Sequence Variations of 31 γ-Chromosomal Short Tandem Repeats Analyzed by Massively Parallel Sequencing in Three U.S. Population Groups and Korean Population

Abstract

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.

Concordance study on Y-STRs typing between SeqStudio™ genetic analyzer for HID and MiSeq™ FGx forensic genomics system

“…An increase of at least 40% in the total number of alleles due to sequence allele variations detected by MPS compared to those identified by CE was also confirmed by other authors [26,46,48,49,[52][53][54], as well as the observation that the most varied allele sequences occurred in DYF387S1 and DYS389II markers [26,34,48,49,51,53]. Concerning the presence of N-1 stutter, the information recovered from other works that had evaluated them agreed with the findings of this study: for example, the highest value of the N-1 stutter ratio was observed at DYS481, which has a trinucleotide repeat [46,51].…”

“…Specifically, poor or inconsistent results for the DYS392 locus, due to a low reads coverage, even when the total sample readings were high, were described and this was also reported by the manufacturer in the manual of the FSSP kit. [11,28,39,46,48,49] Concordance between CE and MPS above 99% in allele calling was confirmed on the basis of their size. The rare discrepancies that have been highlighted in other works at the DYS392, DYS393, DYS481, DYS439, and DY576 markers have been caused by the presence of SNPs in the flanking region, primers binding site mutations, and by the use of different primer sequences employed by CE and MPS systems [26,34,46,[48][49][50][51][52].…”

“…[11,28,39,46,48,49] Concordance between CE and MPS above 99% in allele calling was confirmed on the basis of their size. The rare discrepancies that have been highlighted in other works at the DYS392, DYS393, DYS481, DYS439, and DY576 markers have been caused by the presence of SNPs in the flanking region, primers binding site mutations, and by the use of different primer sequences employed by CE and MPS systems [26,34,46,[48][49][50][51][52].…”

Evaluation of Library Preparation Workflows and Applications to Different Sample Types Using the PowerSeq® 46GY System with Massively Parallel Sequencing

“…An interpretation threshold of 500 reads for homozygotes and 100 reads for heterozygotes was applied to autosomal loci according to Hölzl-Müller et al [51]. Additionally, an interpretation threshold of 100 reads was also applied to Y-STRs according to Moon et al [52]. Reads above 50× coverage but below the interpretation threshold were considered "Below-Threshold" (BT).…”

Y-chromosomal Short Tandem Repeat Variation in Kurd, Assyrian, and Armenian populations in Iraq Kurdistan

“…They have an independent branch of the Indo-European language family. The Armenian and the Kurds historically are living together in one empire or divided between the neighboring empires (Martirosyan, 2014). Numerous DNA-based data are utilized to examine the phylogeography, origins, and demographic history.…”