUC4 for the identification of vaginal secretion by testing eight types of penile swabs taken after different sexual intercourses. All samples (5/5) taken from uncircumcised penis after heterosexual sex were positive for MUC4, but only one sample (1/5) from circumcised penis was positive for MUC4. No detection of any marker of interest was observed in penile swabs taken after homosexual anal sex. Additional buccal swabs from tonsils of men and women and penile swabs from clean penis without sex in past two days, were used as a negative control, while markers statherin and histatin 3, specific for saliva, served as positive markers. Both markers STATH and HTN3 were positive (52/52) in those samples as we expected (saliva) and negative in all others (0/92). The paper demonstrates that the correct identification of biological source of samples as vaginal secretion could be possible only if specific markers for saliva are tested coincidently with marker MUC4, while negative results should be taken with great precaution.

Comparison and optimization of DNA recovery from sperm vs. epithelial cells using laser capture microdissection technology and an immunofluorescent staining system

G. Axler-DiPerte, S. Orans, A. Singh, T. Caragine, M. Prinz, Z.M. Budimlja — 2011-10-07

Abstract: Laser capture microdissection (LCM) is an accurate and robust tool for isolation and separation of pure cell populations from heterogeneous mixtures via direct visualization and collection of elements. In this study, the capability of the P.A.L.M.® LCM for separation of sperm and epithelial cells, combined with immunofluorescent Sperm HY-LITER™ stain was assessed. LCM was evaluated for its sensitivity, specificity, and reproducibility using sperm–epithelial mixture slides with varying sperm counts and ages ranging 1–9 years. In addition, slides stained with immunofluorescent dyes were tested. Separation of sperm and epithelial cells using LCM is a robust method for processing of mixed samples and generates interpretable single source DNA profiles. Immunofluorescent staining method is compatible with the platform.

Mitochondrial genes allow discrimination between three cynegetic species for forensic purposes

D. Gamarra, A. Lopez-Oceja, B.J. Gomez-Moliner, M.M. de Pancorbo — 2011-10-12

Abstract: Efficient tools for consistent species identification are essential to provide information about the implication of wildlife in forensic-related events. The objective of this study was to determine potential markers on the mitochondrial cytochrome b (CYTB), cytochrome c oxidase subunit I (COX1) and 16S rRNA genes for discriminating between three important cynegetic species. Samples included deer (Cervus elaphus), roe bucks (Capreolus capreolus) and wild boars (Sus scrofa). PCR amplification was performed with in-house designed primers followed by separation via capillary electrophoresis. The sequences were edited with Chromas Pro 1.34 and aligned with Clustal X v2.0. Consensus sequences of CYTB, COXI and 16S rRNA genes revealed interspecific mtDNA (p<0.05) to discriminate each of the three game species.

Complete automated DNA procedure to facilitate DNA database collection

F. Jaffredo, F. Freund, S. Godichaud, M. Gaboyard, J.P. Moisan — 2011-11-16

Abstract: To address the requirements of DNA database workflow, a complete automated procedure is necessary. Analysis by direct PCR on FTA® punches is possible. However, since DNA quantity is highly variable from one sample to another, genotyping STR profiling leads to a very high rate of invalidated genetic profiles (20–25%). In order to be in the best amplification conditions, it is important to isolate good quality DNA in a normalized concentration. To meet this requirement in one step, we used Smart D-N-Adem-kit for profiling (ADEMTECH) containing calibrated magnetic nanoparticles suitable with a fast and completely automated procedure, from DNA extraction to injection plate setup. The DNA extraction and normalization procedure was successfully validated on 2047 FTA® cards, leading to 95.5% success rate with av. PHR of 86.5%. DNA quantification is no longer necessary prior to STR typing. Similar results have been shown with buccal sample swabs.

Cold cases: New technologies for DNA analysis allow the reopening and solution of unsolved cases

A. Caglià, P. Stefanoni, A. La Rosa — 2011-10-13

Abstract: In recent years considerable progress in the field of forensic science (latent prints, ballistics and biology) have occurred, causing the reopening investigation of old unsolved murders, called “Cold Cases”, especially when findings can still be analyzed and/or re-analyzed taken into account new technologies and new scientific methodologies.The UDI working group (Unsolved Crimes Unit) of the Italian National Police, which collects scientific and technical expertise and investigators, deals to assess which unresolved cases could be selected in relation to new investigative hypotheses or/and in the presence of not analyzed samples.During last two years about 30 cases, occurred between 1985 and 2005, were selected.In particular, in eight cases the DNA analysis led to the identification of one or more genetic profiles that are considered of great interest from the investigative point of view, allowing the identification of the murderers in three different cases.

A new assay for identifying endangered species in Traditional East Asian Medicine

Shanan S. Tobe, Adrian Linacre — 2011-10-05

Abstract: Despite renewed public interest, education, research and legislation, Traditional East Asian Medication (TEAM) continues to incorporate animal parts from endangered species and can be obtained openly in many countries. Products sold as having medicinal properties include plasters, pills, ointments, tonic wine and others. Due to the low population levels of some endangered species other more common species may be substituted or very small amounts may be used. This results in low levels of DNA which can be difficult to identify. Most species identification tests rely on sequence comparison with a known reference sample. Most TEAM contains mixtures of several different species making this technique unsuitable.We describe a novel single step assay to rapidly and simultaneously identify rhino, tiger, bear, leopard, pangolin, musk deer and several non-endangered mammals often substituted in TEAM. The test targets the mitochondrial genome to amplify species-specific fragments that can be separated easily using a genetic analyzer. Each fragment is of a different size so that none can be confused. The specificity of each primer pair allows for species identification to be made even if a mixture of several species is present. Further, if more than one of the target species is present then all the species will be amplified and identified simultaneously. The test is sensitive to very low levels of DNA equating to several hundred mitochondrial copies (a fraction of single cell).

Family selection study among DNA samples collected from Amerindian ethnic group (Wayuu) in northern Colombia

2011-10-05

Abstract: In the Sonoda-Tajima Cell Collection, one of the cell bank of RIKEN BioResource Center, DNA samples from an Amerindian ethnic minority group (Wayuu) in Colombia analysed for 21 autosomal STRs, 17 Y-STRs and HV-I and -II regions in mtDNA to select the kinships among those samples in order to study on how the kinships influence population genetic analysis. As a result, totally 17 kinships including 4 mother–child, 6 one-parent-two-child, 3 sibling relationships, and 4 kinds of complicated kinships were selected. All the paternal linages were confirmed by the Y-STR haplotypes. The sequence data of almost the maternal linages were concordant with each relationship. When compared between the 98-samples population and the 78-samples population in which the first degree of parentage relationships were removed, no significant difference was observed at allele frequency distributions, and the topology in a NJ-tree with the data from the other 20 ethnic groups in South America was not different. It suggested that about one-fifth of parentage relationship in such population size does not influence population genetic analysis.

Establishment of Italian national DNA database and the central laboratory: Some aspects

R. Biondo, F. De Stefano — 2011-10-17

Abstract: On 27 May 2005 in Prüm was signed a convention between the Kingdom of Belgium, the Federal Republic of Germany, the Kingdom of Spain, the French Republic, the Grand Duchy of Luxembourg, the Kingdom of the Netherlands and the Republic of Austria on the stepping up of cross-border cooperation, particularly in combating terrorism, cross-border crime and illegal migration (hereinafter the Prüm Treaty) improving the exchange of information and data resulting by DNA, dactyloscopic and vehicle data to regulate forms of closer cooperation between law enforcement and judicial authorities.On 23 June 2008 the Council of the European Union adopted the Prüm Treaty under the Treaty on European Union with the Decisions 2008/615/JHA and 2008/616/JHA.On 14 July 2009 the Italian Parliament has passed the act n.85/2009 published on the Official Journal (G.U.) n.160 Supp.Ord.n.108/L G.U. General series with the title: “Adhesion of the Italian Republic to the Prüm Treaty. Establishment of national DNA database (NDNADB) and the central laboratory for the NDNADB” in order to facilitate the identification the perpetrators of crimes.The purpose of this paper is to illustrate some aspects described on the chapters of the law as the organization, the rules, the types of activities of NDNADB and the central laboratory, the categories of the person for inclusion in the DNA database, the methodology of analysis of evidence and reference sample, etc.

Italian population data for the new ENFSI/EDNAP loci D1S1656, D2S441, D10S1248, D12S391, D22S1045. The GeFI collaborative exercise and concordance study

C. Previderè, P. Grignani, S. Presciuttini, the GeFI Group — 2011-10-17

Abstract: A collaborative exercise of the ISFG Italian Working Group GeFI was organised to investigate the five new ENFSI/EDNAP miniSTR loci D1S1656, D2S441, D10S1248, D12S391 and D22S1045. Allele frequencies were determined in a sample of 960 individuals collected by the nineteen participating laboratories. The concordance of the genotypes has been evaluated in duplicate experiments, using different kits. No discordant genotypes were revealed for the five ENFSI/EDNAP miniSTR markers. All the labs participating in the collaborative exercise correctly typed the two blind blood stains sent for proficiency testing.

Likelihood ratio statistics for DNA mixtures allowing for drop-out and drop-in

2011-10-12

Abstract: The likelihood ratio (LR) is the recommended approach for forensic DNA mixture analysis by the DNA commission of the ISFG, as it makes maximum use of available data and parameters for allelic drop-out and drop-in can be incorporated. We have developed and validated a LR method and software for analysis of mixed evidence samples in which drop-out of true contributors′ alleles and drop-in of extraneous alleles may have occurred. This method, the forensic statistical tool (FST), employs empirically determined drop-out and drop-in probabilities for single source samples and mixtures. The LR is computed for pairs of prosecution and defense hypotheses based on sample characteristics specified by the user. Data from up to three evidence amplifications may be considered simultaneously. The performance of the program was evaluated with hundreds of profiles generated from blood and buccal samples, purposefully degraded samples, and touched items with one, two, three, and four known contributors. The validation demonstrated that FST assigns an appropriate weight to all types of comparisons. FST is now being used in casework and results continue to be consistent with qualitative assessments.

Latin-American Society Of Forensic Genetics (SLAGF) results of the Interlaboratory Quality Control Exercise 2010–2011

2011-10-17

Abstract: Latin-American Society of Forensic Genetics (SLAGF) Interlaboratory Quality Control Exercise (2010–2011) included the analysis of three bloodstain samples in FTA Classic Card (three persons, biologically unrelated) and one theoretical exercise. There were 56 participating laboratories from 13 Latin-American countries that belong to society, were reported 70 STRs, including autosomal and sex chromosome markers with consensus in 53 STRs with a rate in reporting errors of 2.3%. Fifty-six laboratories reported results in theoretical exercise with mistakes in calculation of IP for each marker.It is necessary to hold meetings to discuss the results of this exercise to reach conclusions and recommendations on all aspects of DNA forensics analysis and paternity test, to improve results and quality in the results of each laboratory.

Re-evaluation of the identical-by-state method in pairwise kinship inference

T. Tamura, M. Osawa, Y. Inaoka, S. Tanaka, F. Satoh, T. Nakamura — 2011-10-05

Abstract: The identical-by-state (IBS) method in pairwise kinship analysis is based on probabilities of shared allele numbers (0, 1 and 2). The sharing probabilities in parent–child, full siblings and non-relatives depended on the heterozygosity of loci. In the present study, the performance of the IBS method was re-evaluated using the data for 15 short tandems repeat loci in the Identifiler system. Whereas the IBS method generally produced lower values of combined indices of the likelihood ratio, smaller deviations of the distributions were evident. The heterozygosities of the 15 loci were consistent across various population groups. The convenience of fixed values of the likelihood ratio in the IBS method is beneficial for cases with uncertain allele frequencies and rare alleles.

Assigning confidence to sequence comparisons for species identification: A detailed comparison of the cytochrome b and cytochrome oxidase subunit I mitochondrial genes

Shanan S. Tobe, Andrew C. Kitchener, Adrian Linacre — 2011-10-05

Abstract: Species identification is a tool used extensively in forensic science; particularly in the investigation of wildlife crime. The two most commonly used genetic loci in species identification are the cytochrome oxidase I gene (COI) and the cytochrome b gene (cyt b), and identification is generally carried out through the use of DNA sequencing. However, there is currently no standard method to quantify the data from sequence comparisons for presentation in reports and to courts as there have been no detailed studies of the expected levels of inter- and intraspecific variation.For the first time this study provides a detailed comparison of the effectiveness of these two loci. Interspecific and intraspecific variation are assessed and statistical confidence is applied to sequence comparisons. Comparison of 217 different mammalian species revealed that cyt b more accurately reconstructed their phylogeny and known relationships, and gave better resolution when separating species based on sequence data.Intraspecific variation was assessed using three model species and showed variation ranging from 0 to 1.16% (Kimura 2-parameter p-distance (K2P)×100%), indicating that some level of variation should be expected. Interspecific variation was greater in cyt b than in COI. Using a K2P (×100) threshold of 1.5, cyt b gives a better resolution for separating species with a lower false positive rate and higher positive predictive value than those of COI. This study allows, for the first time, application of statistical confidence to sequences comparisons for species identification.

Study of two X-linked microsatellite blocks: Allelic frequencies in mixed and isolated population groups

M. Castañeda, A. Odriozola, V. Mijares, M.T. Zarrabeitia — 2011-10-28

Abstract: Two X-linked microsatellite blocks were studied in Cantabria, a small region in Northern Spain. DNA samples were obtained from two different areas: a coastal area, with a well mixed population, and a mountainous area, with a population that has limited communications for social interaction with people from other communities. Statically significant differences were found in allelic frequency distribution between the populations analyzed, revealing the importance of a proper interpretation when kinship analysis and forensic cases include individuals from relatively isolated populations whose frequencies can differ from the general population data.

Verification of alleles by using peak height thresholds and quality control of STR profiling kits

Linda Albinsson, Johannes Hedman, Ricky Ansell — 2011-10-17

Abstract: In the autumn of 2010 SKL performed in-house validation of PowerPlex ESX 16 System (Promega). As the validation showed that very low amounts of DNA (∼10pg) may provide correct allele callings (peaks above 50rfu), we investigated the linear range, i.e., the interval of DNA amounts where a profile is well balanced and does not contain drop-outs and/or drop-ins. The linear range as indicated by our results is approximately from 0.5ng (manufacturer's recommendation) to 2.0ng of DNA. As minute DNA amounts may be detected using the kit, extra care needs to be taken not to report a contaminant allele as a part of the correct profile. A way to verify the correctness of a single donor profile in routine analysis, without using duplicate analysis, is to use conservative peak height thresholds. We determine STR marker specific peak height thresholds for each new lot of DNA profiling kits, based on the results from three different tests: heterozygote balance, signal intensity and repeatability, and PCR inhibitor tolerance. The tests also serve to verify the quality of the kit lot. Generally, the peak height thresholds vary between 200 and 250rfu for heterozygote alleles, with doubled values used for homozygotes.

False homozygosity at D12S391 locus: A case report

V. Cortellini, A. Agostino, A. Verzeletti, N. Cerri, F. De Ferrari — 2011-10-12

Abstract: STR analysis is currently used in paternity testing. We report a case of an apparent paternal inconsistency due to low discrimination power of heterozygosis status at D12S391 locus.

How to mimic realistic case work samples for small scale in-house validation—A serving suggestion

2011-10-17

Abstract: Validation of in-house and commercial protocols for DNA-extraction and PCR analysis has become a central issue in improving and amending techniques in forensic DNA laboratories, especially those focusing on accreditation requirements. We present a practical procedure for mimicking realistic case work samples and a comparative in-house validation study on different DNA-extraction methods routinely applied in our laboratory.

Molecular analysis of botanical evidence by DNA thermal dissociation temperature

S.S. Tobe, R. Thatcher, K. Gracie, N. Watson — 2011-10-12

Abstract: This work was based upon earlier studies which exploited indels located in the mitochondrial genome to discriminate between different varieties of grass. The nad7 and nad5 indels were amplified from the DNA of nine different varieties of amenity grass species typical of lawn and recreational use. Discrimination was achieved by differences in Tm values as shown by dissociation curve and supported by gel electrophoresis. Experiments were also implemented to determine the applicability of a genetic test using samples that were exposed to different environmental conditions. These were designed to include simulated conditions experienced by real forensic samples and consisted of grass leaves, stored dry inside paper envelopes at room temperature, desiccated grass leaves contaminated with fungal growth, and also stains made from grass leaves on cotton cloth to simulate grass marks that might be found on clothing. These stains were stored in a variety of conditions including, contact with soil, water logged, dry and exposed to sunlight and also stored dry in the dark and at 4°C for comparison. All samples except the soil and the fungus contaminated samples gave amplification products that could be distinguished by means of both gel electrophoresis and by Tm determined by a dissociation curve.

Developmental validation of a fully automated genotyping assay capable of detecting length and sequence variation in the CODIS STR loci

David D. Duncan, John V. Planz, Steven A. Hofstadler, Thomas A. Hall — 2011-10-07

Abstract: Sequence variants have been observed in short tandem repeat (STR) loci, but are not detected with conventional electrophoretic analyses. We have validated a high-throughput assay providing base composition analysis of the thirteen CODIS STR loci plus the amelogenin locus. Alleles are amplified with an 8-well panel and the PCR products are directly analyzed on an automated electrospray ionization-mass spectrometry (ESI-MS) platform. The resulting mass determinations are converted to base compositions specifying the number of each of the nucleotides in the PCR amplicons, and STR profiles are derived from the base compositions. Notably, the accuracy of the mass measurements supports detection of sequence variants of STR alleles. Here we describe the developmental validation of the assay and summarize results from analysis of sample panels characterizing allele frequencies, the inheritance of SNP alleles, and the occurrence of germ line mutations in trio sample sets.

Mutational dynamics of STRs: Analysis of FGA-FIBRA locus

Manuel Paredes L. — 2011-10-17

Abstract: The microsatellite FGA-FIBRA locus, is a STR of complex structure. Because the high polimorphism it represents one of the more useful loci in forensic investigation. In 31,808 cases submitted for paternity or maternity DNA analysis during 39 months, 15 STR loci were studied and 97 mutational events were detected. All of the other analysed markers were compatible with expected segregation pattern and the paternity probability was higher than 99.999%. The origin of mutations was predominantly paternal. In general, 5 alelles are involved on 82% of events. All of them were complete gains or losses. We detect more gains events that losses. The gains increases if the allele size increases too, but this behavior is observed only until the 25 allele. On the other hand, from 27 allele, the loss is ten times more frequent than the gains. The mutation frequency by maternal or paternal origin was calculated. The sequence analysis showed that mutations involved the variable zone of the STR.

Postmortem molecular analysis to SIDS victims

M. Osawa, Y. Inaoka, I. Hasegawa, F. Satoh — 2011-10-10

Abstract: In the present report, our recent molecular analyses to SIDS victims are introduced. The majority of congenital central hypoventilation syndrome patients carried the polyalanine repetitive expansion on exon 3 of PHOX2B. In contrast to the presence of the expansions in all clinically diagnosed CCHS patients, no abnormalities of the gene sequence were evident in a total of 87 SIDS cases. We finally concluded that CCHS should be too rare to be involved in SIDS. For long QT syndrome, a total of six non-synonymous substitutions were detected from direct sequencing of the responsible ion channel genes such as KCNQ1, KCNH2, and SCN5A in specimens from the victims. We therefore considered that SIDS might be partly explainable by fatal arrhythmia. Postmortem molecular analysis is not omnipotent in disease diagnosis, but this approach is effective for searching for disorders in which mainly physiological dysfunction is exhibited.

Tackling poaching: Recovery of human DNA profiles from deer remains

Shanan S. Tobe, James Govan, Lindsey A. Welch — 2011-10-07

Abstract: Poaching is a worldwide crime that can be difficult to investigate due to the nature of the evidence. Previous studies have focused on the identification of endangered species in cases of poaching. Difficulties arise if the poached animal is not endangered. In the UK deer have hunting seasons whereby they can legally be hunted. Therefore, identification of deer alone has little probative value as samples could have originated from legal hunting activities in season. After a deer is hunted it is usual to remove the innards, head and lower limbs. The limbs are removed through manual force and represent a potential source of human ‘touch DNA’.We investigate the potential to recover and profile human autosomal DNA from poached deer remains. Samples from the legs of ten culled deer were obtained (40 in total) using minitapes. DNA from samples was extracted, quantified and amplified to determine if it would be possible to recover human STR profiles. Low quantification data led to the use of an extended PCR cycling protocol of 34 cycles. Samples from five deer gave match probabilities of varying quality.This study demonstrates the recovery of human touch DNA from poached animal remains. This is the first time that human STR profiling has been successfully applied to touch DNA in regards to wildlife crime.

Development of a genotyping assay for the UK, European, and CODIS core STR loci identifying length and sequence variation in the target loci

2011-10-04

Abstract: We have developed a highly automated assay covering twenty one markers spanning the UK, European, and CODIS core short tandem repeat (STR) loci. In this assay, sample DNA is amplified with an 8-well PCR panel, and the masses of the PCR products are determined on an electrospray ionization-mass spectrometry platform. The mass measurements are accurate enough to assign a base composition to the PCR products, specifying the number of their constituent dA, dG, dC, and dT residues. The base compositions in turn define the STR genotypes. Sequence variants have been observed in many of the STR loci, and the accuracy of the mass measurements supports their identification with this assay. Parameters in the initial evaluation of the assay included species specificity, sensitivity, reproducibility, accuracy, and concordance. Results indicate that the assay is concordant with existing assays while providing additional information by virtue of the detection of sequence variants of STR alleles.

Can microbes on skin help linking persons and crimes?

Anu Aaspõllu, Triin Lillsaar, Lea Tummeleht, Jaak Simm, Madis Metsis — 2011-10-20

Abstract: Linking persons to crimes through DNA analysis is a well-established approach for more than 25 years. While enormous numbers of cases all over the world have been solved based on DNA, there is still need for additional tools for improvement in physical evidence choice and collection. Unfortunately not all samples collected from crime scenes, are suitable for linking persons to crimes due to the quantity and quality of the human DNA collected. Recent studies have shown personality of bacterial community on human body surface that may open new perspectives for forensics.The aim of the study was evaluation of variability of bacterial communities on skin of palm and fingers between and within individuals as well as transfer of bacterial DNA during contact to the object and persistence of community parameters during storage. Four volunteers were recruited and samples collected during 5 days and afterwards once per week during 1 month in the morning and in the afternoon by swabbing of palm and fingers after holding sterile object for 1min with persons dominant hand. The samples were also collected from the handled objects, except the first object, in which surface was quartered and sampled by quarter zone with 1-week interval after storing the object at room temperature. DNA was extracted and metagenomic analysis of bacterial community using 16S rRNA gene hypervariable regions was performed on Roche/454 platform. Our preliminary results are promising however foresee need for more elaborative studies to be able to implement this approach into the routine practice.

Success rate of LT DNA analyses in casework

C. Dufva, A. Nilsson — 2011-11-09

Abstract: There is often a demand for statistics to evaluate and improve different analytical methods. This is especially important in forensic DNA analysis, facing a wide variety of items and substrates, from which samples are collected. For LT DNA analysis it is valuable to have data showing what kind of results that are received, readily divided into different categories of items. We have developed a database showing the success rates for different items. During the years 2008–2010 LT DNA results were collected in 417 cases, with a success rate of 38%. Corresponding success rate per sample in the different categories of items (i.e. knives, electronics and bombs, letters and envelopes) vary between 7% and 19%.

Effects of the most common methods for the enhancement of latent fingerprints on DNA extraction from forensic samples

S. Gino, M. Omedei — 2011-10-13

Abstract: The aim of the research was to understand if the use of chemicals compounds used to enhance latent fingerprints, might interfere with the extraction and amplification of DNA from biological samples on crime scenes. Only three methods were used: powders (black and white ones, used on non porous surfaces, and here applied on glass), cyanoacrylate (used on non porous surfaces, and here applied on plastic, silver, plastic-coated paper, panty hose and glass too) and DFO (only used on porous surfaces and here applied on white, colored and recycled papers). The biological samples put on surfaces included blood, saliva and fingerprints applied to all substrates by pressing for 5s. Finds were analyzed not only upon 24h, but also after 7, 30 and 60 days. “Untreated” samples have been used as control. DNA coming from each model was quantified by using three different kinds of techniques: the first is a qualitative one (amplification of the beta-globin gene) and the other two (Real Time PCR and Nano Drop) are quantitative. The obtained results from each sample are very similar for blood and saliva: independently from the presence of chemical compounds or trace age DNA extracted was useful for typing complete genetic profile with STR markers. Only for latent fingerprints, were noticed important differences between spectrophotometer analysis and the techniques based on PCR. To resolve these ambiguities, STR amplification and mtDNA sequencing were performed. Only mtDNA sequencing could be classified as a good technique to extract DNA from this kind of fingerprints.

Kinship analysis based on SNP data from microarray assay

Y. Inaoka, A. Tajima, T. Tamura, F. Satoh, M. Osawa — 2011-10-07

Abstract: Pairwise kinship analysis is the basic approach in forensic examinations. The conventional assessment is achieved after statistical calculation of genetic data from a set of STR markers. However, the combined index of likelihood ratio does not separate siblings from unrelated pairs. In the present study, we ensured the effectiveness of SNP microarray assay to the kinship analysis. Reproducible large-scale genotypes data were obtained by the assay. In the setting of more than 0.05 of minor allele frequency and r2=0.2 in linkage disequilibrium, the SNP data was analyzed using a IBD-based coefficient, π, in addition to IBD values. The minimum numbers of SNP subset for separation of full siblings and second degree pairs from unrelated pairs should be 1000 and 5000, respectively. SNP microarray assay will be useful for the analytic approach for full siblings and the second degree pairs.

The effect of increased cycle numbers using the Quantifiler® Duo DNA Quantification Kit (AB) on the detection of minute amounts of male DNA in mixtures and its application in routine case work

K. Anslinger, B. Bayer — 2011-11-07

Abstract: In this study the effect of increased cycle number conditions for Quantifiler® Duo DNA Quantification Kit (AB) on the detection of minute amounts of male DNA in male/female mixtures was tested. 92 male/female mixtures with minute amounts of male DNA were quantified with the Quantifiler Duo Human Quantification Kit using 40 and 45 cycle conditions. Comparison of the results showed that the minute amount of male DNA was detected more reliably under increased cycle conditions. Therefore 508 mixtures with different male/female ratios from our routine case work samples were quantified twice using the 45 cycle conditions. Depending on the mixture ratio and the overall DNA amount, the samples were grouped and typed with 17 autosomal and/or 16 Y-chromosomal STRs. Each profile was confirmed by a second multiplex PCR. In 8 of 311 samples, in which no male DNA was detected in either quantification reactions, full or partial reproducible Y-chromosomal profiles could be obtained. In conclusion, increasing the cycle number of RT PCR leads to an improved sensitivity but cannot exclude false negative results. Furthermore this study investigated the dependence of the total amount of DNA, the male ratio and the reproducibility of Y-chromosomal marker as well as the drop out frequency of different Y-chromosomal STRs.

Population data for 8 Y-chromosomal STRs (not included in Y-filer™ kit) in a population sample of Czech Republic

2011-10-17

Abstract: 142 unrelated males from the population sample of the Czech Republic were genotyped using the miniSTR pentaplexes I and II in 8 Y-chromosomal STRs not included in Y-filer kit (Applied Biosystems, USA). A total of 142 haplotypes were obtained of which 127 were unique. The most common haplotype were found in 4 samples. We observed the haplotype diversity of 0.998 and the discrimination capacity of 0.894.

Molecular structure and genealogical characterization of the DYS458.2 allelic variants founded in Turkey population samples

A. Serin, H. Canan, B. Alper, D. Kotan — 2011-10-05

Abstract: We found 38 intermediate alleles of DYS458 locus in a 249 Turkish population samples. We have examined the molecular structure of these allelic variants and association of J1 haplogroup, using by sequence analyses. Analysis results showed that all of the samples with DYS458.2 variant have incomplete repeat caused by an AA insertion or GA deletion in front of the third repeat form from the last and have M267 Single Nucleotide Polymorphism (SNP) specific to haplogroup J1. This finding is supportive to common origin for the DYS458.2 allelic variants.We have also observed that all of the 38 samples have been shared the same alleles at some Y-chromosome Short Tandem Repeat (Y-STR) loci when we have examined 17-YSTRs loci in our population data. These alleles are DYS392*11, DYS393*12, DYS438*10 and DYS458.2 variants. Complete dependency was detected noteworthy between DYS458.2 variants and three loci; DYS392*11, DYS393*12 and DYS438*10 respectively, related with the same origin. However, further studies should be done with more additional samples.

Evaluation of the IrisPlex system in admixed individuals

P.R. Prestes, R.J. Mitchell, R. Daniel, K.N. Ballantyne, R.A.H. van Oorschot — 2011-10-05

Abstract: The prediction of externally visible characteristics (EVCs) is an important investigative tool and a growing field in forensic genetics. The IrisPlex system uses six single nucleotide polymorphisms to predict blue and brown eye colour in humans with over 90% precision. However, the accuracy of this system has not been tested in samples with known genetic admixture. We therefore tested the IrisPlex assay in 64 samples with known and varying levels of Asian-European genetic admixture. Self-declared eye colour information was obtained from participants. The overall accuracy rate for eye colour assignment was 94%, however only 50 samples achieved classification above the 0.7 probability threshold employed. The correct eye colour was predicted in 100% of both blue and brown eye colour samples although none of the green eye colour samples were accurately predicted. When the probability threshold is removed, the accuracy decreases to 84.4% for all 64 samples, with only 94.1% and 85.7% of brown and blue phenotypes predicted correctly. There was also a trend for decreasing accuracy with increasing number of generations since admixture, where individuals with levels of admixture 1:1 and 1:3 were predicted correctly in 96.9% and 87.5% of cases, respectively, and over 1:7 level of admixture correctly predicted in 71.4% of cases. Prediction of EVCs in admixed individuals may present certain difficulties but has been shown to be possible. Larger sample sizes and different types and levels of genetic admixture can improve our knowledge and assist the predictive accuracy in admixed samples.

Rapid analysis for confirmation of amelogenin negative males characterized by a Yp11.2 deletion

A. Giuliodori, S. Corato, E. Ponzano, D. Rodriguez, L. Caenazzo — 2011-10-07

Abstract: In humans, the amelogenin gene is present on both the X and the Y chromosomes: there are size differences in this gene between these chromosomes, which have been utilised for sexing in forensic casework and prenatal diagnosis. The assay typically generates a 106bp long fragment from the X chromosome and a 112bp long fragment from the Y chromosome. Several studies have shown that the amelogenin gender test may not always be concordant with true male gender. Deletions of AMELY can result in no amplification product and these null AMELY alleles can occur in different percentages in different population groups. The literature data support that the null allele is the result of a larger deletion on the short arm of the Y chromosome. Considering the consequences of the result obtained using only the amelogenin marker, and potential interpretation difficulties in the few cases where gender misinterpretation may be problematic, in this paper we propose a method for the identification of samples with deleted AMEL based on a small polyacrylamide-gel electrophoresis of a duplex PCR product of a novel marker residing in the SRY gene, which results in a 197bp long PCR product in combination with primers for AMEL. This method can be applied, as an additional assay, in case of doubt regarding the presence of deleted AME in the DNA profile.

A de novo mutation in Caveolin-3 gene may confer genetic susceptibility to Long QT syndrome

2011-10-04

Abstract: Long-QT syndrome (LQTS) is a potentially lethal, inheritable arrhyithmia disorder, stemming from perturbed cardiac repolarization and affecting about 1 in 3000 persons. The pathogenetic basis for LQTS has focused on ion channels. Recently, 2 LQTS-susceptibility genes encoding for two non-ion channel proteins have been discovered. The first gene encodes an adapter protein, ankyrin B, which participates in localization of sodium and calcium channels to the sarcolemma; the second one encodes Caveolin-3, the major scaffolding protein present in caveolae in the heart: mutations in both genes result in secondarily ion channels disruption as consequence of altered localization or function . Caveolin-3 is involved in multiple cellular processes, including vesicular transport, cholesterol and calcium homeostasis, and signal transduction , playing a diverse and critical role in the cardiovascular system . Caveolin-3 functions as chaperons and scaffolds protein recruiting signaling molecules to caveolae, providing direct temporal and spatial regulation of signal transduction . In particular, Caveolin-3 can interact and modulate the activity of ERK 1/2 . In the heart ERK 1/2 control a complex network of regulatory mechanisms that protect cardiomyocytes against different stresses (such as hypoxia, ischemia–reperfusion, hyperosmotic and oxidative stress) that ultimately lead to cellular disfunction and death . Therefore, aberrant cellular stress responses are potentially relevant to the development of arrhythmias and such mechanisms may play a significantly role in LQTS secondarily to Caveolin-3 alteration.

The change in human DNA content over time in the artefacts of the blowfly Lucilia cuprina (Meigen) (Diptera: Calliphoridae)

Annalisa Durdle, R. John Mitchell, Roland A.H. van Oorschot — 2011-10-17

Abstract: Adult blowflies can act as vectors of human DNA by depositing faecal or regurgitation spots (termed artefacts) after feeding on meals of human biological fluid. Given the number of backlog cases in many forensic laboratories, it is important to know the maximum time period any human DNA within a fly artefact can remain unextracted before the DNA degrades to unusable levels. The present study aimed to determine whether or not the human DNA content in fly artefacts deposited by the blowfly Lucilia cuprina changed over time, and also to provide a timeframe for forensic biologists in which the extraction of human DNA from fly artefacts should be attempted. Both the blood and semen data showed that the amount of human DNA that could be extracted increased over the first 400 days, but had decreased to one-month levels by 750 days. The saliva data showed no changes over the 60-day time period in the amount of human DNA that could be extracted.

Peruvian genetic structure and their impact in the identification of Andean missing persons: A perspective from Ayacucho

2011-10-10

Abstract: In the process of identifying around 15,000 missing persons by the armed violence in Peru (1980–2000), the major problem is the high probability of random matches in especial of Andean Ayacucho population which represents the largest number of missing persons. To that end, in a first analysis, we analyzed the relationships and genetic structure in 880 individuals from 20 regions of Peru (including Ayacucho) with IDENTIFILER Kit and in a second analysis; we studied 203 individuals of Ayacucho using the ARGUS-X and ESSPLEX_SE Kit with the aim to confirm the intrapopulation structure found in the first analysis. In the first analysis using the delta–mu parameter, clearly shows three genetic groups at the national level (North, Central and South) with a low variability among groups but significative with AMOVA (0.47%** (P<0.01)). In the tree, Ayacucho population is located in the south group and interestingly showed a significative FIS (0.10** (P<0.01)). The second analysis confirm this FIS but lowest (FIS=0.06) and this FIS could be explained by the evidence of a recent bottleneck found under the IAM, SMM and TPM models (P<0.01). On the other hand, the X-STR show low probability of random match than in Autosomic STR which may be related to content of admixture among native and foreign populations (30% foreign content in Peruvian population). The results in this study are consistent with the demographic history (processes of migration, immigration, inbreeding), which contribute to the increase of IBDs and may be related to the random match DNA identification.

Utility of multilocus variable number tandem repeat analysis as a microbial forensic tool for subtyping Chinese Escherichia coli O157:H7 strains

Libing Yun, Yan Gu, Lagabaiyila Zha, Fengcai Zhu, Yiping Hou — 2011-10-04

Abstract: In microbial forensics arena, the large number of microbes been used as potential bioterrorist agents in a criminal attack, especially many foodborne pathogens such as Escherichia coli O157:H7 caused serious zoonotic disease involved. Further development of high resolution genotyping assays is needed to rapidly and accurately analyze microbiological evidence from a crime for attribution purposes. Some recent approaches support multilocus variable number tandem repeat analysis (MLVA) is a PCR-based subtyping method to discriminate among different strains of a bacterium. In our study, MLVA combined with automated capillary electrophoresis was used to analyze genetic relationships and potential population structure within 31 E. coli O157:H7 isolates from humans and animals in China and standard strains. Alleles of each variable number tandem repeat (VNTR) were validated by sequencing. MLVA resolved 29 distinct genotypes respectively, and were able to largely separate genotypes from humans and all kinds of animals among zoonotic strains. Microvariation events occurred in two VNTR loci. In the light of the advantages of highly discrimination, MLVA can be regarded as powerful tools for detailed tracking of E. coli O157:H7 and could also prove useful in forensic investigations.

C667T mutation in MTHFR: population data in Friuli-Venezia Giulia (North-East Italy)

2011-10-10

Abstract: In this study, a population sample (n=142) born in Friuli Venezia Giulia (FVG), a region of North-East Italy, was studied for C>T 667 mutation in the fourth exon of the 5,10-methylenetetrahydrofolate reductase gene by a PCR-RFLP based method. The genotype and allele frequencies are reported.

Application of insertion/deletion polymorphisms in human gastrointestinal tumour tissues for identification purpose

Shumin Zhao, Suhua Zhang, Tingzhi Que, Zhenmin Zhao, Chengtao Li — 2011-10-05

Abstract: Criteria of Likelihood ratio in traditional human identification were not suitable to the source identification of body for tumour tissue because of high mutation rate of short tandem repeat in cancer. Consequently, it would be extremely necessary to draw a completely new strategy for source identification of body for tumour tissue. In this study, 30 insertion/deletion polymorphisms (InDel), included in an inhouse 31plex PCR genotyping system, were examined in 69 colorectal cancer and 31 gastric cancer fresh samples and their homogenous normal tissue samples. All the samples had been genotyped with Identifiler multiplex system. Consequently, two kinds of InDel mutation type (including pLOH and LOH) and four kinds of STR mutation type (including pLOH, LOH, new allele and additional allele) were found in this group of tumour samples. The frequency of InDel genotypic alteration (IDGA, including LOH) was 0.25%, which was about 1/21 of that of STR genotypic alteration (STRGA, including LOH, new allele and additional allele). At the level of individual, IDGA could be detected in 7.00% of tumour samples, which was about 1/4.57 of that of STRGA. Difference of the frequency or the detectable proportion of the tumour samples between IDGA and STRGA was statistically significant with P values of 2.771×10−33 and 1.056×10−5, respectively. Consistency testing revealed that the mutation of the two different genetic markers, InDel and STR, in gastrointestinal tumour was unrelated (P=1.0000). Results of this study suggested that InDel might be more powerful than STR in source identification of body for tumour tissue.

Contamination monitoring in the forensic DNA laboratory and a simple graphical model for unbiased EPG classification

Pernilla Digréus, Ann-Christin Andersson, Anders Nordgaard, Ricky Ansell — 2011-10-07

Abstract: Monitoring presence and level of background DNA in forensic DNA laboratory environments can be used to control work routines and cleaning procedures and to follow changes in these, as well as being an indicator for increased/decreased contamination risk. Previous monitoring routines as sampling and interpretation have not been standardised, making it difficult to compare between different sampling events and observe potential trends. Factor analysis was used to generate a simple graphical classification model for unbiased ranking of electropherograms, which can be modified according to user's need, taking into account number of detected alleles, markers and peak height.

DNA typing for the identification of eight victims of Spanish Civil War reprisals in the Canary Islands: The case of “the Fuencaliente thirteen” mass graves (Fuencaliente, La Palma)

E. Betancor, R. Fregel, M. Almeida, N.M. Suárez, J. Pestano — 2011-10-07

Abstract: During the Spanish Civil War (1936–1939), the Canary Islands suffered one of the highest levels of repression by the insurgent side, even though there were no battles in the islands. More than 50 people were killed in the island of La Palma between July 1936 and June 1937. The Association for the Recovery of Historical Memory in La Palma, made up of relatives of people who went missing during the Civil War, located the Fuencaliente mass graves in 2004. The excavation process recovered eight skeletal remains. The aim of this work was the genetic identification of these reprisal victims.In general, obtaining nuclear DNA profiles from old skeletal samples is known to be difficult. Due to the age and conservation conditions, this was the case for the Fuencaliente remains. For these reasons, we firstly attempted to analyze the mitochondrial DNA (mtDNA). Although mtDNA control region sequences were obtained for the eight skeletal remains, the limited number of possible maternal relative donors complicated identification based only on mtDNA.Taking into account the problems in establishing identity by using mtDNA, a few years later we were presented with the possibility of analyzing nuclear DNA using the new PrepFiler Express BTA™ Forensic DNA extraction methodology. This extraction protocol, in combination with the new AmpFℓSTR® NGM™ PCR Amplification Kit (Applied Biosystems), allowed us to obtain full nuclear DNA profiles for the eight victims.In conclusion, the use of specific protocols designed for old DNA samples, such as PrepFiler Express BTA™ Forensic DNA extraction and AmpFℓSTR® NGM™ PCR amplification kit seems to be crucial for obtaining full nuclear profiles that allow statistically significant identification of the putative relatives.

Reliable nuclear and mitochondrial DNA quantification for low copy number and degraded forensic samples

R. Fregel, M. Almeida, E. Betancor, N.M. Suárez, J. Pestano — 2011-10-31

Abstract: DNA quantification is a prerequisite for both low copy number (LCN) forensic analysis and ancient DNA (aDNA) studies. Moreover, if nuclear quantification is focused on the amelogenin locus, it also allows sex determination. Some of the problems of these techniques are allelic drop-out phenomenon in amelogenin locus and mitochondrial DNA (mtDNA) quantification biases, due to human intraspecific variation affecting the annealing of primers and/or probes.The method presented here combines two multiplex TaqMan® real-time PCR (qPCR) for nuclear and mtDNA quantification in degraded or limited samples. Nuclear DNA detection is based on the independent amplification of X and Y chromosome specific fragments in the amelogenin locus and an internal PCR control (IPC) to recognize inhibition problems. The small length of the fragments (71bp) favors the quantification of severely degraded DNA, whereas the use of two distinct primer sets for X and Y chromosome amplification is directed to reduce allelic drop-out in LCN analysis. MtDNA quantification is based on the amplification of three PCR fragments located in the mtDNA 16S region. Two of them are amplified with human specific conservative primers and probes, which allows a world-wide application of this technique. Moreover, their length difference (167 and 314bp respectively), provides information about the DNA degradation level. In order to also recognize non-human DNA an interspecific mtDNA fragment (187bp) was also designed.

Identification of skin in touch/contact forensic samples by messenger RNA profiling

E. Hanson, C. Haas, R. Jucker, J. Ballantyne — 2011-10-07

Abstract: The true nature of touch DNA evidence has remained elusive, generally perceived to be the result of DNA obtained from shed skin cells yet never confirmed with scientific certitude. This is largely due to the belief that it is not possible to ascertain the tissue source of origin of the biological material in touch DNA evidence. Thus far, research has failed to provide crime laboratories with feasible methods to identify the tissue source of origin of touch DNA. The aim of the current work was to identify highly sensitive and specific biomarkers for the identification of skin.We have previously demonstrated the use of tissue specific messenger RNA (mRNA) profiling assays for body fluid identification. We therefore utilized mRNA profiling to identify potential biomarkers for the identification of skin. From an evaluation of over 100 potential genes, we identified five mRNA markers that demonstrated a high degree of specificity for skin. Using these markers, we have been able to successfully identify skin using as little as 5–25pg of input RNA. The presence of skin has been successfully identified in swabs of human skin and in a variety of touch samples. One of the markers (LCE1C) is particularly highly sensitive and permits the detection of skin in a majority of known skin containing samples tested. Although further work is needed to produce an assay for routine casework, these initial studies demonstrate that a molecular-based characterization of the biological material recovered from touch samples is possible.

Validation of the hemoglobin (Hb) hypsochromic shift assay for determination of the time since deposition (TSD) of dried bloodstains

E. Hanson, A. Albornoz, J. Ballantyne — 2011-10-07

Abstract: Determining the time since deposition (TSD) of a bloodstain found at a crime scene could prove invaluable to law enforcement investigations, identifying the time frame in which the individual depositing the evidence was present. Although various TSD methods have been proposed, none have gained widespread use due to poor time resolution and weak age correlation. Previously, we examined the UV–visible absorption spectral profile of hemoglobin (Hb) in dried bloodstains of different ages and identified a hypsochromic shift (blue shift, shift to shorter wavelength) of the HbSoret band that demonstrated a high correlation with TSD. While this method demonstrated promise for use with forensic samples, additional validation work was needed.We have further evaluated the effects of temperature and underlying substrate on the ability to obtain reliable TSD measurements using the Hb hypsochromic shift assay. We evaluated bloodstains deposited onto cotton, denim, polyester, and paper incubated at various temperatures for up to 3 months. The effects of temperature were consistent with previous studies and no significant effects from underlying substrate were observed. We further evaluated the use of a portable spectrophotometer which could be used “on-site” at crime scenes. This would allow not only an estimation of the age of the stain but the rapid positive identification of the presence of blood due to the unique visible spectrum of Hb. Good quality DNA profiles can be obtained from the original TSD aqueous extracts.

Optimization of dried stain co-extraction methods for efficient recovery of high quality DNA and RNA for forensic analysis

C. Parker, E. Hanson, J. Ballantyne — 2011-10-17

Abstract: A requirement for the routine implementation of mRNA profiling methods is the ability to co-extract RNA and DNA from the same biological stain. While methodologies exist for the co-isolation of DNA and RNA from biological material, few studies include a thorough evaluation of these methods for use with forensic samples. Frequently, modifications to standard methods need to be implemented in order to accommodate the challenging nature of forensic biological samples. We therefore evaluated several co-extraction methods for ease of use and ability to recover DNA and RNA of sufficient quantity and quality for forensic analysis. From the five methods evaluated, two methods were selected for subsequent optimization (NCFS organic co-extraction and the Qiagen AllPrep DNA/RNA Mini kit). Modification of the incubation temperature and time and the inclusion of additional purification steps were found to improve the efficacy of nucleic acid recovery.

The discrimination power of the hypervariable regions HV1, HV2 and HV3 of mitochondrial DNA in the Brazilian population

2011-10-17

Abstract: Analysis of mitochondrial DNA (mtDNA) has been widely applied in the field of human identification, and features a large number of mtDNA molecules per cell, the exclusively maternal inheritance, lack of recombination and high mutation rate found in the control region, and ensure the high level of polymorphism, mainly in hypervariable regions HV1, HV2 and HV3. However, despite highly polymorphic, one of the limitations of using these regions is that many polymorphisms are highly common, resulting in the presence of sequences shared by more than one individual, or maternal lineages, in different populations. The aim of this study was to evaluate the discrimination power of the analysis of hypervariable regions HV1 (16,024–16,365), HV2 (73–340) and HV3 (438–574) of mtDNA in a Brazilian sample containing 290 unrelated individuals. Sequencing was performed using BygDye Terminator v3.1 and capillary electrophoresis was performed on ABI3130. All samples were validated by EMPOP and were classified into 15 haplogroups. Eighty out of 290 individuals presented European haplotypes, 108 showed Amerindian haplotypes and 102 individuals presented African haplotypes. Sixty-nine individuals (23.8%) could not be discriminated by the analysis of hypervariable regions HV1, HV2 and HV3 of mtDNA, and they were distributed in 24 different groups of common sequences. The most common sequence belonged to European haplogroup R0 with 10 individuals showing the same sequence. Trying to increase the discrimination power of common haplotypes and, therefore, individuals, analysis of SNPs in the coding region has been done.

MiR16 as a microRNA marker applied in species identification

Yan Li, Zheng Wang, Yiping Hou — 2011-10-10

Abstract: MicroRNAs (miRNAs, 18–25 bases in length) are small, non-coding RNAs that regulate gene expression at the post-transcriptional level. MiRNAs expression patterns, including presence and relative abundance of particular miRNA species, provide cell- and tissue-specific information that can be used for body fluid identification. Recently, two published studies reported that a number of body fluid-specific miRNAs had been identified. However, they focused on identifying specific human body fluids and did not consider species specificity. In this study, we selected blood high abundance miR16 that be reported it can distinguish venous blood from other body fluids in human to detect the expression abundance in blood of nine animals (monkey, rabbit, chicken, duck, cattle, sheep, dog, SD rat and mouse) using high-specificity stem-loop reverse transcription (RT) and high-sensitivity hydrolysis probes (TaqMan) quantitative real-time polymerase chain reaction (qPCR). The results showed that miR16 is differentially expressed in nine animals, which need to be further studied and additional work remains necessary search for more miRNA markers to identify human blood stain from animal blood stains.

A new algorithm for mtDNA sequence clustering

Inês Soares, António Amorim, Ana Goios — 2011-10-13

Abstract: We here present a new strategy for mtDNA sequence clustering based on protein coding region analysis and using a new algorithm for a fast calculation of similarities between sequences. The resulting phylogenetic tree showed a good clustering of major haplogroups according to canonical classifications. We propose the use of this method for a first assignment of sequences into major haplogroups, thus avoiding the first step of going through a phylogenetic tree and/or as a quality control for sequences classified manually.

Genetic characterization of six SNPs in Catalonian population

C. Barrot, S. Jiménez, C. Sánchez, E. Bañón, M. Ortega, E. Huguet, M. Gené — 2011-11-21

Abstract: It will only be a matter of time before physicians can screen patients for susceptibility to a disease by analyzing their DNA for specific SNP profiles.The study of SNPs associated with dopamine and serotonin systems has a strong interest because of their roles in controlling behaviour and mental status.According to the increasing understanding of the genetic bases of human behaviour, the possible role of SNPs in developmental psychopathologies could be important in forensic studies. Furthermore, the growing interest of forensic researchers in autosomal SNPs is due to the potential advantages in human identity and paternity testing, because of the low rate of mutation and the possibility of analyzing highly degraded samples. The purpose of this study is to report allele frequency data of a Catalonian population sample (n=408) from Spain for six SNPs, related to a neurotransmitter pathway genes (ANKK1, DRD2B, HTR1B, TPH1, ADRA2A and OPRM1).No significant deviations from Hardy–Weinberg expectation were found for all SNPs. From a forensic point of view the heterozygosity value, power of discrimination and the a priori chance exclusion value were calculated.

Efficient DNA extraction from hair shafts

M. Almeida, E. Betancor, R. Fregel, N.M. Suárez, J. Pestano — 2011-10-10

Abstract: Hairs are common biological samples in crime scene investigation. However, most of this evidence is comprised of hair fragments without the root. As the major part of DNA is located in the root, hair shafts are usually problematic samples in forensic analysis. For these reasons, hair DNA typing is directed at mitochondrial DNA (mtDNA), which is present in high copy number in each cell, instead of nuclear DNA analysis. In our laboratory, we have used the PrepFiler BTA™ extraction method for routinely processing difficult samples such as old bones or cigarette butts, obtaining good quality DNA in all cases. As the use of automatic extraction methods has been progressively introduced in forensic laboratories, we have tested the applicability of the PrepFiler BTA™ extraction method in combination with AutoMate Express™ equipment, to the analysis of hair shafts. In order to determine the efficiency of the method, DNA extractions were quantified using a real-time PCR approach, and mtDNA fragments of different lengths were amplified to determine DNA degradation. We also processed several types of hairs, with different characteristics (thickness, gender, antiquity and hair dyeing) and from diverse ethnical groups. In all cases, the PrepFiler BTA Express™ extraction method showed very reproducible results in obtaining DNA from hair shafts, its application being highly recommendable as a routine protocol in forensic laboratories.

Chromosome X markers DXS6795, DXS9907 and GATA144D04: Repeat structure and allele distribution in a German population

S. Hering, J. Edelmann, C. Augustin, R. Szibor, U.D. Immel — 2011-10-26

Abstract: The STR markers DXS6795, DXS9907 and GATA144D04 are located on the short arm in the region Xp22.11–Xp11.23. These loci were typed from buccal swab samples from unrelated German individuals (161 male, 362 female). Additionally, DNA from 90 males with one ore more daughters and their 135 grandsons were typed to check for mutability of these loci. German population and sequencing data for these three loci as well as statistical parameters of forensic interest are presented. DNA typing patterns of cell lines were used as intra- and inter-laboratory standards to calibrate allelic ladders. We found 8 different alleles in DXS9907 and GATA144D04, whereas DXS6795 exhibited only 6 alleles. Allelic distribution patterns are identical for males and females. There is no evidence for deviation from the Hardy–Weinberg equilibrium in female samples. Furthermore, no mutations were detected within 90 paternal and 135 maternal meioses. The three microsatellites are moderately variable with PIC values of 0.61, 0.56 and 0.70.

Synovial fluid: An alternative source for forensic DNA

2011-11-07

Abstract: In human remains that have undergone decomposition, blood and other biological tissues are recognized as sources for forensic DNA identification, yet the success rate is influenced by the extent of decomposition to the subcellular structures and nucleic acids. Thus, synovial fluid may be considered as an alternative source for forensic DNA recovery, as compared to other decomposed tissues. In this study, 24 samples (12 synovial, 8 blood, and 4 muscle tissue samples) were obtained from 12 decomposed bodies. Our results show that synovial fluid is a suitable source for forensic DNA recovery compared to decomposed blood or muscle tissue. In addition, no mixture or contamination of DNA was detected in synovial sources. Further study is required to determine whether an additional pre-treatment of the synovial sample with hydrolysis can potentially recover more DNA.

A practical model to explain results of comparative DNA testing in court

A.J. Meulenbroek, T. Sijen, C.C.G. Benschop, A.D. Kloosterman — 2011-11-07

Abstract: With ongoing developments in DNA typing more information can be derived from minimal and degraded biological samples, but explaining this information to criminal justice professionals has become increasingly challenging. We use a practical model to help forensic scientists explain and lawyers understand the meaning of reported DNA-based evidence. Using this model, reporting officers classify results of comparative DNA testing in a uniform way. Application of this model to casework met with appreciation from legal professionals.

Population genetic evaluation of 12 X-chromosomal short tandem repeats of Investigator Argus X-12 kit in North-East Italy

Stefania Turrina, Giulia Filippini, Domenico De Leo — 2011-11-21

Abstract: Short tandem repeats (STRs) located on X chromosome are suitable in complex kinship cases when analysis performed using only autosomal loci does not provide informative results. In this work we evaluated the distribution of 12 X-STRs (DXS10103, DXS8378, DXS7132, DXS10134, DXS10074, DXS10101, DXS10135, DXS7423, DXS10146, DXS10079, HPRTB, DXS10148) using Investigator Argus X-12 PCR Amplification kit in a population sample of 207 unrelated healthy individuals (89 females and 118 males) living in North-East Italy. Allele frequencies and parameters of forensic interest were calculated for the twelve X-STRs. Some new microvariant and rare alleles in the loci DXS10148, DXS10101, DXS10079 have been detected. The Polymorphism Information Content (PIC) of the 12 X-STRs ranged from 0.642 (DXS8378) to 0.942 (DXS10135).

Concordance study and allele frequencies for 5 new European standard set (ESS) loci in the North-East Italian population

Stefania Turrina, Giulia Filippini, Domenico De Leo — 2011-11-07

Abstract: In this work we performed a concordance study using four different commercial kits that contain five new short tandem repeat (STR) loci (D1S1656, D2S441, D10S1248, D12S391 and D22S1045), included in the new European Standard Set (ESS). Moreover we determined allele frequencies and statistical parameters of forensic interest in a population sample of 266 unrelated healthy individuals living in the North-East Italy. No significant differences were found in comparison with other European genetic population data. Concordance between the typing results for the four kits was observed in 99.925% (2658 out of 2660) STR allele calls compared.

Forensic evaluation of the Investigator DIPplex typing system

Stefania Turrina, Giulia Filippini, Domenico De Leo — 2011-11-07

Abstract: Insertion/deletion polymorphisms (known as InDels) are short biallelic length markers widely spread throughout the genome. This kind of markers have aroused the interest of the forensic community in recent years, even if population data are limited. In order to increase the information about Indels, we report allele frequencies and statistical parameters of forensic efficiency obtained typing a sample of 200 unrelated healthy individuals living in North–East Italy using a panel of 30 insertion/deletion polymorphisms included in Investigator DIPplex kit (Qiagen, Hideln, Germany).

The future of forensic science standards

Linzi M. Wilson-Wilde, James Brandi, Stephen J. Gutowski — 2011-11-03

Abstract: In 2010 the Australia New Zealand Policing Advisory Agency National Institute of Forensic Science (NIFS) established a project to set up a sustainable mechanism for the development and maintenance of standards, across a broad science and technology base that is relevant to general law enforcement, and the forensic science community. The project includes four core forensic standards covering the collection, analysis, interpretation and reporting of forensic evidence and discipline specific standards. One specific standard identified covers specifications regarding the manufacture of products used to collect biological material for forensic analysis. This is significant as contamination events caused by staff during the manufacturing process have resulted in investigations being hampered by non-case related DNA profiles obtained during case work analysis. The core standards continue to be developed as Australian Standards, however the contamination minimisation standard is being progressed through ISO. The development of the standards and potential impact is discussed.

Comparison study in determination of full sibling with Identifiler multiplex system between ITO method and identity by state scoring method

Shumin Zhao, Suhua Zhang, Chengtao Li — 2011-12-01

Abstract: In this study, 2003 pairs of unrelated individuals (UI group) and 280 pairs of full siblings (FS group) were genotyped with Identifiler system. Cumulative full sibling index (CFSI) and the identity by state (IBS) score of Identifiler system (IBSi) between each pair of individuals were calculated with ITO method and direct counting method, respectively. Difference of Log10CFSI or IBSi between the two groups was statistically significant (P<0.0001). Consequently, two groups of discrimination functions were established with Fisher discriminant analysis based on Log10CFSI and IBSi, respectively. Total false determination rate of the two groups of functions were 3.48% with ITO and 2.98% with IBS method, respectively. High consistency of the two different method was indicated with Kappa index=0.8841 (P<0.0001). Results suggested power of IBS method was similar to that of ITO method in FS determination, with advantages of convenience in calculation and independence on the allele frequency of STR loci.

Identical but not the same: The value of DNA methylation profiling in forensic discrimination within monozygotic twins

Chengtao Li, Suhua Zhang, Tingzhi Que, Li Li, Shumin Zhao — 2011-11-07

Abstract: Monozygotic twins (MZs) show remarkable resemblance in many aspects including behavior and health, because they share identical genomic DNA. However, evidence for epigenetic differences within MZs has been accumulated. DNA methylation differences between MZs could partially account for their phenotypic discordance of behavioral traits and diseases. High throughput epigenomic microarray profiling can be a strategy of choice for identification of epigenetic differences in phenotypically different MZs. In this study, we mapped MZs DNA methylation differences in white blood cells by interrogation of the unmethylated genome on methylation Beadchip. Blood samples were taken from 22 pairs of adult MZs. Genomic DNA was bisulfite modified by EZ DNA methylation-Gold kit according to the manufacturer's protocols, consequently analyzed with Illumina's Human Methylation27 Beadchip including more than 27,000 CpG sites. The results indicated that MZs exhibited remarkable differences among their genome-wide 5-methylcytosine. According to a set of selection criteria, 377 CpG sites with significant differences of methylation status were picked out. Although DNA methylation shows only partial stability, primary results of this study strongly suggested that the CpG methylation could be a perspective biomarker to distinguish MZs from each other.

Morphological and DNA analysis in human skeletal remains exposed to environmental conditions in Brazil

2011-11-07

Abstract: Investigations of genetic kinship and human identification through DNA analysis of human skeletal remains have been required for several types of cases. A molecular study of this kind of sample is a challenge because of the small amount of cells available. In Brazil, there is a dominating humid tropical climate which can exert a direct influence on the microscopic morphology of bones and consequently on the DNA. The objective of this study was to analyze the microstructure of femoral compact bone tissue samples that were exposed to harsh environmental conditions in Brazil, correlating it with the amount of human DNA extracted. Compact bone fragments were used from femoral diaphysis of 20 skeletonized corpses found in the period 1998–2009 in Ribeirao Preto, São Paulo, Brazil. Samples were processed, stained with H&E and from the densest cellular area, 10 consecutive fields were selected by a pathologist and software Image Tool (UTSCH-USA) and Image J (NHI-USA) were used for cellular analysis. DNA extraction was performed through commercial kit and quantification with Quantifiler Duo DNA kit (Applied Biosystems). Osteocytes were observed in all cases ranging from 1 to 40 and DNA amount was correlated with the morphology observed. These results indicate preservation of osteocytes in bone material exposed to tropical environmental conditions, signifying the feasibility of obtaining DNA for genetic studies. A preliminary morphology analysis in skeleton samples can predict success in extracting DNA from these samples, since a significant correlation was found between these two variables.

The distribution of H19HP haplotypes in Asian populations

2011-11-07

Abstract: An autosomal haplotype system, H19HP, is composed of 23 SNPs with high diversity in ethnic groups, and also a parent-of-origin detectable polymorphism. In this study, the distribution of H19HP haplotypes in seven Asian populations (Japanese, Chinese, Korean, Mongolian, Burmese, Thai and Turk) was investigated. Five haplotypes, 2, 6, 10, 15 and 17, were commonly and dominantly observed in each population. Several new population-specific sequences, including haplotype 21, were detected. Interestingly, haplotype 19, which is haplotype 17 with one SNP (rs76162918), was observed almost exclusively in Japanese subjects from four areas, with a significant frequency of about 8% on average. This system might therefore be useful as a forensic tool, not only for human identification purposes, but also for determining ethnicity, especially for Japanese subjects.

Structural polymorphisms at the X-chromosomal short tandem repeat loci DXS10134, DXS10135, DXS10146 and DXS10148

Atsushi Nagai, Yasuo Bunai — 2011-11-02

Abstract: Sequence analyses for the X-chromosomal short tandem repeat loci DXS10134, DXS10135, DXS10146 and DXS10148 were performed in 140 unrelated Japanese males. For the DXS10134 locus, 12 different-sized alleles were identified; a structural polymorphism was found in 1 allele only. For the DXS10135 locus, 16 different-sized alleles were identified; structural polymorphisms were found in 8 different-sized alleles. For the DXS10146 locus, 14 different-sized alleles were identified; structural polymorphisms were found in 2 different-sized alleles. For the DXS10148 locus, 16 different-sized alleles were identified; structural polymorphisms were found in 7 different-sized alleles. Discrepancies between allele designations based on size and repeat number were observed in 2 samples each for DXS10146 and DXS10148. Therefore, sequencing is effective in identifying alleles of DXS10134, DXS10135, DXS10146 and DXS10148 where sequence polymorphisms are observed.

Repeated extraction of DNA from FTA cards

2011-11-07

Abstract: Extraction of DNA using magnetic bead based techniques on automated DNA extraction instruments provides a fast, reliable and reproducible method for DNA extraction from various matrices. However, the yield of extracted DNA from FTA-cards is typically low. Here, we demonstrate that it is possible to repeatedly extract DNA from the processed FTA-disk. The method increases the yield from the nanogram range to the microgram range.

Purification and concentration of PCR products leads to increased signal intensities with fewer allelic drop-outs and artifacts

L.M.I. Pedersen, M. Stangegaard, H.S. Mogensen, N. Morling — 2011-11-07

Abstract: Capillary electrophoresis of amplified DNA isolated from trace evidence samples occasionally results in inadequate STR-profiles due to artifacts caused by, e.g. primers and dNTPs. Removal of artifacts by purification and subsequent concentration of the PCR products may increase the sensitivity and the quality of the DNA profiles without re-amplification of the sample. We have validated and implemented an automated method to purify and 2-fold concentrate PCR products resulting in allelic peaks with higher intensity (a median height across all loci from 130 to 404RFU), fewer allelic drop-outs and a reduced number of artifacts compared to both an increase in injection time and increase in the number of amplification cycles.

Applying a PCR inhibitor tolerant DNA polymerase blend in forensic DNA profiling

J. Hedman, C. Dufva, L. Norén, C. Ansell, L. Albinsson, R. Ansell — 2011-11-07

Abstract: Crime scene samples often contain extraneous compounds that may interfere with PCR-based DNA analysis, resulting in imbalanced, partial or blank/negative DNA profiles. Customising the chemical content of the PCR reaction is a strategy that may increase PCR inhibitor tolerance without manipulating the sample. We have validated a modified version of AmpFlSTR SGM Plus, replacing AmpliTaq Gold DNA polymerase with a customised blend of two alternative polymerases, ExTaq Hot Start and PicoMaxx High Fidelity. Allele calls are identical to standard analysis. Stutter sizes and balance values are indistinguishable. The modified chemistry provides increased resistance to PCR inhibitors, resulting in an elevated number of detected alleles for various problematic crime scene samples.

Application of indels (investigator DIPplex) in mixture samples

Ana Carvalho, Laura Cainé, Raquel Carvalho, Maria F. Pinheiro — 2011-11-07

Abstract: The main goal of this work was to study the application of the Investigator DIPplex kit to interpret mixtures linked with forensic cases, essentially those involved in the analysis of sexual crime samples. The study of these polymorphisms was performed in mixtures of two DNA controls included in commercial kits for the STRs typing, one male and the other female. The analysis was done according to the kit's manufacturer manual. The results were surprisingly better than expected, as the mixture interpretations were straightforward.

Concordance study of direct PCR kits: PowerPlex 18D and Identifiler Direct

Peter M. Vallone, Carolyn R. Hill, Erica L.R. Butts — 2011-10-28

Abstract: The ability to perform robust STR typing of a single-source reference sample while bypassing extraction and quantitation can provide a savings of time and cost. Several commercial direct PCR kits and enzyme systems have been specifically developed for this purpose (e.g. Identifiler Direct and PowerPlex 18D). These newer direct PCR typing kits contain PCR master mix components not typically found in traditional STR kits and are optimized to overcome PCR inhibitors commonly found in blood such as heme, immunoglobin G, and lactoferrin. A cohort of 50 blood samples was spotted on FTA and 903 collection cards and amplified using the direct PCR technique. An aliquot of each blood sample was also extracted and purified with a standard forensic protocol (Qiagen EZ1 Advanced platform) for non-direct genotyping and STR concordance comparisons. Successful STR amplifications (full profiles) were obtained from 1.2mm punches of blood adhered onto FTA and 903 paper substrates without prior extraction. Comparisons between PowerPlex 18D and Identifiler Direct kits revealed a single discordant genotype call for the D2S1338 locus.

The new Standard Reference Material® 2391c: PCR-based DNA profiling standard

M.C. Kline, E.L.R. Butts, C.R. Hill, M.D. Coble, D.L. Duewer, J.M. Butler — 2011-10-28

Abstract: Standard Reference Material® 2391c (SRM 2391c) is the fourth generation certified reference material for PCR-based DNA profiling. The first generation SRM 2391 was released in 1995; the next two subsequent versions have had minor modifications in the type and number of loci certified but always used DNA from the same donors. SRM 2391c has been produced with an entirely new set of genomic DNA samples. In addition to four liquid samples, SRM 2391c has two dry storage matrices including FTA paper as well as 903 paper.SRM 2391c consists of six components: three are single source genomic DNA samples that are labeled A, B, and C, with the fourth genomic sample (component D) as a mixture of 3 parts of components A (female donor) to 1 part of component C (male donor). Component E consists of two 6mm punches of 903 paper that have been spotted with approximately 75,000 cells/spot. Component F consists of two 6mm punches of FTA paper that have been spotted with approximately 75,000 cells/spot of a different cell line.The six components representing five different DNA samples plus the mixture component have been analyzed using 22 commercially available STR typing kits, obtained from three different vendors, as well as the 26plex STR multiplex developed at NIST. In total there are data for 51 autosomal STRs and 17 Y-STRs included in the certificate of analysis.

Short tandem repeat sequencing on the 454 platform

Melissa Scheible, Odile Loreille, Rebecca Just, Jodi Irwin — 2011-11-09

Abstract: To investigate the feasibility of next generation sequencing (NGS) technology for the multiplex detection and sequence production of short tandem repeats (STRs), thirteen STR markers (CSF1PO, D2S441, D3S1358, D5S818, D7S820, D8S1179, D10S1248, D13S317, D16S539, D21S11, D22S1045, TPOX, and vWA) were amplified using an optimized multiplex reaction with primer sequences designed for reduced size amplicons. Each sample multiplex was barcoded with a different sample-specific multiplex identifier (MID) for subsequent parallel tagged sequencing on the GS Junior System (454 Life Sciences, Branford, CT).

New rapid ABO genotyping using direct extraction kit

2011-11-09

Abstract: Since ABO genotyping has been a helpful tool for DNA profiling, several kinds of method had reported. We developed a new ABO genotyping by TaqMan assay using the TaqMan Sample-to-SNP Kit. Eight SNPs sites were selected to detect 26 genotypes controlled by 10 alleles. The fast PCR was demonstrated to finish the whole progress in less than 60min by using a little volume of DNA. In addition, the genotypes of forensic samples such as blood, bloodstain, tissue and buccal swab were precisely determined and the affection of post-mortem changes of the blood and tissue collected from one day to 4 months and 2 months passed deposed body were so small. This developed method offers the advantages of rapid, cost efficiency and available genotyping that sufficiently used for forensic cases.

Mutational analysis of the mitochondrial DNA detected in sudden death cases with cardiac hypertrophy

2011-11-03

Abstract: Comprehensive screening of mitochondrial DNA (mtDNA) was performed in sudden death cases with cardiac hypertrophy (SDCH), in order to evaluate the prevalence of mtDNA mutations in sudden death. Missense mutations detected only in SDCH were 18 alterations in 16 cases and 8 mutations were new. It was considered that these missense mutations in turn affected the ability of the essential components of the oxidative phosphorylation complexes. It may possibly be that these mutations are tended to have increased risk of SDCH compared to the controls.

Is it always possible to avoid exhumation in particular defective paternity cases by increasing autosomal STRs number?

2011-11-07

Abstract: Genetic markers represent a very important tool in forensic identification caseworks, in family relationships as well as in criminal analysis. The discrimination power of current genetic polymorphisms is so high that the inferential process can be efficiently used even in cases where direct knowledge on the genetic data of one of the terms in comparison is lacking. However in some cases despite the use of Probabilistic Expert Systems (FINEX) it is not always possible to achieve an acceptable percentage of paternity probability. Certainly one of these cases is the request to verify the relationship between two half-siblings of different gender in the absence of data from parents. In these cases it is not possible to use important tools such as polymorphisms of sexual chromosomes, so that the only possible approach is to increase the number of autosomal STRs. Therefore the authors decided to investigate 36 pairs of half-siblings with known relationships from different parts of Italy, using a high number of autosomal STRs. The aim of this study is to verify whether, increasing the number of autosomal STRs analyzed, the application of PES allows to achieve an acceptable value of paternity probability without availability of parents’ profiles.

Development of a 12-plex X chromosomal STR loci typing system

2011-11-07

Abstract: X-chromosomal short tandem repeat (X-STR) loci can play increasingly important roles in some complex kinship cases. In our paper, 360 samples (191 males and 169 females) unrelated individuals from Guangdong province were successfully genotyped using a new multiplex PCR system which can co-amply 12 X-STR markers (GATA172D05, DXS6789, DXS10074, DXS10078, GATA165B12, DXS6797, DXS6803, DXS6804, GATA31E08, DXS7130, DXS9895 and DXS6810). 110 alleles were observed totally. Hardy–Weinberg equilibrium tests showed no significant deviation for all of the 12 X-STR loci. DXS6789–GATA165B12 and DXS7130–DXS9895 were found to be in linkage disequilibrium. The results show that the 12-plex X-STR multiplex system is highly polymorphic and sensitive. It may be useful for forensic personal identification, paternity testing, especially for the kinship testing.

DNA typing strategy to overcome post-mortem bone maceration

C. Franchi, E. Pilli, F. Barni, S. Potenza, A. Berti — 2011-10-26

Abstract: Chemical maceration is a useful procedure to remove soft tissues and to bleach bones facilitating the forensic anthropologist examination. However, prolonged exposure to chemicals often compromises DNA recovery and its amplification via PCR. Our purpose was to test the new generation multiplexes to type nuclear DNA and contemporarily to verify the possibility of mt-DNA sequencing. Our work demonstrates the ability of NGM kit (Applied Biosystems, Foster City, CA) to give a result when applied after an extraction strategy that implies large amounts of bone powder, a high affinity column separation and a “small meshed molecular size” concentration. Finally, we suggest not to use chemicals to macerate human bones for the purpose of identity or at least to save a fragment of femur from the treatment but if this is not the case, we indicate an analytical strategy that consider as the best method to obtain a nuclear-DNA profile in highly degraded DNA condition.

Y-SNP analysis in an Angola population

2011-10-28

Abstract: The aim of the present work was to study the origin of paternal lineages in Angola population, namely in the three main ethnic groups, inferred by phylogeographic analyses of Y chromosome defined haplogroups. To determine the male lineages present in the Angola population studied, 112 unrelated males were typed using a PCR multiplex – minisequencing SnaPshot based method including 29Y-SNPs, which characterize the majority haplogroups in sub-Saharan Africa populations.

Amplification of non-FTA samples with AmpFlSTR® Identifiler® Direct PCR Amplification Kit

2011-10-28

Abstract: The AmpFlSTR® Identifiler® Direct PCR Amplification Kit is a short tandem repeat (STR) multiplex assay, optimized to amplify 16 loci directly from biological material (blood or buccal cell samples) stored in FTA paper, without resorting to DNA extraction. However, preliminary experiences have shown the versatility and robustness of this amplification kit, when applied to other biological sample supports. The aim of this research work is to analyze the results of this kit when applied to non-FTA paper or other support for biological samples such as a buccal swab.

Estimating coancestry from genotypes using a linear regression method

N. Pinto, L. Gusmão, P.V. Silva, A. Amorim — 2011-11-07

Abstract: The estimation of the coancestry coefficient associated with a pair of individuals, given their genetic types, has been a matter of great theoretical and practical importance, namely in forensic kinship tests. Generally considered as a condensed measure of relatedness, it is quite often assumed that there is a loss of information when this measure is taken in consideration instead of Jacquard's identity-by-descent partitions. Nevertheless, considering non-inbred individuals, it can be proved that, excluding parent–child and full-siblings pedigrees, both measures are equivalent for genealogies that relate two individuals by exactly one path. In this work, the coancestry coefficient between two (not necessarily non-inbred) individuals is straightforwardly inferred from their genetic types through the probability of finding two identical alleles, each one randomly chosen from each individual, or, alternatively, through the expected homozygosity of their virtual offspring. The presented method is rooted in the algebraic expression for the probability of finding a homozygous individual in the population. It is shown that the coancestry coefficient of two individuals related by a given kinship can be mathematically modeled by a linear function depending on (a) the expected homozygosity of their virtual offspring, and (b) the average homozygosity in the population; the slope and the y-intercept of the straight line carrying information about the gene diversity of each database.

Influence of presumptive reagents on DNA typing

K. Tsukada, Y. Harayama, M. Shimizu, Y. Kurasawa, K. Kasahara — 2011-12-01

Abstract: We generally use luminal (LM) and leucomalachite green (LMG) in presumptive tests at crime scenes. LM is used to search for scattered blood traces or blood stains. LMG is used to detect fingerprints in latent blood stains and blood stains in laboratory settings. When only trace specimens are available, DNA extraction must be performed from LM- or LMG-treated specimens. No reports to date have been published on the actual effects of LMG on DNA extraction, although some reports suggest the potential influence of LM on DNA extraction.This study examined the effects of LM and LMG on DNA typing.

Preliminary validation of Prepfiler Express™ Extraction kit in AutoMate Express DNA Extraction System

2011-10-28

Abstract: A variety of challenging biological samples, including blood stains, saliva, semen, hair, bones, finger nails, among others, are often a part of our casework investigation. In this study, semen, blood samples and saliva swabs were extracted by several methods in order to optimize and validate the Prepfiler Express™ Extraction kit and the AutoMate Express DNA Extraction System. Results obtained with the robot (using silica-coated magnetic beads) were compared with methods based on a chelating resin, silica membranes and paramagnetic resin.

Complex casework using single nucleotide polymorphisms

Paulo Dario, Teresa Ribeiro, Deodália Dias, Francisco Corte-Real, Helena Geada — 2011-11-07

Abstract: Complex kinship analyses are normally a challenge for forensic laboratories, especially in cases in which the individuals involved can have criminal responsibilities. This paper presents two complex relationship caseworks studied with routine STRs and autosomal SNPs as supplementary markers. In the first case, to exclude trafficking of children, maternity investigation of a child was requested involving two alleged mothers – a 39-year-old woman, the alleged grandmother, and her absent daughter. The second one was a possible incest case with a young girl with Trisomy 21 where her father was also the alleged child's father. The individuals of these cases were typed for 17 autosomal STRs with AmpFlSTR Identifiler or IdentifilerPlus and Powerplex 16. Twenty autosomal SNPs were also typed using SNaPshot® methodology, with two 10-plex previously revealed useful in paternity testing. Both cases gave low likelihood ratio values with STRs and a genetic incompatibility was also detected in the first case. SNP studies strongly indicated that the alleged grandmother was not the child's mother but indeed the grandmother in a real complex immigrant kinship case, while in the second casework reinforced the incest relationship. Therefore, SNPs revealed useful as additional markers in complex kinship testing.

Primary teeth as DNA reference sample in disaster victim identification (DVI)

2011-11-07

Abstract: The identification of disaster victims through the use of DNA analysis is an integral part of DVI response, regardless of the scale and nature of the disaster. DNA analysis is performed to assist in the identification of victims through kinship (familial matching to relatives) or direct (self source sample) matching of DNA profiles. The exfoliated primary teeth can be an alternative self-sample used as an antemortem record for direct matching in DVI context.The main purpose of the study is to evaluate the possibility of DNA extraction in primary teeth with probative value for identification, despite being stored for long periods of time (until 18 years).

Two 16S rRNA mitochondrial markers for species identification in forensic science

S. Corato, A. Giuliodori, E. Ponzano, E. Novelli, D. Rodriguez, L. Caenazzo — 2011-10-28

Abstract: In the recent years, with the development of molecular biology technology, new methods for human identification to forensic purposes, based on genetic differences among the animal species have been proposed. The analysis of cytochrome b (cyt b) showed a good feasibility to species determination and individual human identification.The use of cytochrome b is well-known for species detection, even if sequences analysis and comparison in BLAST made this analysis troublesome in case of degraded samples from which it is difficult obtain a good sequence.In this paper we propose a method for the identification of human samples based on the amplification of a duplex PCR corresponding to two 16S mitochondrial fragments, an universal fragment of 236bp and a human-specific fragment of 157bp. A rapid analysis of the PCR products can be performed in a mini poliacrylamide gel.The primers for both 16S rRNA fragments were designed by us. Since these two 16S rRNA fragments are small, they amplify easily even if the presence of old and highly degraded specimens in a single round PCR: for these reasons, this method could result very useful for forensic purposes in case of human species identification.

Forensic analysis of magnetic stripe skimmer devices

C. Dufva, J. Bengtsson, M. Svensson, A. Nilsson — 2011-11-07

Abstract: At SKL, the number of skimmer cases has increased since 2009. Hence it is important to have a good workflow for the forensic analysis. A successful multidisciplinary collaboration has been developed between DNA, fingerprints and IT forensics. Members of staff from these disciplines perform an initial examination deciding about which parts of the skimmers that are to be analysed for DNA or fingerprints. The combined experience from the different disciplines increases the chances of receiving successful results. For DNA analysis the success rate per case is 69%, which is a relatively high success rate.

Ancestry proportions in urban populations of Argentina

U. Toscanini, L. Gusmão, G. Berardi, A. Gómez, R. Pereira, E. Raimondi — 2011-11-03

Abstract: In this work we aimed to evaluate the African, European and Native American ancestral contributions to different Argentinean populations. A set of 46 ancestry-informative insertion–deletion polymorphisms (AIM-Indels) was used to type 279 unrelated individuals from urban populations of six provinces located in distant geographical regions of Argentina. The STRUCTURE analysis showed that the African contribution presented the lowest values, with no significant differences among the studied regions (between 2 and 3%). As expected, the population from Buenos Aires appeared to have the highest European ancestry contribution (86.8%). In contrast, the Native American component is better represented in urban samples from the northern and southern regions of the country, reaching almost 40% in both Tucuman and Santa Cruz. These differences are also revealed by the Fst analysis results, where most of the significant genetic distances are between Buenos Aires and the other populations. The results are in agreement with previous reports based on other type of genetic markers (e.g. uniparental) and supported by the demographic history of Argentina.

Development of a Long QT-Syndrome mutation detection method

M. Nastainczyk, R. Lessig, D. Husser-Bollmann, J. Dreßler, J. Edelmann — 2011-11-07

Abstract: Sudden cardiac death (SCD) especially in younger adults with no previous symptoms is a challenging problem in forensic diagnostics, occurring with an incidence of about 80,000 per year in Germany. Long QT-Syndrome (LQTS) and other cardiac disorders associated with abnormality of cardiac rhythm triggered by mutations of cardiac ion channels have to consider if no cause of death is detectable during autopsy. A molecular genetic screening can help to ensure. Meanwhile twelve genes are known in which more than 700 different mutations are associated with LQTS. The three major LQTS-susceptibility genes are KCNQ1, KCNH2 and SCN5A. They are responsible for 75% of all congenital LQTS cases. We developed a rapid, sensitive and reasonable method for the simultaneously screening of the most common LQTS mutations, focused on these three genes. Using the SNaPshot® minisequencing primer extension assay a total of 91 Single-Nucleotide Polymorphisms (SNPs) were examined in six multiplex assays. The data suggest that this technique is applicable, solid, flexible and a good alternative to complete sequencing.

Semen detection: A retrospective overview from 2010

Beate B. Hellerud, Mariam Bouzga, Per Hoff-Olsen, Bente Mevåg — 2011-11-09

Abstract: The literature on sexual assault cases describing the persistence and detection of spermatozoa in combination with acid phosphates (AP) test results on samples from different female anatomical locations is limited. Samples from the victim's genitals were collected for the detection of semen in 181 of the 371 cases of alleged rape examined by our laboratory in 2010. All the swabs from vulva, vagina and cervix, in addition to samples from the female lingerie, were tested with AP and underwent Christmas tree staining followed by microscopy to confirm the presence of spermatozoa. In 67 cases (37%) spermatozoa from a presumed perpetrator were detected in one or more samples, including 9 cases in which spermatozoa were found only in the female garment. For a selection of samples differential extraction and quantification (Quantifiler® Duo) were performed prior to the DNA analysis (AmpFℓSTR® SGM Plus®) in order to determine the DNA profiles. The average time between the alleged assault and the medical examination was 12.4h, with some variation between the three different sampling areas. 74 samples (46%) with microscopically verified spermatozoa displayed a negative AP reaction, and the results indicate that vulva samples more frequently produce a negative reaction despite verified spermatozoa compared to samples from vagina and cervix.

First application of the Investigator DIPplex indels typing kit for the analysis of ancient DNA samples

C. Hollard, F. Mendisco, C. Keyser, E. Crubézy, B. Ludes — 2011-11-09

Abstract: Kinship testing and identification of human remains in forensic and anthropological fields is commonly based on the analysis of short tandem repeats (STRs) or more seldom single nucleotide polymorphisms (SNPs). In this study, another type of genetic variations, insertion/deletion polymorphisms (indels) was used through the DIPplex indels typing kit (Qiagen) for such purpose. The objective was to evaluate the performance of the assay for the analysis of ancient DNA samples as well as the informativeness of the kit on kinship investigation. Although some limitations have been noticed, this work shows that indels are well suited for the analysis of degraded samples and that they might be an interesting strategy in addition to STRs typing.

Allele frequencies of the new European Standard Set (ESS) loci in the population of Czech Republic

2011-11-07

Abstract: 120 unrelated individuals from the population sample of the Czech Republic were genotyped using the AmpFℓSTR® NGM™ PCR amplification kit (Applied Biosystems). The population study was conducted to evaluate the usefulness of the five new STR loci (D10S1248, D22S1045, D2S441, D1S1656, and D12S391) included in the new European Standard Set and to establish the allele frequencies.

Genomic DNA extraction protocols for bone samples: The comparison of Qiagen and Zymo Research spin columns

2011-11-07

Abstract: The aim of this study was to develop an extraction protocol for bone samples based on ZR Genomic DNA Tissue MicroPrep kit and perform a quantitative comparison with the existing silica extraction protocol based on Qiagen columns and evaluate the effect of demineralization on the quantity of extracted DNA.

Capillary electrophoresis analysis of DNA primary structure: Toward a quality control test for the reliability of the STR-typing from forensic specimens

C. Previderè, G. Marrubini, S. Sorçaburu-Cigliero, P. Grignani, P. Fattorini — 2011-11-07

Abstract: A capillary electrophoresis (CE) method was used to study the DNA primary structure of artificially degraded DNA samples and aged/forensic specimens. CE investigated the ratio between the pyrimidinic and the purinic canonical moieties still bound on the DNA backbone and identified the presence of additional compound (i.e. un-canonical DNA bases). The DNA samples were then analysed by using molecular assays such as agarose gel, UV spectrophotometry and qPCR. All these data have been concurrently considered for a critical evaluation of the outcome of the STR typing.

Molecular characterisation of a mummified body found in a glacier

C. Previderè, P. Grignani, G. Peloso, P. Fattorini, L. Andrello, A. Osculati — 2011-11-07

Abstract: A complete genetic profile of a mummified male body that had emerged from the melting ice of an Alpine Glacier was achieved. The body could be dated back to the late 1920s, due to the finding of newspaper cuttings in an internal pocket of the clothes. DNA was recovered from still well preserved internal organs and nail clippings. Autosomal STRs, Y chromosomal STRs and SNPs and mtDNA HV1 region were determined in order to provide a complete genetic survey of the mummified body. In a future perspective, after having collected general information on the possible identity of the body, all these genetic data, and especially the ones from the lineage markers, could be used to get a positive identification.

Automated extraction of DNA from clothing

2011-11-07

Abstract: Presence of PCR inhibitors in extracted DNA may interfere with the subsequent quantification and short tandem repeat (STR) reactions used in forensic genetic DNA typing. We have compared three automated DNA extraction methods based on magnetic beads with a manual method with the aim of reducing the amount of PCR inhibitors in the DNA extracts and increasing the proportion of reportable DNA profiles.

Y-STR diversity in the Swedish population and its implication on forensic casework

Andreas O. Tillmar, Helena Nilsson, Gunilla Holmlund — 2011-11-07

Abstract: 17 Y-chromosome STR loci included in the AmpFlSTR® Y-Filer kit were typed for 302 males originating from seven geographically different regions in Sweden. In the complete dataset, 287 different haplotypes were identified of which 274 were unique. The most common haplotype was found 3 times corresponding to a population frequency of 1.0%. The haplotype diversity was computed to be 0.9996 in the total dataset, and was comparable among the different regions. The overall FST value among the different regions was 0.0016, and the highest pairwise FST value was observed between the regions Västerbotten and Uppsala (FST=0.003, P<0.01). The diversity increased substantially and the interregional differences decreased using Y-Filer compared with the earlier used Y-STRs included in the PowerPlex® Y System.

The Investigator® ESSplex Plus Kit: Fast, sensitive, and robust amplification of the European Standard Set of loci

2011-11-07

Abstract: Forensic DNA laboratories need to provide results in a very short time. Therefore, in addition to crucial quality parameters (e.g., sensitivity and robustness), speed is an increasingly important feature of STR PCR assays. QIAGEN has developed the Investigator ESSplex Plus Kit, which combines the features necessary for fast and reliable analysis of demanding forensic samples. Based on our fast-cycling multiplex PCR technology, we have introduced a novel reaction mix that enables a standard 30 cycle amplification in ∼90min, setting a new level of speed in STR analysis. Well-balanced full profiles are reliably obtained with 125pg of DNA template. Even a single genomic DNA copy gives rise to peak heights easily detectable with commonly used analysis thresholds. The assay is very robust and can tolerate PCR inhibitors of up to 200ng/μl humic acid or 1000μM hematin without allelic dropout, higher than any other kit on the market. The Investigator ESSplex Plus Kit provides a clean baseline, without artifacts that may interfere with the analysis of low-copy-number samples. These features help to reduce the number of samples that require reanalysis and contribute to more streamlined and efficient laboratory workflow.

Doing more with less: Implementing direct amplification with the PowerPlex® 18D System

2011-11-07

Abstract: Time and labor savings continue to be key goals for database and paternity labs. Increasing sample numbers and tightening government budgets necessitate that database laboratories seek process improvements. Similarly, efficiency gains are key for commercial paternity testing organizations to remain competitive. Recently, the STR-typing community has begun to adopt the concept of “direct amplification” from unpurified samples on FTA® cards. Significant time and labor savings can be achieved by eliminating DNA purification and amplifying samples directly from buccal or blood samples on FTA® cards. Another time-consuming process is the PCR itself—often three hours or more. The duration of this step often dictates daily workflow for laboratories, and when combined with DNA purification and capillary electrophoresis, the process often requires more than one day. Although thermal cycling is a hands-off step, decreasing sample time on an instrument with rapid amplification allows samples to be processed in a single day. The PowerPlex® 18D System allows direct amplification and reduced thermal cycling time to provide a simpler and faster genotyping system for database and paternity laboratories. Additionally, the 18 loci amplified represent all markers commonly typed in CODIS laboratories.A validation of the PowerPlex® 18D System was completed according to SWGDAM guidelines to demonstrate effectiveness and performance of the assay. This testing included sensitivity, concordance, variation of thermal cycling parameters, variation of reaction component concentrations, and tolerance to contaminating substances. The results support that the PowerPlex® 18D System is a robust and reliable identification assay fit for human identification purposes and the kit has subsequently been approved for use by NDIS CODIS-participating laboratories.

Development of a SNP multiplex assay for the inference of biogeographical ancestry and pigmentation phenotype

Charmain Castel, Anita Piper — 2011-11-07

Abstract: SNPs exhibiting strong associations with specific populations and/or pigmentation phenotypes have been identified in mitochondrial, Y chromosome and autosomal DNA. Ancestry and phenotype inferences gained from DNA may assist forensic investigations of unknown perpetrators or aid identification of missing persons and disaster victims. To test the predictive power of combining SNPs from all three DNA types, 21 coding region mitochondrial SNPs, 28 Y chromosome SNPs and 14 autosomal SNPs were selected and analysed in a hierarchical multiplex assay.

Investigator® Quantiplex Kit: For reliable quantification of human DNA in forensic samples

2011-11-07

Abstract: Commonly, short tandem repeat (STR) analysis is performed for human identification, although alternative approaches, such as the analysis of deletions/insertions polymorphisms (DIPs/Indels) are now available. However, these multiplex assays used for human identification are complex and require a defined range of template input. Accurate quantification (even at low concentration) and assessment of the presence of PCR inhibitors are key to ensure successful genotyping at the first attempt. Quantitative real-time PCR is the standard method for quantification of DNA in forensic samples. However, there is a need for advanced solutions that further streamline the forensic workflow by increasing the accuracy of and reducing the time to results. The Investigator Quantiplex Kit provides fast and accurate quantification of human DNA in forensic database and casework samples. The assay provides sensitivity down to 0.3pg/μl, with highly accurate quantification in the linear range of standard curve of 4.9pg/μl. A balanced internal amplification control ensures detection of PCR inhibitors. The Investigator Quantiplex assay uses PCR fast-cycling technology for rapid results. When used with the Rotor-Gene Q real-time PCR system, quantification is achieved in 48min. To further streamline the workflow and to minimize time-consuming and error-prone manual steps, it is possible to combine the Investigator Quantiplex Kit with the QIAgility instrument, a bench-top instrument allowing automation of routine procedures in the forensic laboratory workflow, such as quantification and STR setup. Combining this innovative real-time PCR-based quantification tool with liquid-handling instrumentation significantly shortens hands-on time and overall time to result, with increased accuracy and sensitivity. In addition, overall process safety is enhanced.

Mitochondrial control region sequences of the Czech Republic population and a comparison to other populations

2011-11-07

Abstract: The correct use of mitochondrial DNA (mtDNA) testing in the forensic context requires appropriate population databases to determine the relative rarity of the haplotype of the tested sample. The aim of this study was to evaluate the results of full HVRI and HVRII mtDNA sequences of 255 unrelated individuals and to compare the data to the previously published Czech and European population data The results indicate that the full control region sequencing can bring more precise population information that is useful for the comparison with other data sets and also for the forensic identification purposes.

An investigation into the protective capabilities of nucleosomes on forensic STRs

Phuvadol Thanakiatkrai, Lindsey Welch — 2011-11-07

Abstract: Partial DNA profiles are often obtained from degraded forensic samples due to allelic and locus dropout, particularly at the high molecular weight loci. It is hypothesized that nucleosomes could offer protection to the bound DNA by limiting access to enzymes. Choosing and incorporating STRs that are protected into a “high nucleosome-binding” multiplex could mean that a better DNA profile could be obtained from degraded samples. 58 STRs were previously evaluated for their nucleosome-forming potential (NFP) using two computer programs – NXSensor and nuScore. Ten mini-STR primer sets for amplifying ten loci with varying nuScores were designed and optimized. They were used to amplify degraded saliva samples and simulated casework samples to determine if there was any correlation between nuScore and DNA survivability, indicated by DNA concentration. The effect of nucleosome protection was not observed for both sample types (ρ=−0.166, p=0.647). High-nuScore loci did not perform better than low- or medium-nuScore loci. It was possible that nucleosome protection might only work in living cells and not in forensic stains, as histones could be removed during necrosis by lysosomal proteases, and subsequently DNA would be degraded by endo- and exonucleases. Other processes such as chromatin remodeling and DNA methylation could shift nucleosomes away from the predicted sites, thereby changing nuScores. It was concluded that nucleosome protection did not exist for degraded saliva samples, given that nuScores accurately represented the probability of finding a nucleosome.

Population data for 38 autosomal insertion/deletion (InDels) and 50 SNPS polymorphisms in Argentinean population

2011-11-07

Abstract: Forensic laboratories must add as much genetic information as possible in order to solve the broadened variety of post-mortem paternity and kinship cases now routinely presented. Often, use of STR autosomal markers alone is not enough to reach a high discrimination power, especially when dealing with degraded post-mortem material or complex family pedigrees. In order to expand the set of genetic markers used to solve challenging cases, and since reference databases are required in forensic application, allele frequencies and forensic parameters for 38 InDel and 50 SNP binary markers were calculated from a sample of thirty (for SNPs) and fifty (for InDels) unrelated individuals from the two most populous cities of Argentina: Córdoba and Buenos Aires. DNA was extracted with a modified salting out procedure and amplification products were detected on an ABI 3130 Genetic Analyzer (Applied Biosystems). No significant deviations from Hardy–Weinberg expectations were found. In the analyzed sample, no pairwise linkage disequilibrium between InDels and SNPs was found neither among the 88 binary markers nor the 17 STR markers from commercial kits. Comparisons were also made with previously studied populations. The present database will be useful for forensic and paternity purposes for the above mentioned cities of Argentina. Moreover, these additional markers can help forensic laboratories to solve parentage testing as well as to improve the analysis of degraded DNA samples.

RNA can do better—An improved strategy for RNA-based characterization of different body fluids and skin

J. Wobst, R. Banemann, I. Bastisch — 2011-11-07

Abstract: RNA-based identification has proven to work for cellular material from various sources of body fluids. This study addressed the questions whether simultaneous co-extraction of DNA and RNA yields similar amounts of DNA compared to established DNA extraction methods, whether RNA tests have an at least comparable sensitivity as conventional characterization tests, whether also skin epithelial cells can be characterized by specific transcripts and whether all used markers show specificity or cross-reactivity. Results showed that modification of the AllPrep DNA/RNA Mini co-extraction protocol to overnight lysis led to an at least comparable DNA amount to that obtained from QIAamp® DNA Mini extraction. In comparison to conventional tests the RNA-markers HBB, PBGD, HTN3, STATH, PRM1, TGM4, MMP11 and GASS revealed comparable sensitivity and tissue specificity. Evaluation of suitability and specificity of three novel skin markers (CDSN, CST6 and DSC1) demonstrated cross-reaction of those markers with vaginal secretion and CDSN additionally with menstrual blood. Three established multiplexes were tested on 57 mock samples. 27 of them gave a complete result on the first try.

Genetic analysis of the 11 X-STR loci in Uigur and Northern Han populations from China

Chengtao Li, Suhua Zhang, Zhenmin Zhao, Li Li, Yan Liu, Shumin Zhao — 2011-11-07

Abstract: X-chromosomal short tandem repeats (X-STRs) loci are used for forensic practice in recent years which play increasingly important roles in some complex kinship cases. In this paper, a total of 1250 samples obtained from 941 Northern Hans Chinese and 309 Uigur were successfully analyzed using a homemade new multiplex polymerase chain reaction (PCR) system which can analyze simultaneously11 X-STR markers including DXS8378, DXS6795, DXS7132, DXS6803, DXS9898, DXS6801, DXS7133, GATA165B12, HPRTB, DXS8377 and DXS7423. A total of 103 alleles for all the loci were observed. Hardy–Weinberg equilibrium tests demonstrated no significant deviation from expected values (P>0.05) for all of the 11 X-STR loci. The results show the 11 loci in the multiplex systems may provide high polymorphism information for kinship testing and relationship investigations, and it is useful for forensic analysis for the two nationality populations from China.

Revision and implementation of the SAFE kit used in the Netherlands

Corina C.G. Benschop, Titia Sijen — 2011-11-07

Abstract: In this study we revised and effectively implemented the sexual assault forensic examination (SAFE) kit used in the Netherlands to improve the forensic investigation of sexual offences. The former version was developed over a decade ago, and since then new and advanced methods have been introduced in forensic research that warrant revision of the SAFE kit. We briefly describe the steps that were undertaken, the products that were chosen and/or developed and the benefit(s) regarding the former version of the SAFE kit. Collaboration and dialogue between the parties involved were of great importance during both the revision and the implementation process.

Implementation and first case results of a rapid DNA profiling service

2011-11-07

Abstract: Recently, we implemented a “DNA-6hours” service that aims to provide an investigative lead early in the police investigation. Within the DNA-6hours procedure DNA information is rapidly derived from an evidentiary trace, the STR profile is searched against the DNA profiles of known persons in the DNA database and a brief report containing information about match or no match is given to prosecution. In the first ten months of its existence the DNA-6hours service was able to give useful information in 12 out of 15 cases. In one case, the results of the DNA-6hours service resulted in an arrest of a suspect within 24h after the crime took place.

Effect of common fingerprint detection techniques on subsequent STR profiling

Bryan Bhoelai, Bas J. de Jong, Marcel de Puit, Titia Sijen — 2011-11-07

Abstract: DNA profiling of latent fingerprints can be compromised by fingerprint detection techniques. We found that cyanoacrylate (CA) fuming and/or vacuum metal deposition (VMD) did not affect subsequent STR typing. Treatments that involved washing steps like basic yellow or safranin staining reduced DNA quantities. Methods that rely on immersion of items like 1,8-diaza-9-fluorenone (DFO) and ninhydrin staining were found to present the risk of introducing DNA contamination from the staining solution even though the fingerprint DNA was not negatively affected. The use of physical developer was deleterious for the DNA results. When items are handled before a fingerprint is placed, contaminating alleles occur at the fingerprint area. The fingerprint DNA can outstand this background, but due to the large variation for DNA quantities in fingerprints this is not certain and cautious interpretation is appropriate.

Linkage disequilibrium analysis of 67 SNP loci on X chromosome

Li Li, Shumin Zhao, Yan Liu, Chengtao Li, Yuan Lin, Suhua Zhang — 2011-11-07

Abstract: In order to assess the patterns of linkage disequilibrium (LD) about 67 SNP loci on X chromosome, genomic DNA samples extracted from blood fluids from 213 unrelated Chinese Han female individuals were analyzed through multiplex amplification followed by MALDI-TOF MS. LD was analyzed by SNPAnalyzer 2.0. Slight LD were found at two pairs and tight LD were observed at six pairs. From the 67 X-SNP loci, 56 informative X-SNPs showing independent inheritance could be chosen out. Both of the combination discrimination power and the cumulative exclusion probability of the 56 markers were above 0.999999. It may be concluded that the panel of 56 X-SNPs loci is valuable in forensic identification.

Tooth portion profile in criminology

A. Corte-Real, A. Serra, M.J. Anjos, M. Carvalho, J. Gamero, D.N. Vieira — 2011-11-03

Abstract: Up to date the mineralized tissue is the only option, in many extreme forensic conditions, to achieve a genetic profile. In several samples, both tooth type and portion were studied in order to optimize the methodology and to reduce time and costs.Results from a wisdom tooth (a natural degraded sample), a case of the National Institute Legal Medicine of Portugal, and the topographic drawings of Gaytmenn and Sweet's study from 2003, are presented.DNA from the root tooth (apical and remaining portions) was extracted by using the ArchivePure DNA Tissue kit (5Prime®). Total DNA quantification was performed by real time PCR, by using the Human Quantifiler kit (Applied Biosystems®). Depending on the DNA quantification value, the most relevant polymorphisms for genetic identification, autosomic STRs, were amplified with AmpFℓSTR® Identifiler™ by Applied Biosystems and PowerPlex® by Promega.The two root tooth portions (apical and remaining) presented different results (in quantity and quality), with respect DNA quantification and genetic profile. The apical portion allowed an autossomic profile while in the remaining portion was not allowed.Our results can be explained based on the fact that apical canicular obliteration through the formation of terciary dentin can preserve some of the pulpar contents in the topographic region, on the other hand cementogenesis in the apical portion can occur by invagination into the canalicula and canaliculi in a rapid and disorganized fashion enabling the trapping of cementocites in its lacunae. Furthermore we must add that it is the apical portion that through its cellular content potentially presents us with the best results in genetic analysis.

An automated approach for generating consensus profiles from low template STR typing results

R. Decorte, A. Gilissen, B. Bekaert — 2011-11-07

Abstract: The ‘biological’ approach of reporting STR alleles in replicate PCRs has been implemented by many forensic laboratories for reporting STR typing results of low template DNA samples. Until now, this method relied mostly on manual analysis of GeneMapper® data which is therefore very laborious and prone to error. In order to overcome this, we developed a Microsoft Access 2007 database to interpret GeneMapper® peak height data and export a table with a consensus profile of the STR typing results. This method has the advantage to automate the interpretation and generation of a consensus profile based on the peak height of the alleles (major and minor contributors) and the number of observations of the alleles in the different replicates.

Genetic diversity of 10 X-STR markers in a sample population from the region of Murcia in Spain

2011-11-09

Abstract: The purpose of this study was to apply autosomal STRs and X-STRs to confirm paternity and sibship in a sample of 315 female twins and triplets from the region of Murcia in Spain. X-STR amplification was performed using GEP-ISFG X-STR decaplex primers. Deviations from Hardy-Weinberg equilibrium were not observed at any loci. Acceptable levels of power of discrimination and mean exclusion chance were determined. The highest locus diversity and power of discrimination was determined for loci GATA172D05 and GATA31E08. Combined power of discrimination was 0.9999999997 (females) 0.999998548 (males). Mean exclusion chance was 0.999993172 (trios) and 0.999708265 (duos). Pairwise genetic distance (Fst) of X-STR frequencies suggest that Murcia can be closely grouped to other Iberian populations, and genetically furthest from Pas Valley, Navarra and Basque Country.

Forensic entomology: Molecular identification of blowfly species (Diptera: Calliphoridae) in Portugal

A.R. Oliveira, A. Farinha, M.T. Rebelo, D. Dias — 2011-11-07

Abstract: Insects, in particular Calliphoridae species have a very important role in decomposition process and are the earliest insects to infest a corpse. An accurate morphological identification is essential but very difficult or even sometimes impossible to do. So, molecular identification provides a rapid and reliable method that can be done in all development stages. The potential of mitochondrial cytochrome oxidase subunit I (COI) is very well established. But in some species this gene is not effective. In this work, we used the ribosomal internal transcribed spacer 2 (ITS2) to complement COI data, demonstrating ITS2 effectiveness in insects’ identification. Blowflies of the family Calliphoridae (Calliphora vicina, Calliphora vomitoria, Lucilia caesar and Lucilia sericata) were collected in Portugal. COI fragments permitted correct specimens identification using BLAST search for all blowflies, except for L. caesar because of the high similarity with Lucilia illustris. For these species, we used ITS2 sequences for species determination. This genetic marker analysis facilitated the differentiation of these two species.Our results indicate that it would be of great importance to increase the sequences collection to prevent incorrect identification and reinforce results validity.

X-STR admixture analysis of two populations of the Basque Diaspora in America

M.J. Illescas, J.M. Aznar, A. Odriozola, D. Celorrio, M.M. de Pancorbo — 2011-11-07

Abstract: The Basque Diaspora began to spread worldwide in the last century, mainly due to wars and first-born privileges over family land. The aim of this study was to explore the genetic composition and admixture of two populations of the Basque Diaspora located in Argentina and the United States. Samples were collected from individuals with Basque descendency living in the California, Nevada and Reno, United States and Buenos Aires, Argentina. PCR amplification was performed using GEP-ISFG X-STR decaplex primers. No deviations from Hardy–Weinberg equilibrium were observed. Admixture analysis revealed a high genetic contribution (33.3±1.9%) from Buenos Aires population to the admixed population of the Basque Diaspora in Argentina. A high genetic contribution (21±1.9%) from US Hispanic population was determined for the admixed population of the Basque Diaspora in the United States.

Forensic performance of insertion–deletion marker systems

2011-11-11

Abstract: The ability to improve amplification and analysis of degraded DNA extracts has been a long-standing area of research in forensic genetics. One of the latest approaches is the single multiplex typing of insertion–deletions (InDels), short biallelic length polymorphisms.InDels share most of the properties of single nucleotide polymorphisms (SNPs) that makes them ideal markers for forensic analysis of degraded DNA. The short amplicon size ranges, high multiplexing capability, and low mutation rate make them an attractive complement to mini-STRs. In addition, as length polymorphism markers, InDels can be analyzed with the same simple end-labeled PCR primer methods as STRs, thus avoiding the multi-step protocols required of SNP typing single base extension assays, as well as providing a more direct relationship between input DNA and peak height ratios. InDel genotyping should be considered a serious candidate for incorporation into the forensic marker battery. In order to assess the utility of such assays to the forensic community we have conducted a thorough analysis of a set of U.S. population samples with a commercial Indel assay, a 30 marker multiplex ‘DIPplex’ produced by Qiagen.

Comparison of two STR multiplexes for the analysis of chimerism after hematopoietic stem-cell transplantation

2011-11-07

Abstract: Short-tandem repeat (STR) genotyping is widely used for forensic purposes and it is also used to determine the proportions of donor and recipient cells after hematopoietic stem-cell transplantation. New STR multiplexes have recently been developed to analyze the complete CODIS plus D19S433 and D2S1338, such as AmpFℓSTR® Identifiler® Plus PCR Amplification kit (Applied Biosystems) and Investigator™ IDplex® (Qiagen). The objective of this study was to compare the sensitivity of both kits in the analysis of chimerism. The Investigator™ IDplex® allowed quimerism detection only in 10 (67%) of the 15 cases with chimerism detected by using the Identifiler® Plus kit, suggesting that the latter may have higher sensitivity.

Assessing the potential of next generation sequencing technologies for missing persons identification efforts

Jodi Irwin, Rebecca Just, Melissa Scheible, Odile Loreille — 2011-11-09

Abstract: To assess the utility of next generation sequencing (NGS) technologies for missing persons applications, we have recently initiated a study of various platforms and target enrichment strategies for sample types regularly encountered in our large-scale identification efforts. Specific laboratory workflows based on target marker enrichment and NGS platform are being considered for different sample types, and the overall effort is being undertaken with a strong emphasis on both raw data and final consensus sequence quality. The current study is first and foremost a general evaluation of these data and technologies from the standpoint of forensic application, yet the strategy we are pursuing is ultimately intended to facilitate NGS integration into standard casework laboratories. We are, therefore, evaluating NGS workflows and data for the typical nuclear and mitochondrial DNA markers used in forensics, while still allowing for future work that may take greater advantage of the strengths of these technologies. Here, we present an overview of our NGS strategy.

Human autosomal SNP profiling using fully automated electrospray ionization time of flight mass spectrometry

2011-11-21

Abstract: Single nucleotide polymorphisms (SNPs) represent a simple yet powerful tool for individual identification. Efforts by Pakstis and Kidd et al. to produce an ideal panel of genetically unlinked binary SNPs with high heterozygosity, low population bias, and uniform distribution over global populations have resulted in a 40-SNP panel analyzed across 40 global populations and a more-recent highly unlinked 45-SNP panel analyzed across 44 global populations. A fully automated PCR/Electrospray ionization mass spectrometry (ESI-MS) assay that genotypes the first 40-marker SNP panel has been developed for the Ibis PLEX-ID™ platform and developmentally validated. A 64-SNP assay that incorporates the union of the two SNP panels has been developed for the Ibis PLEX-ID™ and is undergoing validation. Concordance with standard TaqMan assays for a panel of samples has been demonstrated for all loci. The assay has been characterized for sensitivity, reproducibility, species specificity, and the ability to detect when genotyping results indicate a pure sample or a mixture/contaminated sample. Validation studies suggest sensitivity close to 100pg DNA per reaction. A convenient software interface has been developed for visual review of automated data analyses. The Ibis PLEX-ID™ ESI-MS platform is capable of running Y-STR, autosomal STR, mitochondrial DNA, and SNP analyses on a single instrument within the same automated run.

Human Y-STR profiling using fully automated electrospray ionization time of flight mass spectrometry

2011-11-21

Abstract: Polymorphic Y-chromosomal markers are useful for studying the male-specific complement of human DNA. In forensic analyses, Y-chromosomal short tandem repeats (Y-STRs) are typed to produce a haplotype profile that is shared among male members of a lineage and can be useful in parentage and relationship testing of male family members. Y-STRs can also be valuable in cases involving a male suspect's DNA with an excess of female DNA, such as rape cases. An automated Y-STR profiling assay has been developed for the Ibis Biosciences PLEX-ID™ mass spectrometry platform that reveals sequence polymorphisms within Y-STR alleles and requires only DNA template is added to a pre-fabricated plate prior to thermal cycling. The assay analyzes sixteen loci: DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, and Y-GATA-H4. Samples from Caucasian, African American and Hispanic population groups were analyzed via PCR followed by direct ESI-MS. Correct allele assignments were confirmed for a subset of samples by comparison to truth data produced using standard Y-STR typing techniques. Allelic variants differing by sequence polymorphisms were revealed, expanding the allele base for several loci. Preliminary validation has shown sensitivity to approximately 125–250pg of DNA per reaction. The assay has been characterized for sensitivity, reproducibility, species specificity, and specificity to male DNA in the presence of a 100- to 1000-fold excess of female DNA. The ESI-MS platform is capable of running Y-STR, autosomal STR, mitochondrial DNA, and SNP analyses on a single instrument within the same automated run.

Validation of the NGM SElect™ kit

C. Gehrig, S. Vuichard, A. Teyssier, V. Castella — 2011-11-09

Abstract: The Swiss national database was launched August 2000 based on the 10 SGM Plus loci. With the aim of addressing the needs of the next-generation European STR genotyping systems in Switzerland, we validated the NGM SElect™ kit. In this study, we present the results of forensic validation studies including the following aspects: sensitivity, heterozygote peak height ratio calculations, performance with simulated PCR inhibition, proficiency tests and Swiss population data.

Sequencing of mtDNA HV1 and HV2 regions from samples with trace amount of DNA

S. Erdem, H. Altunçul, G. Filoğlu, A.M. Ölçen, Ö. Bülbül — 2011-11-03

Abstract: Some biological samples cannot be genotyped with routine STR analyzes, if they contain trace amount of DNA. At these cases, mitochondrial DNA analyzes can be carried out. The aim of this work was optimizing the sequencing of mtDNA's HV1 and HV2 regions from samples like hair, nails, earrings, toothbrushes, q-tips, glass edge swabs, gums, razors and cigarette butts. At this work 5 unrelated individual's samples mentioned above were sequenced and other 11 unrelated individual's blood samples were sequenced for the reliability of the procedure. In this examined Turkish population, most common polymorphic positions were 263, A>G point mutation, and 315.1 C insertion. The HV2 profile “263, A-G 315.1 C” observed on 3 individuals and “152, T-C 263, A-G 309.1 C 309.2 C 315.1 C” profile observed on 2 individuals. Two individual also shared a profile same as rCRS on their HV1 regions. Every analyzed person has unique HV1+HV2 profiles together.

Evaluation of three new forensic DNA profiling kits on PCR-inhibitory crime scene samples

J. Hedman, L. Albinsson, L. Norén, R. Ansell — 2011-11-09

Abstract: In 2009–2010, several forensic DNA profiling kits customised for Europe and the Prüm Treaty were commercially released. The manufacturers have made efforts to increase the PCR inhibitor tolerance compared to previous kits, as shown by their increased resistance to known molecular inhibitors such as humic acid and haematin. We have evaluated three new 15 STR-marker profiling kits (AmpFlSTR NGM, PowerPlex ESI16 and PowerPlex ESX16) on various PCR-inhibitory crime scene samples. All three kits produced usable DNA profiles from most samples. However, the kits were affected by inhibitory compounds from some of the samples, resulting in partial DNA profiles.

Import and direct processing of capillary electrophoresis analysis data in forensic casework

S. Köhnemann, J. Kretzschmann, K. Mittmann, H. Pfeiffer — 2011-11-03

Abstract: The processing and tabulation of analysis data in forensic casework is time consuming and has a lot of sources for possible errors. To eliminate known sources of errors and to reduce the time of work, we established a Microsoft Access database relying on Visual Basic programming. The database is based upon the direct data import from the capillary electrophoresis, after that the data will be processed immediately. The criteria for processing are variable and may depend on the experts’ opinion. A check for contamination against the DNA-characteristics of the laboratory personnel and/or the DNA-characteristics stored in the database will help to find false or contaminant DNA-profiles.

Genetic polymorphism of the new PowerPlex® ESI 17 system in a Tibetan population from Dharamsala (India)

L.N. Riccardi, S. Ceccardi, M. Falconi, D. Luiselli, C. Bini, S. Pelotti — 2011-11-07

Abstract: Alleles frequencies for the 17 short tandem repeats (STRs) loci included in the PowerPlex® ESI 17 kit were investigated in a sample of 80 unrelated individuals from a Tibetan ethnic group living in Dharamsala in the state of Himachal Pradesh (Northern India). Due to the relative novelty of this kit, including the 5 new ENFSI recommended loci (D22S1045, D2S441, D10S1248, D1S1656, D12S391) together with SE33 locus, no information regarding all these new loci is available for the Tibetan population. The aims of the present study were to determine the genetic polymorphism of forensic validated markers and to increase the ethnical population database.

Determination of siblings: A special case report from Halle

U.D. Immel, S. Lutz-Bonengel, J. Edelmann, R. Lessig — 2011-11-09

Abstract: A case of three female children, which were found in a baby hatch of a hospital in Halle during the last three years will be reported. The youth welfare office in cooperation with the adoptive parents wanted to know, if the three girls are full siblings, half siblings or unrelated.We present the results of all mentioned investigations as well as the results of the calculation of sibling (DNAVIEW – Ch. Brenner).

DNA typing in single cell analysis: Single sperm cells outperform whole genome pre-amplified samples

T. Kroneis, I.E. Pickrahn, A. El-Heliebi, G. Schmidt-Gann — 2011-11-09

Abstract: We recently developed a method in single cell analysis allowing for multiple molecular genetic and cytogenetic analyses of one and the same cell. In the actual study we compared direct DNA typing to DNA typing of whole genome amplified single sperm cells. Showing a PCR efficiency of 62.8%, DNA typing of non-preamplified single sperm cells turned out to be superior to single sperm cell samples pre-amplified by means of isothermal whole genome amplification (7.5%).

Analysis of 9 mitochondrial SNP's from samples with trace Amount of DNA

A.M. Ölçen, G. Filoğlu, H. Altunçul, S. Erdem, Ö. Bülbül — 2011-11-09

Abstract: Forensic scientists often use nuclear STR loci for identification. But sometimes it is difficult to get successful results from nuclear DNA, especially when the DNA in a sample is degraded or the amount is not sufficient. In these cases forensic scientists use mitochondrial DNA (mtDNA). The aim of this work is to genotype of the 9 mitochondrial SNP sites (3010, 5004, 6776, 8592, 10394, 10754, 11864, 15340, 16519) and to identify polymorphic sites within these SNP's in Turk population, while validating a routine procedure that is optimized to analyze samples trace amount of DNA. After optimization, blood samples from 30 volunteers and trace amount of DNA containing samples from 5 of these volunteers were analyzed successfully except hanky with mucus on it. These samples were hair, nails, earrings, toothbrushes, qtip, glass edge swabs, gum, razors and cigarette butts. The result of 30 volunteers showed that the SNP sites 3010 and 16519 were polymorphic sites among Turks, and the 15340. SNP site was different than the rCRS (revised Cambridge Reference Sequence) at all volunteers.

Genetic markers for body fluid and tissue identification in forensics

I. Gomes, F. Kohlmeier, P.M. Schneider — 2011-11-03

Abstract: Many studies have focused on the identification of human fluids and tissues that are often left behind in forensic settings but only a few have, so far, analysed potential markers for human skin determination. The current work presents initial data on the comparison of two recently reported methods for skin tissue determination for forensic applications using DNA- and mRNA-based assays, aiming towards the implementation of an identification assay for human skin tissue.

The recovery of DNA profiles from saliva and touch evidences after postal bomb explosion

A. Berti, F. Barni, A. Virgili, C. Colozza, F. Maiorino, M. Tocca — 2011-11-03

Abstract: Terrorist crime usually makes use of improvised explosive disposals (IEDs) which, especially in national environments, consist of postal or pipe bombs often assembled by using plastic or paper envelopes, adhesive tapes, electric components such as switches, batteries, cables, etc. To date it has been considering quite complex to recover DNA in quality and quantity proper for STRs typing aimed at the identification of the manufacturer of the IED especially due to the very serious damage degree of the evidence (exposed to high temperatures). In this work we tested saliva and touch evidences (perspiration), usually found on a postal package, deposed inside the postal bombs charged with deflagrating or detonating explosives in different quantities and we evaluated the possibility to achieve DNA profiles useable for identification purposes from such kind of post-explosion biological evidences. We demonstrated that with new generation multiplexes it is possible to gain STRs profile useful for identification purposes even from highly burned and degraded bomb debris.

NEW miniSTR loci D10S1248, D14S1434, D22S1045, D4S2364, D2S441, D1S1677 validation and optimization on blood samples

Tuğba Ünsal, Gönül Filoğlu, Elif Sipahi, Havva Altunçul, Gülten Rayimoğlu — 2011-11-11

Abstract: In this study; validation and optimization study of 6 new miniSTR was made to use these loci on forensic laboratory in Turkey. Coble and Butler's method was based in this study but several modifications were made on this PCR method. Primer dye set should be FAM-TET-HEX rather than FAM-VIC-NED. Primer concentrations were increased. Top Taq™ Master Mix Kit (Qiagen) was used for PCR rather than use of single by single PCR solution. 6 loci were studied as two triple multiplex sets. After the method was optimized with these conditions, validation studies were carried out by working the sensitivity and reproducibility of this procedure and analyzing mixed samples.

Test of the rapid PCR method using AmpFlSTR Identifiler kit

Chong Min Choung, Dong Sub Lee, Ki Won Park, Myun Soo Han — 2011-12-01

Abstract: Current forensic DNA typing is conducted in approximately 8–9h. Whole steps included DNA extraction step, Quantification, PCR amplification step, electrophoresis process through capillary separation with fluorescence detection, data analysis and DNA profile interpretation. Among them, we have tested rapid PCR method of AmpFlSTR Identifler PCR to reduce running time. We have altered several PCR conditions of ordinary AmpFlSTR Identifler PCR method and used 9947A control DNA to cut the time for the PCR reaction. In the results of this study, the critical step of the PCR reaction was the annealing step and also we reduced PCR running times by 1/3 to 2/3 (approximately 60–90min) with complete concordance of STR allele calls using standard reference material 9947A.

Validation of the PowerPlex® ESX17 and ESI17 kits for STR typing of telogen hair roots in forensic casework

Verena Brune, Birgit Bayer, Katja Anslinger — 2011-11-09

Abstract: Telogen hair roots are usually characterized by only a small amount of nuclear DNA which is frequently also degraded. STR analysis of telogen hair roots is therefore often complicated. However, significant improvement has been achieved. Due to the requirements to analyze the new 16 STR markers of the European Standard Set of loci (ESS) and SE33 in forensic casework, we investigated the new PowerPlex® ESX17 and ESI17 kits from Promega for their application to telogen hair roots in comparison to the genRES® MPX SP-4 kit from Serac and the AmpF/STR SEfiler Plus™ kit from Applied Biosystems. Apart from the advantage to be able to analyze 17 loci in one multiplex PCR to implement the new European Standard Set of loci into the routine investigation of telogen hair roots in forensic casework, the PowerPlex® ESX17 and ESI17 kits from Promega yielded reproducible results and showed the tendency to be even more sensitive than the genRES® MPX short-plex SP-4 kit from Serac and the AmpF/STR SEfiler Plus™ kit from Applied Biosystems. Considering the possible amount of detectable allels, “allelic drop out” did not appear increasingly in PCR systems with amplicon sizes more than 200 base pairs.

A SNaPshot™ assay for the identification of forensically important blowflies

J.A. Smith, H. Godfrey — 2011-11-10

Abstract: A dead body is an attractive habitat for many different insect species throughout the process of decomposition, but it is members of the blowfly family (Calliphoridae) that are usually the first to arrive, using the body as an oviposition site and food source for developing larvae. The stage of larvae found on a body can be a useful indicator of time since death but in order for species specific life cycle data to be applied, accurate species identification is critical. Damaged, unviable or immature specimens can be difficult to identify morphologically and recent work has focussed on genetic identification. The aim of this study was to assess the cytochrome oxidase I (COI) gene within the mitochondrial genome as a suitable marker for the Calliphoridae and develop a simple assay for species identification. We present a 6-plex SNaPshot™ assay that differentiates seven common blowfly species found in the UK.

Allele frequency distribution of twelve X-chromosomal short tandem repeat markers in four U.S. population groups

2011-11-11

Abstract: A total of 853 samples from the four major U.S. population groups (African American, Asian, Caucasian, and Hispanic) were typed using the Qiagen® Investigator Argus X-12 kit. Allele frequency distributions are reported here for each of the 12 X-STR markers (DXS10103, DXS8378, DXS7132, DXS10134, DXS10074, DXS10101, DXS10135, DXS7423, DXS10146, DXS10079, DXS10148, and HPRTB).

Investigative DNA databases that preserve identification information

Mark W. Perlin — 2011-11-03

Abstract: At the heart of the science of genetics is the genotype, a genetic type comprised of allele pairs at a set of loci. Since the time of Mendel in the 19th century, genotypes have been understood to be uncertain quantities represented by probability. Forensic DNA has uprooted that scientific tradition, seeking genotype certainty where none exists in the evidence. The result is a tremendous loss of identification information through the application of misguided scientific models. That information loss extends to forensic DNA databases, where genotypes are incorrectly stored as allele pairs or lists, rather than preserving their full identification power through a standard probability representation. The consequences to society are severe, since the loss of DNA database investigative information leads to the needless victimization of innocent citizens by crimes that could have been prevented. The solution is to deploy investigative DNA databases that properly preserve identification information using probabilistic genotypes.

mixsep: An R-package for DNA mixture separation

Torben Tvedebrink — 2011-11-11

Abstract: An implementation of a mixture separating algorithm based on a statistical model for STR peak intensities is presented . The implementation is freely available in the open source software R. A graphical user interface (GUI) eases the data importing, specification of known profiles and analysis of the STR data .The statistical model formulated for the peak intensities satisfy intrinsic proportions of the STR peak data, and assumes that the contribution to shared alleles is additive in terms of the total amount of DNA. The model and algorithm for separating DNA mixtures was validated and gave similar results as those of three experienced forensic geneticists. The advantages of the model-based approach are consistency in case reports within a laboratory, increased speed and objective measures for comparing different proposed combinations of DNA profiles. Furthermore, the statistical model can be used to compute expected peak intensities, which plotted together with the observed peak intensities provide a powerful tool for communicating the conclusions of an analysis.

Statistical model for degraded DNA samples and adjusted probabilities for allelic drop-out

Torben Tvedebrink, Poul Svante Eriksen, Helle Smidt Mogensen, Niels Morling — 2011-11-11

Abstract: DNA samples found at a scene of crime or obtained from the debris of a mass disaster accident are often subject to degradation. When using the STR DNA technology, the DNA profile is observed via a so called electropherogram (EPG), where the alleles are identified as signal peaks above a certain level or above a signal to noise threshold. Degradation implies that these peak intensities decrease in strength for longer short tandem repeat (STR) sequences. Consequently, long STR loci may fail to produce peak heights above the limit of detection resulting in allelic or locus drop-outs.In this paper, we present at method for measuring the degree of degradation of a sample and demonstrate how to incorporate this in estimating the probability of allelic drop-out. This is done by extending an existing method derived for non-degraded samples. The performance of the methodology is evaluated using data from degraded DNA, where cases with varying amounts of DNA and levels of degradation are investigated.

Genetic variants related to nicotine dependence

B. Corradini, P. Sánchez-Diz, M. Alù, A. Estany-Gestal, A. Carracedo, G. Ferri — 2011-11-10

Abstract: Large-scale population studies have proved that genetic factors contribute to individual differences in smoking behavior. Genes responsible for nicotine's pharmacokinetics and pharmacodynamics seem mainly involved, although a significant fraction of variance remains unexplained. In this study we examined 10 SNPs from 8 candidate genes with positive previous reports of association with smoking. A total of 454 Italian unrelated subjects were genotyped by a multiplex minisequencing assay through the SNaPShot kit. Cases were chosen as current and former nicotine dependent (FTND≥4 and SQ≥15), while controls were smoking-exposed but non-dependent and never smoker individuals (FTND=0 and SQ≤10 and FTND=0 and SQ=0, respectively). Preliminary results shows that the SNPs CHRNA5-rs16969968 and CHRNA3-rs1051730 could be associated with risk of developing nicotine dependence. Factors as age, sex, and exposition to smoke were also found as possible factors of risk of nicotine addiction. The identification of susceptibility loci for individual response to substance abuse is particularly motivating for medicine for the global epidemic dimension of addictions and the urgent need of effective preventive and therapeutic strategies.

Analysis of SNPs involved in central nervous system in completed suicide victims

G. Ferri, B. Corradini, A. Estany-Gestal, P. Sánchez Diz, E. Radheshi, M. Alù — 2011-11-10

Abstract: Suicide is a devastating psychopathological trait with familial transmission. According to epidemiological data suicide is partly under genetic influence. Despite the extensive body of association studies, to date genetic factors linked to suicide diathesis are largely unknown. In this study a set of 8 SNPs and a 43bp ins/del was genotyped in an Italian sample of suicide completers (n=65) and control subjects (n=77). The selected markers are spread across multiple brain pathways: serotonergic, stress-related HPA axis, catecholamine metabolism and nitric oxide (NO) neurotransmission. Basic analysis at single loci shows significant differences in allele and genotype distributions between victims and controls for TPH1-rs1800532(allele A) and NOSI-rs693534(allele A), pointing to an association with the group of suicides. Presented results are intended to be exploratory, requiring further investigation in a larger sample size. Systematic efforts in suicide research is quite important in order to empower understanding of risk factors and developing preventive strategies in clinical practice.

Implementation of the SNPforID multiplex on the Sequenom® MassARRAY® analyzer 4 system

L. Poulsen, C. Børsting, N. Morling — 2011-11-10

Abstract: An assay was designed for typing 29 of the 52 SNPs in the SNPforID multiplex on the Sequenom® MassARRAY® analyzer 4 system. The dedicated TYPER 4 software was used for automatic allele calling. The sensitivity of the assay was tested using 0.5–40ng pristine template DNA in the PCR reaction. Overall, no difference was observed in the sensitivity, measured as call rate, for the different amounts of template DNA. Typing of paternity case work samples resulted in lower call rates (54%) as compared to the sensitivity test (89%), and more incorrect calls, 0.4% as compared to 0.1%. Finally, the ability of the software to detect 1:1, 1:3 and 1:5 DNA mixtures was investigated. Compared to the sensitivity test, the call rates of the 1:1 and 1:3 mixtures were low (64–74%), whereas the call rates of the 1:5 mixtures were similar (88–90%). However, in comparison with the case work samples, the call rates of the mixtures were not unusual and mixtures may be overlooked.

Molecular “eyewitness”: Forensic prediction of phenotype and ancestry

Katherine Butler, Michelle Peck, Jessica Hart, Moses Schanfield, Daniele Podini — 2011-11-09

Abstract: When an STR DNA profile obtained from crime scene evidence does not match identified suspects or profiles from available databases, further DNA analyses targeted at inferring the possible ancestral origin and phenotypic characteristics of the perpetrator (i.e. hair color, skin color and eye color) could yield valuable information. Single Nucleotide Polymorphisms (SNPs), the most common form of genetic polymorphisms, have alleles associated with specific populations and/or correlated to physical characteristics. We have used Single Base primer Extension (SBE) technology to develop panels which include 103 ancestry and phenotype markers selected from recent literature. DNA samples, along with corresponding ancestry/phenotype survey information and spectrophotometric skin color data, have been collected from 276 anonymous volunteers of varying ethnicity, gender and age. These samples, and additional samples of known ancestry, have been screened with the SBE panels to assess the predictive value of the candidate SNPs, with the goal of identifying the optimal panel of SNPs to efficiently assess an unknown individual's characteristics. STRUCTURE software analysis showed that individuals are classified in the expected groups. Also Principal Component Analysis was performed for pigmentation (eye, hair, and skin) and for biogeographic ancestry of individuals. Results show that the different categories in which individuals were classified for each trait could be graphically separated with a reduced number of selected SNPs. Several SNPs provide information for both pigmentation and ancestry; thus, we expect that it will be possible to generate informative inferences with a panel of 30–35 SNPs.

A SNP multiplex for the simultaneous prediction of biogeographic ancestry and pigmentation type

2011-11-09

Abstract: DNA analysis of ancestry informative markers (AIMs) and physical trait markers from biological stains can help provide investigative leads in cases without suspects. To enhance the resolution and informative value of two previously developed single nucleotide polymorphism (SNP) multiplexes, the 34-plex and Eurasiaplex assays (Phillips et al. , Phillips et al. ) differentiating European, South Asian and Middle East populations, we have selected an additional 22 AIM-SNPs. The selected markers focus on the differentiation of Europeans and Asians and we supplemented this AIM set with 10 recently published pigmentation markers informative for eye, hair and skin colour (Walsh et al. , Sturm ). Comparisons of reference SNP data from HGDP-CEPH and 1000 Genomes population panels with 7 Eurasian study populations were made using STRUCTURE and principal component analysis (PCA) indicating that this multiplex can improve the differentiation of populations within East Asia. In a small pilot study using voluntary donors from Turkey, the IrisPlex SNP markers (Walsh et al. ) built into our multiplex were successfully used to make predictions about eye colour.

SE33 variant alleles: Sequences and implications

2011-11-07

Abstract: U.S. population sample sets have been tested at the SE33 locus with PowerPlex ESX 17 and ESI 17 as well as AmpFlSTR NGM SElect, Investigator ESSplex SE, and the widely used “Polymeropoulos” primers to explore any concordance issues between kits possessing primers in different positions. A G→A mutation 68bp downstream of the repeat region has been detected in several samples that can cause a mobility shift in PowerPlex ESI 17 and ESSplex SE relative to PowerPlex ESX 17 and NGM SElect SE33 alleles. In addition, a C→T mutation 60bp downstream of the repeat region created the same mobility shift and allele dropout with the ESX 17 kit.

Evaluation of automatable silica-based extraction methods for low quantity samples

Kimberly Sturk-Andreaggi, Toni Marie Diegoli, Rebecca Just, Jodi Irwin — 2011-12-01

Abstract: This study evaluated four commercial silica-based extraction chemistries using serum and mock forensic samples to determine which automatable method is best suited to isolate DNA from low quantity samples. Data from quantitative polymerase chain reaction assays identified the PrepFiler™ Forensic DNA Extraction kit as the superior extraction method with average yields equal to or better than those obtained with organic and other commercial silica-based methods tested.

Human sex determination by amelogenin padlock probes

Piyarat Thaijaroen, Korapin Srisiri, Rapee Boonplueang, Nathinee Panvisavas — 2011-11-03

Abstract: In this study, we utilized a technique called ligation-mediated rolling circle amplification (L-RCA) that combined padlock probe ligation and rolling circle amplification techniques to determine human sex. Amelogenin X and Y specific padlock probes were designed based on polymorphisms and the 6-bp indel of the amelogenin X and Y alleles. Lengths of DNA target for padlock probe detection were 40 and 33 bases of amelogenin X and Y alleles, respectively. Analysis of female DNA samples produced one L-RCA product from the AmelX padlock probe, whereas 2 different types of products were amplified from male DNA samples, each generated by AmelX and AmelY padlock probes. Utilization of padlock probes via L-RCA technique would provide an alternative tool for human sex determination in highly degraded DNA analysis in the future.

20 SNPs as supplementary markers in kinship testing

2011-11-07

Abstract: Single Nucleotide Polymorphisms (SNPs) are having an increasingly role in Forensic Genetics due to very low SNP mutation rates and the possibility to multiplex a great number of loci. The purpose of this study was to evaluate the use of 20 autosomal SNPs as additional markers in the resolution of kinship casework where the alleged father was not available for testing and close relatives were used instead. A total of six caseworks which included alleged paternal grandparents, alleged uncles or alleged brothers were studied. All individuals studied in these cases were typed before with 17 autosomal STRs using Identifiler(Plus)® and Powerplex 16® systems. Twenty SNPs were typed using SNaPshot® methodology with two 10-plex, previously shown useful in paternity testing. LRs were calculated with “Familias” using South Portugal STR and SNP frequency databases. This study confirms that even as few as 20 autosomal SNP loci can be very useful in kinship analysis as a complement to standard methodologies. Moreover, SNaPshot® methodology can be easily implemented in any Forensic Laboratory.

Combining DNA evidence for greater match information

Mark W. Perlin — 2011-11-03

Abstract: Statistical science studies multiple experiments in order to assess their implications and variation. Forensic science is often limited to a single experiment, and so cannot employ statistical inference. With DNA, however, there can be multiple items and assays from crime scene evidence. Since STR data have quantitative peak heights obtained from DNA sequencer signals, they can be statistically analyzed in a likelihood function. Moreover, multiple STR experiments can be mathematically combined using a joint likelihood function. Statistics tells us that using more data should yield more information. We find that computing with a joint likelihood function to combine DNA evidence can infer more identification information, as measured by the likelihood ratio match statistic.

Results of the 2011 Relationship Testing Workshop of the English Speaking Working Group

L. Poulsen, S.L. Friis, C. Hallenberg, B.T. Simonsen, N. Morling — 2011-11-07

Abstract: We present the results of the 2011 Relationship Testing Workshop of the English Speaking Working Group of the International Society for Forensic Genetics. A total of 62 laboratories participated. The exercise included relationship testing of blood samples from a man and two children. Assuming that the man is the biological father of both children, the laboratories were asked to investigate if the results were in favour or against that the children were half or full siblings. Furthermore, the laboratories filled out a questionnaire concerning the laboratory strategies and routines. Finally, the laboratories were encouraged to do a paper challenge with statistical calculations. The relationship testing exercise revealed that the use of mtDNA and X-STR was informative and excluded the possibility of full siblings. In the questionnaire, most laboratories (77%) indicated that a single autosomal STR kit was used in all paternity cases. However, >60% of the laboratories had three or more autosomal STR kits available for paternity testing. The paper challenge showed large variations in the biostatistical calculations in cases of rare events such as a rare allele and when a single genetic inconsistency was observed.

A comparison of AmpFlSTR Identifiler™ Kit versus AmpFlSTR Identifiler Plus™ Kit in challenging bone samples by using normal and increased PCR cycle number

C. Romanini, M. Romero Ferrer, M.L. Catelli, C. Vullo — 2011-11-11

Abstract: With the aim to increase the chance of obtaining DNA profiles from challenging forensic samples, several strategies are tested. One of the most powerful tools used in forensic DNA typing are commercial amplification kits. Enhanced buffers are provided with these kits allowing amplification of highly degraded or inhibited samples. For low DNA samples, sensitivity can be increased by raising the PCR cycle number.This study presents the results of the comparison made between Identifiler™ versus Identifiler Plus™ by using normal and increased PCR cycle number.Fifteen samples of 30–40 years post-mortem belonging to missing persons during military governments in Argentina were tested with Identifiler™ using 28 and 34 PCR cycles and with Identifiler Plus™ using 29 and 32 PCR cycles in order to compare both amplification kits.A high percentage of samples showed higher number of amplified loci with Identifiler Plus™ than with Identifiler™. This effect is more evident when increased PCR cycle number was used.Also, there were samples that exhibited identical number of amplified loci by using both kits. These samples showed high degraded DNA characteristics and the amplified loci were not the same for each amplification kit.For a group of samples that displayed flat profiles using normal PCR cycles for both kits, increased PCR cycles allowed a profile improvement that was higher using Identifiler™ with 34 PCR cycles than using Identifiler Plus™ with 32 cycles. Characteristics of highly degraded DNA or low DNA concentration were found in these samples.Furthermore, Identifiler Plus™ showed a lower percentage of locus drop-out than Identifiler™ for most of the analyzed loci and also an improved amplification success of the larger loci.On the basis of our results, Identifiler Plus™ offers a more increasing chance of DNA typing than Identifiler™ does, based on the new buffer which mainly allows overcoming PCR inhibition.

Floods and mudslides in the State of Rio de Janeiro and a plane crash in the Brazilian Amazon rainforest: A study of two different experiences in disaster victim identification (DVI)

2011-11-03

Abstract: In mass fatality incidents there are critical variables that will shape the response to the events. These variables will determine different strategies of action and will require specific approaches for the appropriate disaster management and identification of victims. Magnitude and nature of the disaster, number of victims, if it is an open or closed event, degree of fragmentation and decomposition of bodies, accessibility of ante-mortem data, availability of DNA reference samples and kinds of post-mortem samples for DNA testing are some critical variables in disaster victim identification (DVI). In this study, we will discuss how some of these variables shaped the response and the results of the methods of identification by DNA, fingerprint and dental analysis in two different disasters that occurred in Brazil: Floods and mudslides in the mountainous region of the State of Rio de Janeiro, in January 2011, in which 895 people died, and a plane crash in the Brazilian Amazon rainforest, with 154 fatal victims, in September 2006.

Mitochondrial DNA analysis of formalin-fixed paraffin-embedded tissue samples: Effect of formalin on DNA stability and its implications in genetic studies

2011-11-07

Abstract: Formalin-fixed paraffin-embedded tissue (FFPET) samples are widely employed in Molecular Epidemiology and Forensic Genetics. However, the effects of formaldehyde on mitochondrial DNA (mtDNA) still remain unexplored. Our aim was to determine the presence of alterations in mtDNA caused by the process of fixation with formalin. FFPET, blood samples and fresh tissue samples were collected from autopsies. Segment HVSIa within the displacement loop (Dloop) and a segment of the coding region of the mtDNA were amplified and sequenced. Changes were not observed in the coding region. However, analysis of HVSIa revealed the existence of numerous differences between FFPET samples and their corresponding reference sequences from blood and/or fresh tissue. These results point to readdress the use of FFPET samples in studies of the Dloop of the mtDNA and urge to act with caution in the resolution of practical cases in Forensic Genetics.

An Italian Jean Jacques Rousseau: A complex kinship case

S. Inturri, C. Robino, I. Carboni, U. Ricci, S. Gino — 2011-11-10

Abstract: We report the case of four women and a man, all born in an Italian village during and immediately after WWII, that recently contacted our laboratory in order to perform kinship analysis. According to their claim, the propositi were the illegitimate offspring of a country gentleman and a peasant woman, given in adoption immediately after birth. A story that curiously reminded us of Jean Jacques Rousseau, Thérèse Levasseur and their five children. Problems connected with DNA analysis in cases where all stated relationship are questioned, and a wide range of different pedigrees could be used as hypotheses in LR calculations are discussed.

Discrimination of ‘fiber-type’ and ‘drug-type’ Cannabis sativa L. by fluorescent duplex PCR

Arpaporn Sutipatanasomboon, Nathinee Panvisavas — 2011-11-11

Abstract: A fluorescent duplex-PCR test was developed based on polymorphisms of the THCA synthase gene in order to discriminate the fiber- and drug-type Cannabis sativa L. and to indicate the presence of Cannabis trace in suspected materials by the numbers and sizes of PCR-amplified products. DNA analysis of drug-type Cannabis resulted in two different PCR-amplified DNA fragments of 94 and 158bp, whereas only the 94bp PCR product was amplified from the fiber-type DNA. DNA test results of another 6 Cannabis sativa L. collected from the field agreed with chemotype determined by GC-MS. However, it was noted that the only intermediate drug-type sample tested gave a drug-type result for DNA testing. Specificity of the duplex PCR was shown by testing with DNA from species that may be related to Cannabis abuse, i.e., common hop (Humulus lupulus L.), 2 narcotic plants (Papaver somniferum and Mitragyna speciosa), tobacco (Nicotiana tabacum) and human. Sensitivity of detection was as low as 100pg of genomic DNA.

DNA typing from fluorescent powder dusted latent fingerprints

2011-11-03

Abstract: DNA profiles were successfully typed from four latent fingerprints deposited on glass plate, glossy magazine paper, and plastic surface that were dusted with red, green, and yellow fluorescent powder. No or partial DNA profile was generated from one latent fingerprint samples. In addition, two DNA extraction methods, i.e., QIAamp® DNA Mini Kit and Chelex® 100 were compared. Experiments demonstrated that the absorbance measurements by spectrophotometery were interfered by colors of the fluorescent powder extracts, although these did not affect DNA profiling and detection of the PCR-amplified products by the genetic analyzer instrument. Results demonstrated that QIAamp® DNA Mini Kit was more suitable than Chelex® 100 for recovery of DNA from fluorescent powder dusted fingerprints. The quality of partial DNA profiles obtained from fluorescent powder dusted fingerprints was improved through the application of low copy number (LCN) typing, by increasing the number of PCR cycles from 28 to 34. Surface type had an effect on the number of loci obtained, and fluorescent fingerprint powders used had subtle effect on the profile quality obtained.

Analysis of the sarcomere protein gene mutation on cardiomyopathy—Mutations in the troponin complex genes

2011-11-03

Abstract: Developments in the molecular genetic studies of cardiomyopathy (CM) have led to discovery of a large number of mutations in the genes encoding the sarcomeric proteins. In this study, comprehensive screening of TNNT2, TNNI3 and TNNC1 was performed in 36 consented autopsy cases diagnosed as CM, in order to evaluate the prevalence of gene mutations in sudden death caused by CM. A total of 12 mutations and 15 single nucleotide polymorphisms (SNPs) were detected. It was indicated that this study contribute to genetic based diagnosis, risk stratification and prevention of sudden death caused by CM.

Improved performance for forensic casework: Extraction and isolation updates for the Maxwell® 16 instrument

2011-11-03

Abstract: The DNA IQ™ System is an established chemistry for DNA recovery from casework samples. Successful DNA recovery from most casework samples depends on the efficiency of extraction, which refers to removal of DNA from a solid support such as a swab or fabric cutting, and isolation, which refers to the recovery of DNA once it is extracted from the solid support.We have recently improved the performance of the DNA IQ™ System on the Maxwell® 16 Instrument by increasing extraction and isolation efficiencies. First, we designed a new LEV plunger using a proprietary material that increases isolation efficiency. Second, we improved extraction efficiency by introducing an optimized DNA extraction buffer. In this article, we demonstrate the resulting increase in DNA yield across a variety of samples, and compare these results to data generated by organic extraction.

Visualization of latent biological traces via 5-methylthioninhydrin (5-MTN) staining for forensic DNA typing

M.M. Schulz, V. Brune, M. Maierthaler, M. Graw — 2011-11-03

Abstract: Advance in forensic DNA analysis has made it possible to analyze even biological traces invisible to the naked eye, such as skin abrasions. As latent traces include the risk of being overlooked, often large areas are sampled. Unfortunately, this approach includes the danger of a contamination and very often generates mixed DNA profiles, which are associated with disadvantages. This study presents a new dyeing technique which enables a controlled analysis of latent biological traces. By a series of experiments the limitations and possible effects on subsequent STR analysis were examined. On staged exhibits the efficiency of the screening aid was again tested and the usability of the new procedure demonstrated.

Where does this tiger come from?—A robust molecular technique for simultaneous identification of endangered species and subspecies

2011-11-04

Abstract: The Convention on the International Trade in Endangered Species of Wild Fauna and Flora (CITES) monitors the international trade in endangered animal and plant species; a high profile example is the tiger, Panthera tigris. We report on the application of a SNaPshot multiplex technique to simultaneously identify tiger species and subspecies; this test is based on identification of SNPs within the tiger mitochondrial genome. Mitochondrial DNA sequences from four of the five extant putative tiger subspecies were obtained and combined with DNA sequence data from 492 tiger and 349 other mammalian species. A total of 11 SNP loci were identified: five specific for tiger; three specific to Panthera tigris sumatrae and; three specific to P. t. tigris. The multiplex assay was able to reliably identify 15 voucher tiger samples. The sensitivity of the test was 15,000 mitochondrial DNA copies, indicating that it will work on trace amounts of tissue, bone or hair.

Genetic structure of Moroccan population using 15 STRs of NGM kit

H. El Amri, D. Squalli, B. Gazzaz, A. El Harrak Hajri, H. El Ossmani — 2011-11-17

Abstract: Polymerase chain reaction (PCR) amplification using the AmpFℓSTR® NGM™ Kit (“NGM,” an acronym for “Next Generation Multiplex”) was performed in random sample of 180 unrelated individuals from three Moroccan population groups (Arab Speaking, Berber Speaking, and Sahrawi). Allele frequency and other forensically relevant statistics data were generated for the NGM™ multiplex kit includes the original 10STR loci from the AmpFℓSTR SGM Plus® kit (D3S1358, vWA, D16S539, D2S1338, D8S1179, D19S433, TH01, FGA, D21S11, D18S51) together with five additional STRs (D10S1248, D22S1045, D2S441, D1S1656 and D12S391) and the Amelogenin sex-determination locus. Population study was conducted to evaluate usefulness of the loci (especially the five new microsatellite systems) in forensic genetic identification examinations. All Fifteen autosomal STR loci were found to be in Hardy–Weinberg equilibrium. Discrimination power was particularly high in case of D18S51 and D12S391 STR loci.

DNA degradation in post-mortem soft muscle tissues in relation to accumulated degree-days (ADD)

M.S. Nazir, J.A. Smith, W. Goodwin — 2011-11-16

Abstract: In order to assess DNA degradation in the model organisms chosen (pig and rabbit), two nuclear genes, Connexin 43 and RAG-1, were aligned to identify conserved regions. Primers were designed to amplify 70bp, 194bp, 305bp and 384bp amplicons. The primers were also designed to amplify human DNA, which allowed the use of commercially purchased DNA standards to be used as controls. Following DNA extraction PCR analysis was performed using the four primers sets in a multiplex (4-plex): the PCR was optimised so that it worked over a wide range of template amounts (0.1–75.83ng). The multiplex (4-plex) PCR was found to work efficiently in triplicate samples with all three species down to 0.3ng of DNA template. This multiplex has been used to assess whether DNA degradation can be predicted by accumulated degree-days (ADD), which provides a measure of both time and temperature. Full 4-plex profiles were generated until day 7 (112 ADD) from whole carcasses and body fragments. Future work will include; development of real-time PCR quantification assays, DNA fragment analysis and DNA preservation.

Allele frequencies of Nc02 multiplex Str loci (D1s1677, D2s441, D4s2364) in Turkey

Elif Sipahi, Gönül Filoğlu, Tuğba Ünsal, Havva Altunçul — 2011-12-01

Abstract: In this study, the aim is to determine allele frequencies of D1S1677, D2S441, D4S2364 miniSTR loci and to provide these 3 loci for using routinely in Turkish Criminal Laboratories. Allele frequencies and forensic parameters for the three miniSTR loci were investigated in a sample of 200 unrelated healthy Turkish individuals. We use Powerstats Excel workbook, in order to use a simple, quick and reliable method to analyze the race-based population statistics of potentially useful loci. This population data suggests that these three miniSTR loci will serve as useful complements to the CODIS loci to aid in the forensic analysis of degraded DNA, as well as missing persons work and parentage testing with limited next-of-kin reference samples, with a validated method in Turkish Criminal Laboratories. In Turkish population, powers of discrimination for these three loci are more than 0.837. Information content of polymorphism results are 0.75, 0.83 and 0.63 respectively.

Application of next generation sequencing technologies to the identification of highly degraded unknown soldiers’ remains

O. Loreille, H. Koshinsky, V.Y. Fofanov, J.A. Irwin — 2011-11-10

Abstract: Complete genome studies performed with next generation sequencing technologies are becoming more and more abundant. The potential such technologies could have for DNA identification purposes are obvious, but so far, very few forensic laboratories have tested these new instruments. We decided to evaluate the Illumina GAIIx platform for sequencing mitochondrial DNA extracted from an ancient human bone. We show that using standard Illumina protocols, we obtained a very small coverage of the mtDNA genome and observe a high error rate of 1.44%. We therefore tested various methods to improve the quality and quantity of the data. This report will describe our results when we incorporated a DNA repair and a primer extension capture step in the protocol.

The potential impact of secondary transfer and persistence of deoxyribonucleic acid (DNA) on forensic casework

L.M. Walton, A.R. Jackson, H.A. Mountain — 2011-12-01

Due to increases in the sensitivity of DNA analysis it is now possible to recover DNA profiles from handled objects. However, alongside the improvements in the level of detection comes the potential increase in contamination, from known and unknown sources. The aim of this research was to undertake a series of experiments, designed to examine the potential for secondary transfer of DNA. DNA was extracted using the Qiagen Microkit followed by amplification with the AmpFlSTR® Identifiler™ or SGM Plus™ systems. These studies have indicated that secondary transfer does exist both when the vehicle for transfer is an object or another person's hand. Perhaps more importantly, the results indicated that when an object was handled by two different individuals, and partial profiles were retrieved, the dominant profile was not necessarily that of the final handler. This may suggest that the predominance of a subjects’ profile from a handled object is dependent on how well the individual sheds their DNA, and not solely on the order in which the subjects handled the object. This could have a major impact on the information gained from seized objects at crime scenes, as it disputes the theory that the final person to handle an object would be the major contributor to a mixed DNA profile. This presentation will discuss the results of these experiments, further research into DNA transfer and persistence variables, as well as broadly examining the potential impact the findings may have on Forensic Casework.

Australian marsupial species identification

2011-11-03

Abstract: Wildlife crime, the illegal trade in animals and animal products, is a growing concern and valued at up to US$20 billion globally per year. Australia is often targeted for its unique fauna, proximity to South East Asia and porous borders. Marsupials of the order Diprotodontia (including koala, wombats, possums, gliders, kangaroos) are sometimes targeted for their skin, meat and for the pet trade. However, species identification for forensic purposes must be underpinned by robust phylogenetic information. A Diprotodont phylogeny containing a large number of taxa generated from nuclear and mitochondrial data has not yet been constructed. Here the mitochondrial (COI and ND2) and nuclear markers (APOB, IRBP and GAPD) are combined to create a more robust phylogeny to underpin a species identification method for the marsupial order Diprotodontia. Mitochondrial markers were combined with nuclear markers to amplify 27 genera of Diprotodontia. Data was analysed using a likelihood method. The combined data set resolved two suborders: Vombatiformes and Phalangeriformes. Phalangeriformes was subsequently split into two clades. The first clade contained the Macropodiformes and Burramyidae. The second clade contained Petauridae, grouping with Phalangeroidea. Of the markers tested, ND2 provided the greatest level of diagnostic accuracy and could be used as a forensic species identification tool for Diprotodonts, with appropriate validation.

GHEP-ISFG Proficiency Test 2011: Paper challenge on evaluation of mitochondrial DNA results

2011-11-11

Abstract: In this GHEP-ISFG exercise, participating labs were invited to evaluate a forensic case in which the mtDNA haplotype from a hair shaft in the victim's hand matched the suspect's haplotype. 31 forensic labs participated in the exercise. Although all except one used the EMPOP database to estimate the haplotype frequencies different final likelihood ratios (LRs) were reported. The main factors affecting these differences were: the origin of the reference population, the approaches to correct sampling errors, the LR formula, the source of EMPOP data (forensic/literature), the type of search (pattern or literal and “disregard Indels” option) and the selected edition range to perform the queries. This demonstrates that further efforts are needed in order to standardize the statistical evaluation of the mtDNA evidence.

Prenatal samples used as DNA evidence in rape cases

A. Kondili, P. Miniati — 2011-11-10

Abstract: DNA typing of forensic evidence necessary to prosecute rape cases is a routine analysis in criminal forensic casework. Prenatal samples can be used as DNA evidence for such cases by establishing their paternal origin. Herein we report two cases of sexual abuse. The first case regards a thirteen years old girl's rape that resulted in pregnancy. The girl reported the assault after four months and the only available evidence for DNA typing, at that time, was the amniotic fluid. The second case regards a sixteen years old girl's rape by her alleged father. The rape resulted in pregnancy that was terminated three to four weeks after conception. In order to extract DNA from the amniotic fluid sample, the laboratory's protocol established for DNA isolation from tissues was slightly modified. In this presentation we report the modified protocol. In addition we present our approach to process the “abortion specimen” as DNA evidence and the results of DNA analysis.

STR analysis in bones exposed to Brazilian tropical climate

2011-11-10

Abstract: Brazil has one of the highest homicides rates in the world. In this context, many cases of post mortem human bones that have been exposed to adverse environmental conditions and contaminants are the only materials available for analysis. Humid tropical climate with high rainfall and temperature has a direct influence on bone material that has been exposed to those conditions resulting in cell loss and DNA degradation. This study aimed to extract DNA using a commercial kit and organic extraction to evaluate their success in amplifying 15 STR markers from human skeletal remains exposed to tropical conditions. Compact bone fragments were used from the femoral diaphysis of 20 skeletonized corpses, found in the period 1998–2007 in Ribeirão Preto, São Paulo, Brazil. Fragments were sanded and pulverized and DNA was extracted from 150mg of bone powder using commercial kit and phenol chloroform with alcohol precipitation. Samples were quantified with Duo DNA Quantifiler kit (Applied Biosystems) and amplified by PowerPlex® 16 HS System (Promega). DNA could be quantified in 60% of samples by employing the commercial kit extraction. Nevertheless, a complete profile was not obtained in any case, using organic or commercial extraction methods. Partial profiles were obtained in 60% of cases and markers with up to 264 base pairs were amplified. Our results show that is possible to obtain short amplicons, demonstrating DNA degradation and that there is a need of mini STR analysis in these types of sample.

87 DNA markers for a paternity testing: Are they sufficient?

2011-11-07

Abstract: We report a judicial paternity testing with two exclusions at D2S1338 and vWA loci. Since these results suggested that the true father should be a close male relative of the tested man, mother was included into analysis. Subjects were also typed for 24 validated STRs, 11 STRs for linkage analysis, 8 X-STRs and 30 DIPs, for a total of 87 markers. No further exclusions were found. Paternity index, taking into account mutation rates for D2S1338 and vWA, was 1.45×1013 (W=0.99999999999993). The final odds that the true father should be the untyped brother of the alleged father, that refused DNA profiling, was 1:192.

Non-medical applications of non-invasive prenatal diagnosis: Ethical issues

P. Tasinato, M. Montisci, G. te Kronnie, G. Basso — 2011-11-10

Abstract: Non-invasive prenatal diagnosis (NIPD) is becoming increasingly important and its application in prenatal diagnosis is reaching consensus in the scientific research community. We discuss the opportunities and ethics of non-invasive prenatal testing for non-medical purposes, including forensic genetics. A number of ethical issues arise from non-medical applications of NIPD, such as sex determination and paternity testing in earlier gestational age and subsequent offspring selection. NIPD provides a source of information about the genetic make-up of the foetus, avoiding the small but significant risk of pregnancy loss related to invasive testing such as amniocentesis or chorionic villi sampling. NIPD is characterized by: safety, early detection and easy sampling. These features of NIPD increase the opportunity of prenatal testing also for non-medical reasons. Even if NIPD can be qualified as a good practice prenatal diagnosis tout court remains a topic of ethical judgements. The non-medical use of NIPD will benefit from an informed and open debate involving both pregnant women and physicians.

Population assignment in seven Portuguese dog breeds and Iberian wolves

Barbara van Asch, Rui Pereira, João Carneiro, António Amorim — 2011-11-14

Abstract: Seven Portuguese dog breeds (n=344) and Iberian wolves (n=44) were analysed using a previously characterized set of 9 autosomal STRs. The total dataset was used to estimate genetic diversity and parameters of forensic interest among and within populations. Clustering analyses were performed to investigate the genetic similarity of individuals belonging to the same morphological population. Dog breeds showed relatively heterogeneous genetic constitutions and individual dogs could not be readily assigned to their breeds of origin on the basis of these genotypes alone. Nevertheless, genetic discrimination between wolves and dogs was possible with a high probability (>98%) of correct assignment of each individual to its population of origin.

An automated integrated system for pre-PCR punching and PCR-setup

Stefan Mauch, Reto Menzi, Nando Giovanoli, Stefan Glükler, Laurent Baron — 2011-12-01

Abstract: Sampling buccal swabs with downstream STR analysis is a common technique for generating DNA profile databases. One of the bottlenecks of current workflows for this type of analysis are the individual systems required for punching and liquid handling, impairing process robustness and throughput. To overcome these challenges, Hamilton Robotics has developed an automated system integrating punching of sampling cards and subsequent PCR-setup into one workflow.

A production system to generate reference genetic profiles from Buccal Swab cells on FTA® cards

L. Baron, E. Suzanne, A. Calletier, C. Pardo, L. Pène — 2011-12-01

Abstract: Many countries are implementing systematic sampling for burglaries or car theft and other minor crimes also called volume crimes, resulting in a major jump in the number of samples to analyse. For the Laboratoire de Police Scientifique de Lyon the throughput will have to increase from 35.000 casework samples/year today to about 70.000 samples/year within the next 2 years. To deal with such major increase in throughput we looked at ways of optimising the sample workflow and chemistry without changing equipment and staff needed for the actual throughput. We describe how the lysis process was optimised and DNA extraction and putification improved with ABI PrepFiler Chemistry fully automated on a milton Robotics Microlab Starlet.

Forensic application of autosomal STR analysis in Lithuanian population

2011-11-18

Abstract: The data presents the first comprehensive autosomal STR analysis in the Lithuanian population for the purpose to compile an autosomal STR DNA database of natives from Lithuania, evaluate autosomal STR diversity, introduce population reference data for forensic and population genetic issues. Autosomal STR data were collected from the blood samples of 300 unrelated individuals distributed throughout the country. The amplification of 15 autosomal STRs was performed in one multiplex PCR by the use of AmpFlSTR® Identifiler® PCR Amplification Kit for human identity and parentage testing. Statistical analyses were carried out to determine the basic parameters of population genetics and forensic efficiency for 15 autosomal STRs. The results indicated that autosomal STR profiles enable to achieve high-resolution essential for forensic DNA casework.