Research paperSequence-based diversity of 23 autosomal STR loci in Koreans investigated using an in-house massively parallel sequencing panel
Introduction
Compared to the conventional short tandem repeat (STR) analysis with capillary electrophoresis (CE), massively parallel sequencing (MPS) of STRs has the advantage of producing a massive quantity of sequencing data with only a single reaction [1], [2]. In addition, the reduced cost of sequencing through MPS has encouraged many forensic investigators to apply this technology to both casework and databasing. Moreover, several publications have emphasized the primary gains of using MPS related to an STR analysis (i.e., larger multiplexing and detection of sequence variation) [3], [4], [5], [6], [7], [8], [9], [10]. Most of the published studies validate the new MPS system for forensic markers, including STRs [6], [7], [8], [9], and have been performed on low-throughput samples and/or interrogated loci [10]. Therefore, a high-throughput approach is required to demonstrate the superior application of MPS to forensic DNA databasing in association with bioinformatics methods, which will enable the new database to maintain back compatibility with the CE-based method and existing forensic DNA databases.
Several commercial MPS assays, such as the PowerSeq Auto system (Promega, Madison, WI, USA) [6], ForenSeq™ DNA Signature Prep Kit (Illumina, San Diego, CA, USA) [7], and Precision ID GlobalFiler NGS STR Kit (Thermo Fisher, Waltham, MA, USA), are now available for STR analysis. These assays include 22, 27, and 29 forensic autosomal STRs, respectively, and are all inclusive of the expanded US CODIS core loci and the 12 core European Standard Set loci. Such expanded STR loci are expected to increase the discriminatory power and inter-database compatibility, and are ready for study at the population level using an MPS method. To date, population genetic analysis has been performed on the MPS data obtained from 68 African Americans, 70 Caucasians, and 45 Hispanics using a prototype version of the PowerSeq Auto system [10]; however, there is a lack of MPS reports on the expanded STR loci of Asians.
In this study, we present the sequencing results from 250 Korean samples at 23 autosomal STR loci (D1S1656, TPOX, D2S441, D2S1338, D3S1358, FGA, CSF1PO, D5S818, D6S1043, D7S820, D8S1179, D10S1248, TH01, vWA, D12S391, D13S317, Penta E, D16S539, D18S51, D19S433, D21S11, Penta D, and D22S1045) using an in-house multiplex assay designed for MPS. In addition to 22 commonly used STRs, our assay includes D6S1043, which is frequently studied in Asians, especially Koreans [11] and Chinese [12]. With the use of two bioinformatics pipelines and SNP annotation software, we identified sequence variations in both the repeat and flanking regions in association with allele frequencies and performed a population genetic analysis to provide forensic parameters for Koreans.
Section snippets
DNA samples
A total of 250 DNA samples from our previous report [13] were used for this study. Among the 300 DNA samples extracted from buccal swabs using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) around 2001, samples with small quantities and/or low concentrations (<1 ng/μL) through DNA quantification using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) were excluded and the 250 selected Korean DNAs were normalized to 1.0 ng/μL for the following assay. This study was
Multiplex PCR system for the MPS analysis of 23 autosomal STR loci
The developed multiplex PCR system for the MPS analysis of 23 autosomal STRs is the upgraded version based on our previous work, which included 17 commonly used autosomal STRs and amelogenin [9]. In the new version, the primers for several STRs were redesigned to remove minor PCR interference and to increase the PCR yield. In addition, the primer used for the amplification of D13S317 was redesigned to avoid possible allele drop-out because the primer binding area was found to include
Conclusion
Using samples from 250 unrelated Koreans, we examined the repeat region structures and flanking region of 23 autosomal STRs (D1S1656, TPOX, D2S441, D2S1338, D3S1358, FGA, CSF1PO, D5S818, D6S1043, D7S820, D8S1179, D10S1248, TH01, vWA, D12S391, D13S317, Penta E, D16S539, D18S51, D19S433, D21S11, Penta D, and D22S1045). Compared to the conventional CE-based method, 156 additional alleles and 439 additional genotypes were observed in 13 STRs (D1S1656, D2S441, D2S1338, D3S1358, CSF1PO, D6S1043,
Acknowledgments
This study was supported by the research project for practical use and advancement of forensic DNA analysis of Supreme Prosecutors’ Office, Republic of Korea (1333-304-260, 2014).
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2021, Forensic Science International: GeneticsCitation Excerpt :Allele counts and frequencies are summarized in Supplementary Table S2. Core repeat sequences and all observed alleles for 14 sequenced STR loci (CSF1PO, D13S317, D16S539, D17S974, D18S51, D19S433, D1S1656, D6S1043, D7S820, D8S1179, Penta D, Penta E, TH01 and TPOX) had been previously documented [22,25,32–36]. The repeat structure of the D12S391 locus could be described as [AGAT]m[AGAC]n, or [AGAT]p[AGAC]q[AGAT], where m, n, p and q are integers.
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2020, Forensic Science International: SynergyCitation Excerpt :Based on experience with examining many STR allele sequences, a revised sequence guide for forensic STRs used in MPS has been prepared [48] and new approaches for compacting sequence information are being developed [49]. MPS sequence population data have been published for 23 autosomal STR loci in Koreans [50], for the Yavapai Native Americans from West-Central Arizona using the MiSeq FGx system [51], and for U.S. population data across 22 autosomal STR loci [52], across 27 autosomal STR loci [53] and with the complex locus SE33 [54]. Illumina’s MiSeq FGx Forensic Genomics System has been the subject of a developmental validation study [55], an assessment of forensic STR and SNP kits [56], the validation of a mitochondrial DNA sequencing method [57], and an evaluation for use in casework [58].