Abstract
Sexual assault samples are among the most difficult sample types encountered by forensic laboratories. Typically, a sexual assault sample has multiple challenges including small quantity of male DNA, relatively high quantity of female DNA, and presence of PCR inhibitors. These factors make it difficult to obtain an interpretable male profile. Therefore, there is a need for a more robust, highly sensitive, and faster method for the assessment (i.e. quality and quantity) of DNA extracts to determine optimal downstream processing methods, as well as an improve Y-STR amplification system for profiling these difficult samples.
We tested newly developed tools, including a DNA quantification kit (Quantifiler® Trio) that exhibits high sensitivity higher inhibitor tolerance and includes additional useful tools for the determination of DNA quality to inform downstream processing methodology. We also tested a new Y-STR kit (Yfiler® Plus) with enhanced capabilities. This 27-marker kit permits rapid amplification of single source and complex casework samples. These two next generation systems can generate an improved workflow for obtaining interpretable profiles from sexual assault samples. We have successfully used the Quantifiler® Trio and Yfiler® Plus systems to obtain informative Y-STR profiles from challenging sexual assault sample types, including low quantity male DNA, extended interval post coital samples and samples containing high amounts of female DNA. We evaluated more routinely encountered post-coital samples collected 1–3 days after intercourse. Based on the Quantifiler® Trio results, observed outcomes were as expected after subsequent Yfiler® Plus and GlobalFilerTM analysis.
Keywords
1. Introduction
Male DNA-containing samples collected from female crime victims can contain very low levels of cellular male DNA admixed with a large number of female epithelial cells. This often results in failure to obtain an autosomal STR profile from the male DNA donor. Particularly for extended interval post coital samples, differential extraction may still result in no male profile due to a combination of premature sperm cell lysis into the non-sperm fraction and to sperm loss during the physical manipulations required during DNA extraction. Therefore, the use of Y-STR analysis can be used to overcome these challenges as it targets only the male fraction in admixed samples [
1
, 2
]. Despite the improvement in semen donor profile recovery in sexual assault evidence using Y-STR analysis [3
, 4
], the analytical detection limit of the individual typing system or kit used impacts the success rate. Therefore, there is a need for a more robust, highly sensitive, and faster method for the assessment (i.e. quality and quantity) of DNA extracts to determine optimal downstream processing methods, as well as an improved Y-STR amplification system for profiling these difficult samples which includes additional discriminatory power to resolve coincidental matches between unrelated male participants and possibly to distinguish males within the same lineage [[5]
].The new Quantifiler® Trio DNA quantification kit and the new Yfiler® Plus amplification kit were designed to address these needs. This study describes an evaluation of both kits, specifically assessing the utility of the new workflow in processing challenging sexual assault samples (e.g. samples collected 4, 7 and 9 days after intercourse). Despite the presence of low quantities of male DNA and female DNA in vast excess, probative Y-STR profiles were obtained from these samples with mostly full profiles obtained if >100 pg of male DNA was present. While the analysis of these samples was limited to Y-STR typing, we also demonstrate the utility of the new workflow with samples collected 1–3 days after intercourse, including the use of next generation autosomal STR kits (GlobalFiler™).
2. Materials and methods
2.1 DNA extraction
DNA was extracted from the samples using a standard non-differential organic extraction (4, 7 and 9 day samples) with a 75 μl elution volume or a standard differential organic extraction with a 50 μl elution volume (TE−4) (1–3 day samples). The cervix and fornix as well as standard and concentrated (MinElute (QIAGEN, Germantown, MD, USA) purification and concentration) fractions were combined for the 4, 7 and 9 day samples.
2.2 DNA quantitation
All DNA quantitation was performed using the Quantifiler® Trio DNA quantitation kit (Life Technologies, Foster City, CA, USA) on a 7500 Real Time PCR instrument (Life Technologies) according to the manufacturer's instructions.
2.3 STR amplification and detection
Y-STR amplification was performed using the Yfiler® Plus amplification kit according to the manufacturer's recommendations. Autosomal STR amplification was performed using the GlobalFiler™ amplification kit according to the manufacturer's instructions. All amplifications were performed using a GeneAmp® PCR system 9700 thermal cycler. PCR products were detected on a 3130 Genetic Analyzer (GeneScan® 600 LIZ® size standard, POP-7™ polymer; GeneMapper IDX v1.4).
3. Results and discussion
Previously we developed optimized strategies for improved profile recovery (e.g. use of cervical swabbing and post PCR purification) that resulted in the ability to routinely obtain Y-STR profiles from samples collected 3–5 days after intercourse, with partial success with 6 and 7 day samples [
[4]
]. We subsequently developed a Y-chromosome nested PCR pre-amplification multiplex for selective enhancement of male DNA in admixed samples which permitted recovery of male profiles (Y-STR) from samples collected 9 days after intercourse. However, since the development of the pre-amplification method, next generation Y-STR (NGY) amplification kits have become available and our initial testing suggested that improved profile recovery may be achieved with these NGY kits without the need for additional pre-amplification steps. Initial evaluation studies indicated that the Yfiler® Plus amplification kit demonstrated a high sensitivity (17/25 locus profiles with 31/62 pg of male DNA) and, unlike Yfiler®, were not inhibited by high concentrations of inhibitors. Full male profiles were also obtained with 1:1000 and 1:4000 male/female admixtures. We therefore next evaluated whether our analysis of extended interval post coital samples could be improved using the Quantifiler® Trio and Yfiler® Plus systems.We evaluated samples from 28 different donor couples which were collected 4, 7 and 9 days after intercourse. Total Male DNA yields of <150 pg were observed for most samples (∼15 μl extracts, Quantifiler® Trio). Female:male ratios ranged from 333:1 to 4,685,117:1 indicating that autosomal STR analysis would not be possible for any of these samples. The maximum amount of male input DNA was used for Yfiler® Plus amplification (up to 1 ng, majority of samples <1 ng). Full male profiles were obtained for 32%, 7% and 11% of the 4, 7, and 9 day samples, respectively. An excellent correlation between male quantitation and profile recovery was obtained using the Quantifiler® Trio with negative quantitation values resulting in negative or unusable Yfiler® Plus results. The included ‘quality index’ tool indicated no degradation. Yfiler® Plus results were obtained with low amounts of male DNA and with high (μg) quantities of background female DNA (“usable profiles” with F:M ratios ranging from 333:1 to 100,000:1).
We performed additional studies to evaluate more routinely encountered post-coital samples collected 1–3 days after intercourse. All expected outcomes based on the Quantifiler® Trio results were observed with subsequent Yfiler® Plus and GlobalFiler™ analysis. For GlobalFiler™, single source profiles from the female donor were obtained in all non-sperm fractions. Admixed male:female profiles were obtained from the 1- and 2-day sperm fractions, with a very minor female component in the 1 day sperm fraction this permitting recovery of a full male autosomal STR profile.
Overall, the results of these studies demonstrate the utility of the new Quantifiler® Trio and Yfiler® Plus systems with challenging sexual assault samples.
Role of funding
Portions of this work were supported Award #2009-DN-BX-0023, awarded by the National Institute of Justice, Office of Justice Programs, U.S. Department of Justice. The opinions, findings and conclusions or recommendations expressed in this presentation do not necessarily reflect those of the Department of Justice. The funding agency had no role in the study design; in the collection, analysis and interpretation of the data; in the writing of the report; and in the decision to submit the paper for publication.
Conflict of interest
None.
Acknowledgements
We would like to acknowledge Pat Speck and the UTHSC team in Memphis, TN for the 4–9 d post coital samples. We would also like to acknowledge members of the Life Technologies team: Jonathan Tabak, Andrea Carbonaro, Sheri Olson, Jeff Sailus, Joanne Sgueglia, Lisa Calandro, Ariana Wheaton, Melissa Kotkin and Shelly Guerrero.
References
- Y-chromosome short tandem repeats.in: Jamieson A. Moenssens A. Wiley Encyclopedia of Forensic Science. Wiley, Chichester, UK2009: 2677-2682
- The forensic application of Y-chromosome short-tandem repeats.in: Forensic Science Handbook. vol. III. Prentice Hall, Upper Saddle River, NJ2009: 436-466
- Performance characteristics of commercial Y-STR multiplex systems.J. Forensic Sci. 2007; 52: 1025-1034
- Y-STR profiling in extended interval (> or =3 days) postcoital cervicovaginal samples.J. Forensic Sci. 2008; 53: 342-348
- A new future of forensic Y-chromosome analysis: rapidly mutating Y-STRs for differentiating male relatives and paternal lineages.Forensic Sci. Int. Genet. 2012; 6: 208-218
Article info
Publication history
Published online: October 28, 2013
Accepted:
October 2,
2013
Received:
September 4,
2013
Identification
Copyright
© 2013 Elsevier Ireland Ltd. Published by Elsevier Inc. All rights reserved.
