Instituto de Biologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
Center for Human Identification, University of North Texas Health Science Center, Fort Worth, TX, USA.
Forensic Sci Int Genet. 2021 Jul;53:102516. doi: 10.1016/j.fsigen.2021.102516. Epub 2021 Apr 6.
Forensic DNA typing typically relies on the length-based (LB) separation of PCR products containing short tandem repeat loci (STRs). Massively parallel sequencing (MPS) elucidates an additional level of STR motif and flanking region variation. Also, MPS enables simultaneous analysis of different marker-types - autosomal STRs, SNPs for lineage and identification purposes, reducing both the amount of sample used and the turn-around-time of analysis. Therefore, MPS methodologies are being considered as an additional tool in forensic genetic casework. The PowerSeq™ Auto/Y System (Promega Corp), a multiplex forensic kit for MPS, enables analysis of the 22 autosomal STR markers (plus Amelogenin) from the PowerPlex® Fusion 6C kit and 23 Y-STR markers from the PowerPlex® Y23 kit. Population data were generated from 140 individuals from an admixed sample from Rio de Janeiro, Brazil. All samples were processed according to the manufacturers' recommended protocols. Raw data (FastQ) were generated for each indexed sample and analyzed using STRait Razor v2s and PowerSeqv2.config file. The subsequent population data showed the largest increase in expected heterozygosity (23%), from LB to sequence-based (SB) analyses at the D5S818 locus. Unreported allele was found at the D21S11 locus. The random match probability across all loci decreased from 5.9 × 10 to 7.6 × 10. Sensitivity studies using 1, 0.25, 0.062 and 0.016 ng of DNA input were analyzed in triplicate. Full Y-STR profiles were detected in all samples, and no autosomal allele drop-out was observed with 62 pg of input DNA. For mixture studies, 1 ng of genomic DNA from a male and female sample at 1:1, 1:4, 1:9, 1:19 and 1:49 proportions were analyzed in triplicate. Clearly resolvable alleles (i.e., no stacking or shared alleles) were obtained at a 1:19 male to female contributor ratio. The minus one stutter (-1) increased with the longest uninterrupted stretch (LUS) allele size reads and according to simple or compound/complex repeats. The haplotype-specific stutter rates add more information for mixed samples interpretation. These data support the use of the PowerSeq Auto/Y systems prototype kit (22 autosomal STR loci, 23 Y-STR loci and Amelogenin) for forensic genetics applications.
法医 DNA 分型通常依赖于含有短串联重复序列（STRs）的 PCR 产物的基于长度的（LB）分离。大规模平行测序（MPS）阐明了 STR 基序和侧翼区域变异的额外水平。此外，MPS 能够同时分析不同的标记类型——常染色体 STR、用于血统和鉴定目的的单核苷酸多态性，减少了样本的使用量和分析的周转时间。因此，MPS 方法被认为是法医遗传学案件工作中的附加工具。PowerSeq™ Auto/Y 系统（Promega Corp）是一种用于 MPS 的多重法医试剂盒，可分析来自 PowerPlex® Fusion 6C 试剂盒的 22 个常染色体 STR 标记（加 Amelogenin）和来自 PowerPlex® Y23 试剂盒的 23 个 Y-STR 标记。从巴西里约热内卢的混合样本中，从 140 名个体中生成了群体数据。所有样本均按照制造商推荐的方案进行处理。为每个索引样本生成原始数据（FastQ），并使用 STRait Razor v2s 和 PowerSeqv2.config 文件进行分析。随后的群体数据显示，在 D5S818 基因座上，从 LB 到基于序列（SB）的分析，预期杂合度增加最大（23%）。在 D21S11 基因座发现了未报告的等位基因。所有基因座的随机匹配概率从 5.9×10⁻⁹降低到 7.6×10⁻⁹。使用 1、0.25、0.062 和 0.016ng 的 DNA 输入进行了灵敏度研究，并进行了三次重复分析。在所有样本中均检测到完整的 Y-STR 谱，并且在输入 DNA 为 62pg 时未观察到常染色体等位基因丢失。对于混合物研究，以 1:1、1:4、1:9、1:19 和 1:49 的比例，从男性和女性样本中分析了 1ng 的基因组 DNA，进行了三次重复分析。在男性与女性贡献者比例为 1:19 的情况下，获得了可清晰分辨的等位基因（即无堆积或共享等位基因）。负一突峰（-1）随着最长无中断延伸（LUS）等位基因大小读数的增加而增加，并且根据简单或复合/复杂重复。单体特异性突峰率为混合样本解释提供了更多信息。这些数据支持使用 PowerSeq Auto/Y 系统原型试剂盒（22 个常染色体 STR 基因座、23 个 Y-STR 基因座和 Amelogenin）进行法医遗传学应用。
