J Korean Med Sci.  2022 Feb;37(6):e40. 10.3346/jkms.2022.37.e40.

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

• Moon MH
• Hong SR
• Shin KJ

• KMID: 2526029
• DOI: http://doi.org/10.3346/jkms.2022.37.e40

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.

Keyword

Y-STR; RM Y-STR; Massively Parallel Sequencing; Sequence Variation

Figure

• 

  Fig. 1 Comparison of the number of observed alleles by CE and MPS analysis of 31 Y-STRs across four populations (n = 220). The blue bar presents the number of alleles by size-based method and the red bar presents the number of alleles observed by repeat region variations resulted from MPS method. The green bar presents the number of alleles observed by flanking region SNP resulted from MPS method. The nine RM Y-STRs used in this study are listed at the top of the figure.CE = capillary electrophoresis, MPS = massively parallel sequencing, SNP = single nucleotide polymorphism, STR = short tandem repeat, RM = rapidly mutating.

• 

  Fig. 2 Alignment of three sequence fragments of multi-copy DYF399S1 locus and SNP variation annotation. Bracketed motifs in repeat regions are counted for allele designation and lowercase letters in repeat regions are not counted as repeats. The left side shows the coordinates of the sequence in the Y chromosome. Gray boxes indicate nucleotides with sequence differences between fragments. The red letters indicate the SNPs that differentiate them into various alleles in the sequence-based analysis of DYF399S1. The rs number of each SNP is also shown (GRCh38 coordinates).SNP = single nucleotide polymorphism.

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