Hwa Hsiao-Lin, Chung Wan-Chia, Chen Pei-Lung, Lin Chih-Peng, Li Huei-Ying, Yin Hsiang-I, Lee James Chun-I
Department and Graduate Institute of Forensic Medicine, College of Medicine, National Taiwan University, No. 1, Sec. 1, Jen Ai Rd., Taipei 100, Taiwan; Department of Obstetrics and Gynecology, National Taiwan University Hospital, No. 7 Chung Shan S. Rd., Taipei 100, Taiwan; Department of Medical Genetics, National Taiwan University Hospital, No. 7 Chung Shan S. Rd., Taipei 100, Taiwan.
Yourgene Bioscience, No. 376-5 Fuxing Rd., Shulin Dist., New Taipei City 238, Taiwan.
Forensic Sci Int Genet. 2018 Jan;32:94-101. doi: 10.1016/j.fsigen.2017.11.002. Epub 2017 Nov 6.
Massively parallel sequencing (MPS) technology enables the simultaneous analysis of a huge number of single nucleotide polymorphisms (SNPs) and insertion-deletion polymorphisms (indels). MPS also enables the detection of the alleles of minor contributors in a highly unbalanced DNA mixture. In this study, we established a 1204-marker panel optimized for MPS consisting of 987 autosomal markers (964 SNPs and 23 indels), 27 X-chromosome SNPs, 61 Y-chromosome markers (56 SNPs and 5 indels), and 129 mitochondrial SNPs. The DNA samples of six unrelated individuals (two men and four women), 26 nondegraded DNA mixtures (with minor to major ratios of 1:29, 1:39, 1:79, and 1:99), and eight highly artificially degraded DNA mixtures (with minor to major ratios of 1:29, 1:39, 1:79, and 1:99) were analyzed through MPS by using the panel. A scoring system was developed to determine the minor contributors in DNA mixtures based on the genotypes identified using MPS. The genotypes of the 1204 markers were successfully profiled through MPS by using the custom-designed panel. The efficiency of MPS for analyzing these highly degraded samples was lower than that for analyzing nondegraded samples. All minor contributors in the 26 nondegraded and 8 degraded DNA mixtures were accurately assigned using this scoring system based on 964 autosomal SNPs. An association between the observed reads ratio and theoretical ratio of the minor component was noted for nondegraded mixtures. In conclusion, we established a 1204-marker individual identification panel for MPS that successfully analyzed autosomal, X-chromosome, Y-chromosome, and mitochondrial SNPs and indels simultaneously. In combination with the newly developed scoring system, the panel can accurately identify minor contributors in nondegraded and highly degraded DNA mixtures.
大规模平行测序（MPS）技术能够同时分析大量的单核苷酸多态性（SNP）和插入缺失多态性（indel）。MPS还能够检测高度不平衡DNA混合物中次要贡献者的等位基因。在本研究中，我们建立了一个针对MPS优化的1204个标记的面板，其中包括987个常染色体标记（964个SNP和23个indel）、27个X染色体SNP、61个Y染色体标记（56个SNP和5个indel）以及129个线粒体SNP。使用该面板通过MPS分析了6名无关个体（2名男性和4名女性）的DNA样本、26个未降解的DNA混合物（次要与主要比例为1:29、1:39、1:79和1:99）以及8个高度人工降解的DNA混合物（次要与主要比例为1:29、1:39、1:79和1:99）。开发了一种评分系统，以根据使用MPS鉴定的基因型来确定DNA混合物中的次要贡献者。通过使用定制设计的面板，通过MPS成功地对1204个标记的基因型进行了分析。MPS分析这些高度降解样本的效率低于分析未降解样本的效率。基于964个常染色体SNP，使用该评分系统准确地确定了26个未降解和8个降解DNA混合物中的所有次要贡献者。对于未降解的混合物，观察到的次要成分读数比率与理论比率之间存在关联。总之，我们建立了一个用于MPS的1204个标记的个体识别面板，该面板成功地同时分析了常染色体、X染色体、Y染色体和线粒体的SNP和indel。结合新开发的评分系统，该面板可以准确识别未降解和高度降解DNA混合物中的次要贡献者。
