Background Rapidly mutating (RM) Y-chromosomal short tandem repeats (Y-STRs) have been demonstrated to increase the possibility of distinguishing between male relatives due to a higher mutation rate than conventional Y-STRs. Massively parallel sequencing (MPS) can be useful for forensic DNA typing as it allows the detection of sequence variants of many forensic markers. Here, we present sequence variations of 31 Y-STRs including nine RM Y-STRs (DYF387S1, DYF399S1, DYF404S1, DYS449, DYS518, DYS570, DYS576, DYS612, and DYS627), their frequencies, distribution, and the gain in the number of alleles using MPS. Methods We constructed a multiplex MPS assay capable of simultaneously amplifying 32 Y-chromosomal markers, producing amplicons ranging from 85–274 bp. Barcoded libraries from 220 unrelated males from four populations—African Americans, Caucasians, Hispanics, and Koreans—were generated via two-step polymerase chain reaction and sequenced on a MiSeq system. Genotype concordance between the capillary electrophoresis (CE) and MPS method and sequence variation of Y-STRs were investigated. Results In total, 195 alleles were increased by MPS compared to CE-based alleles (261 to 456). The DYS518 marker showed the largest increase due to repeat region variation (a 3.69-fold increase). The highest increase in the number of alleles due to single nucleotide polymorphisms in the flanking region was found in DYF399S1. RM Y-STRs had more diverse sequences than conventional Y-STRs. Furthermore, null alleles were observed in DYS576 due to primer-binding site mutation, and allele drop-outs in DYS449 resulted from low marker coverage of less than the threshold. Conclusion The results suggest that the expanded and discriminative MPS assay could provide more genetic information for Y-STRs, especially for RM Y-STRs, and could advance male individualization. Compiling sequence-based Y-STR data for worldwide populations would facilitate the application of MPS in the field of forensic genetics and could be applicable in solving male-related forensic cases.
Background—Single nucleotide polymorphisms (SNPs) have become popular in forensic genetics as an alternative to short tandem repeats (STRs) due to low mutation rates and small amplicon sizes. The Precision ID Identity Panel (Thermo Fisher Scientific), consisting of 90 autosomal SNPs and 34 Y-SNPs, was introduced for human identification by next-generation sequencing (NGS), enabling many studies on the global population; however, few reports are available on the Southeast Asian population.Methods and Results—A total of 96 unrelated male samples from Myanmar (Yangon) were analyzed with the Precision ID Identity Panel on a MiSeq (Illumina) using an in-house TruSeq compatible universal adapter. The sequencing performance was evaluated by locus balance and heterozygote balance, and the results were comparable to those of the Ion Torrent platform. For 90 autosomal SNPs, minor allele frequencies ranged between 0.068 and 0.500, and combined match probability (6.994×10−34) was lower than that of 22 PowerPlex Fusion autosomal STRs (3.130×10−26). Moreover, we identified 51 cryptic variations around the target SNPs using a custom variant caller, Visual SNP. For 34 Y-SNPs, 14 Y-haplogroups were observed—mostly O2 and O1b groups. Interpopulation analysis revealed that the Myanmar population is genetically closer to the East and Southeast Asian populations than the South Asian population.Conclusions—We demonstrate that the Precision ID Identity Panel can be successfully analyzed on a MiSeq using a custom data analysis pipeline and provide high discrimination power for human identification in the Myanmar population, while extending the accessibility of NGS analysis for SNPs in forensics.
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