Department of Forensic Medicine, Yonsei University College of Medicine, Seoul, Korea.
Graduate School of Medical Science and Brain Korea 21 Project, Yonsei University, Seoul, Korea.
J Korean Med Sci. 2022 Feb 14;37(6):e40. doi: 10.3346/jkms.2022.37.e40.
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.
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.
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.
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.
快速突变（RM）Y 染色体短串联重复序列（Y-STR）由于突变率高于常规 Y-STR，因此被证明可以增加区分男性亲属的可能性。大规模平行测序（MPS）可用于法医 DNA 分型，因为它允许检测许多法医标记物的序列变异。在这里，我们展示了 31 个 Y-STR 的序列变异，包括 9 个 RM Y-STR（DYF387S1、DYF399S1、DYF404S1、DYS449、DYS518、DYS570、DYS576、DYS612 和 DYS627），它们的频率、分布以及使用 MPS 获得的等位基因数量的增加。
我们构建了一个能够同时扩增 32 个 Y 染色体标记物的多重 MPS 检测，产生的扩增子长度为 85-274bp。通过两步聚合酶链反应从来自四个群体（非裔美国人、白种人、西班牙裔和韩国人）的 220 个无关男性的文库中生成了条形码，并在 MiSeq 系统上进行了测序。通过毛细管电泳（CE）和 MPS 方法的基因型一致性以及 Y-STR 的序列变异进行了研究。
总共，MPS 比基于 CE 的等位基因增加了 195 个等位基因（261 至 456）。由于重复区变异，DYS518 标记的增加最大（增加了 3.69 倍）。侧翼区单核苷酸多态性导致等位基因数量增加最多的是 DYF399S1。RM Y-STR 比常规 Y-STR 具有更多的序列多样性。此外，由于引物结合位点突变，在 DYS576 中观察到无效等位基因，而在 DYS449 中由于标记覆盖率低于阈值导致等位基因丢失。
结果表明，扩展和有区别的 MPS 检测可以为 Y-STR 提供更多的遗传信息，特别是对于 RM Y-STR，并可以促进男性个体化。为全球人群编译基于序列的 Y-STR 数据将有助于 MPS 在法医遗传学领域的应用，并可适用于解决与男性有关的法医案件。
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