Hussing C, Bytyci R, Huber C, Morling N, Børsting C
Section of Forensic Genetics, Department of Forensic Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Frederik V's Vej 11, 2100, Copenhagen, Denmark.
Int J Legal Med. 2019 Mar;133(2):325-334. doi: 10.1007/s00414-018-1854-0. Epub 2018 May 24.
Some STR loci have internal sequence variations, which are not revealed by the standard STR typing methods used in forensic genetics (PCR and fragment length analysis by capillary electrophoresis (CE)). Typing of STRs with next-generation sequencing (NGS) uncovers the sequence variation in the repeat region and in the flanking regions. In this study, 363 Danish individuals were typed for 56 STRs (26 autosomal STRs, 24 Y-STRs, and 6 X-STRs) using the ForenSeq™ DNA Signature Prep Kit to establish a Danish STR sequence database. Increased allelic diversity was observed in 34 STRs by the PCR-NGS assay. The largest increases were found in DYS389II and D12S391, where the numbers of sequenced alleles were around four times larger than the numbers of alleles determined by repeat length alone. Thirteen SNPs and one InDel were identified in the flanking regions of 12 STRs. Furthermore, 36 single positions and five longer stretches in the STR flanking regions were found to have dubious genotyping quality. The combined match probability of the 26 autosomal STRs was 10,000 times larger using the PCR-NGS assay than by using PCR-CE. The typical paternity indices for trios and duos were 500 and 100 times larger, respectively, than those obtained with PCR-CE. The assay also amplified 94 SNPs selected for human identification. Eleven of these loci were not in Hardy-Weinberg equilibrium in the Danish population, most likely because the minimum threshold for allele calling (30 reads) in the ForenSeq™ Universal Analysis Software was too low and frequent allele dropouts were not detected.
一些短串联重复序列（STR）基因座存在内部序列变异，而法医遗传学中使用的标准STR分型方法（聚合酶链反应（PCR）和毛细管电泳（CE）片段长度分析）并未揭示这些变异。利用下一代测序（NGS）对STR进行分型可发现重复区域和侧翼区域的序列变异。在本研究中，使用ForenSeq™ DNA Signature Prep试剂盒对363名丹麦个体的56个STR（26个常染色体STR、24个Y染色体STR和6个X染色体STR）进行分型，以建立丹麦STR序列数据库。通过PCR-NGS检测，在34个STR中观察到等位基因多样性增加。在DYS389II和D12S391中发现增加幅度最大，其中测序等位基因的数量比仅通过重复长度确定的等位基因数量大约多四倍。在12个STR的侧翼区域鉴定出13个单核苷酸多态性（SNP）和1个插入缺失（InDel）。此外，发现STR侧翼区域的36个单一位点和5个较长片段的基因分型质量存疑。使用PCR-NGS检测时，26个常染色体STR的联合匹配概率比使用PCR-CE时大10000倍。三联体和二联体的典型父权指数分别比PCR-CE获得的指数大500倍和100倍。该检测还扩增了94个用于人类身份鉴定的SNP。在丹麦人群中，这些基因座中有11个不符合哈迪-温伯格平衡，很可能是因为ForenSeq™通用分析软件中等位基因检出的最低阈值（30次读数）过低，未检测到频繁的等位基因缺失。
