The analysis of Y-STR markers is used to identify victims from natural disasters, rapes or murder, sexual assault cases; moreover it provides information about genetic diversity and can determine genetic differences in population. Yfiler™ Plus PCR Amplification kit (Thermo Fisher Scientific, Waltham, MA, USA) has 6-dye technology and can amplify 27 Y-STR loci including 7 rapidly mutating markers (RM Y-STRs) and 11 mini-Y-STRs. This study reports on the genotyping of 254 individuals from Lithuania were analysed using Yfiler™ Plus PCR Amplification kit (Thermo Fisher Scientific, Waltham, MA, USA). Forensic and population genetic parameters were calculated using relative computer equipment, statistical approaches and computational tools. Population comparison with other European populations from YHRD was performed. Our results revealed that 27 Y-STR loci provides high haplotype diversity, high discrimination power and is an effective tool in forensic genetics. After comparing data of allelic percentage distribution in each examined Y chromosome locus and genetic diversity in these microsatellite areas between Lithuania’s and other world’s populations, Lithuanian population is not very polymorphic or unique and isolated from other populations. When comparing genetic distances, an observation was made, that Lithuanian population with its Y chromosome genetically is the closest to Russian populations. In conclusion, the Yfiler™ Plus PCR Amplification kit (Thermo Fisher Scientific, Waltham, MA, USA) is very useful in daily casework in forensic laboratories.
Y chromosome short tandem repeats are repetitive regions, which repetition motive is 2–13 bp and the frequency of recurrence range from seven to forty [
]. The number of short tandem repetitions can be very variable among individuals, which makes these markers effective for human identification. These polymorphisms are likely formed due to strand-slippage replication and most often this DNA polymerase slippage generates one repeat unit [
- Ham S.K.
- Kim S.Y.
- Seo B.Y.
- Woo K.M.
- Lee S.H.
- Choi C.Y.
Differential pre-amplification of STR loci for fragmented forensic DNA profiling.
Electrophoresis. 2016; 37: 3002-3009
]. Y-STRs have the following advantages: small amount of DNA is enough; suitable for a degraded sample; eligible results with samples with a lot of feminine DNA and scarcely masculine DNA; the results are obtained quickly within 1–2 business days [
- Brunstein J.
STR typing: method and applications.
Primer. 2014; 4: 40-42
]. The analysis of Y-STR markers is used to identify victims from natural disasters, rapes or murder, sexual assault cases, especially on samples with higher female:male DNA ratio. The aim of the study was to evaluate aspects of 27 Y-STR loci application in Lithuanian court genetics and examine Lithuanian population genetic relationships.
- Gill P.
- Haned H.
- Bleka O.
- Hansson O.
- Dorum G.
- Egeland T.
Genotyping and interpretation of STR-DNA: low-template: mixtures and database matches-twenty years of research and development.
FSI Genet. 2015; 18: 100-117
2. Material and methods
2.1 Population samples and DNA extraction
Buccal cell swabs were collected from 254 unrelated Lithuanian male individuals. Genomic DNA was extracted using the Chelex-100 method [
- Walsh P.S.
- Metzger D.A.
- Higuchi R.
Chelex 0 as a medium for simple extraction of DNA for PCR-based typing from forensic material.
Biotechniques. 1991; 10: 506-513
Samples were amplified in duplicates using the Yfiler™ Plus PCR Amplification kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocols in a ProFlex™ PCR System (Thermo Fisher Scientific, Waltham, MA, USA).
Samples were typed using 1 μl of amplified products on an ABI3500 Genetic Analyser (Applied Biosystems, USA) with formamide solution and GeneScan™ 600 LIZ Size Standard v2.0 (Thermo Fisher Scientific, Waltham, MA, USA) following the recommendations of the kit. Allele location was carried out with GeneMapper® ID-X Software Version 1.4 (Applied Biosystem, USA). The DNA typing was performed at the State Forensic Medicine Service. The laboratory is accredited according to the ISO 17025 standard.
2.2 Quality control
All 254 samples of Lithuanian unrelated male population were submitted to the Y chromosome haplotype reference database (YHRD) (www.yhrd.org), passed YHRD quality control as recommended in the guidelines of Forensic Science International Genetics [
] and included in the YHRD database under accession number YA004262.
- Gusmao L.
- Butler J.M.
- Carracedo A.
- et al.
DNA Commission of the International Society of Forensic Genetics (ISFG): an update of the recommendations on the use of Y-STRs in forensic analysis.
Forensic Sci. 2006; 157: 187-197
2.3 Data analysis
Forensic parameters such as haplotype and alleles frequencies were estimated using the counting method. Genetic and haplotype diversities were calculated according to Nei [
]. Multidimensional scaling (MDS) plots were represented from FST values using Analysis of molecular variance (AMOVA). Discrimination capacity (DC) was established as the ratio between the number of different haplotypes and the total numbers of haplotypes whereas match probability (MP) was defined as the sum of the haplotypes frequencies.
- Nei M.
DNA polymorphism detectable by restriction endonucleases.
Genetics. 1981; 97
3. Results and discussion
3.1 Analysis of genetic diversity and forensic genetic indicators
In this study we tested suitability of the Yfiler™ Plus PCR Amplification kit (Thermo Fisher Scientific, Waltham, MA, USA) for application in Lithuanian population. We assessed genetic diversity values which are shown in Supplementary material Fig. S1. The analyses of the results showed that DYS437 locus has a low value – 0.3, but in all other loci genetic diversity value is higher than 0.5. As expected, in all RM Y-STR loci, the genetic diversity values were extremely high – reached more than 0.7.
Moreover, we estimated haplotype diversity, match probability and power of discrimination for Yfiler™ Plus Kit, Yfiler kit and European minimal haplotype. The results are shown in Supplementary material Table 1.
3.2 Comparative analysis of populations
A comparative analysis of Lithuanian population Y-STR haplotypes with other populations was performed using the AMOVA program to find out significant differences in populations. The pairwise genetic distances (RST) were determined, which are significant (P< 0.05) and results are shown in Supplementary material Table 2.
According to the genetic distance matrix, RST values of Lithuania and other countries populations are arranged and visualized on a multi-dimensional scale (Fig. 1). A phylogenetic tree was drawn from a distance matrix, choosing a quantitative metric Euclidean distance measure and neighbour-joining method. Thus, in the dendrogram, it can be seen that Russian and Lithuanian population are sister group, who have a common ancestor and belong to the monophyletic group together with Polish and Hungarian populations and are shown in Supplementary material Fig. S2.
The distribution of Lithuanian male’s individuals haplogroup frequencies for each haplotype was computed. The most commonly occurring haplogroup is R1a, which accounts for 54.7%, while the second highest prevalence haplogroup is N, which constitutes 12.6%. The third, fourth and fifth places occupy Q (7.9%), R1b (5.5%) and I1 (4.7%) haplogroups respectively and are shown in Supplementary material Fig. S3. The most commonly found haplogroups are R1a and N, hence it can be argued that Lithuanians originate from Pakistan/Northwest India and East China/Taiwan.
The Lithuanian population is homogeneous in respect of Y chromosome, but not isolated from other populations. The effectiveness, suitability and usefulness of the Yfiler™ Plus Kit for assessing the Lithuanian population in forensic genetics, which will help to identify people more reliable, has been approved. Ascertained that the closest to Lithuania is the Russian population, thus they share their common ancestor and are close not only geographically.
We thank Nijole Sestakauskiene and Daiva Sosnovskiene for technical assistance.
Appendix A. Supplementary data
The following is Supplementary data to this article:
- Differential pre-amplification of STR loci for fragmented forensic DNA profiling.Electrophoresis. 2016; 37: 3002-3009
- STR typing: method and applications.Primer. 2014; 4: 40-42
- Genotyping and interpretation of STR-DNA: low-template: mixtures and database matches-twenty years of research and development.FSI Genet. 2015; 18: 100-117
- Chelex 0 as a medium for simple extraction of DNA for PCR-based typing from forensic material.Biotechniques. 1991; 10: 506-513
- DNA Commission of the International Society of Forensic Genetics (ISFG): an update of the recommendations on the use of Y-STRs in forensic analysis.Forensic Sci. 2006; 157: 187-197
- DNA polymorphism detectable by restriction endonucleases.Genetics. 1981; 97
Published online: October 17, 2017
Accepted: October 16, 2017
Received in revised form: October 16, 2017
Received: September 9, 2017
© 2017 Elsevier B.V. All rights reserved.