With the increasing number of complex forensic cases in recent years, it's more important to combine the different types of genetic markers such as short tandem repeats (STRs), single nucleotide polymorphisms (SNPs), insertion/deletion polymorphisms (InDels), and microhaplotypes (MHs) to provide more genetic information. In this study, we selected totally 201 genetic markers, including 24 autosomes STRs (A-STRs), 24 Y chromosome STRs (Y-STRs), 110 A-SNPs, 24 Y-SNPs, 9 A-InDels, 1 Y-InDel, 8 MHs, and Amelogenin to establish the HID_AM Panel v1.0, a Next-Generation Sequencing (NGS) detection system. According to the validation guidelines of the Scientific Working Group on DNA Analysis Methods (SWGDAM), the repeatability, accuracy, sensitivity, suitability for degraded samples, species specificity, and inhibitor resistance of this system were assessed. The typing results on 48 STRs and Amelogenin of this system were completely consistent with those obtained using capillary electrophoresis. This system accurately detected 79 SNPs as parallelly confirmed by a FGx sequencer with the ForenSeq™ DNA Signature Prep Kit. Complete allele typing results could be obtained with a DNA input of no less than 200 pg. The detection success rate of this system was significantly higher than that of the GlobalFiler™ kit when the degradation index of mock degraded sample was greater than 15.87. When the concentration of hematin in the amplification system was ≤40 µmol/L, indigo blue was ≤2 mmol/L, or humic acid was ≤15 ng/µL, amplification was not significantly inhibited. The system barely amplified the DNA extract from duck, mouse, cow, rabbit, and chick. The detection rate of STRs on routine samples of this panel is 99.74%, while all the SNPs, InDels, and MHs were successfully detected. In summary, we setup a NGS individual typing panel including 201 genetic markers with the high accuracy, sensitivity, species specificity, and inhibitors resistance, which is applicable for individual identification of degraded samples.
近年来，法医实践中复杂案件数量逐渐增多，需要联合使用短串联重复序列(short tandem repeat, STR)、单核苷酸多态性(single nucleotide polymorphisms, SNP)、插入缺失多态性(insertion/deletion polymorphism, InDel)、微单倍型(microhaplotype, MH)等不同类型的遗传标记，为案件提供更多的参考信息。本研究筛选了24个常染色体STR(autosomes STR, A-STR)、24个Y染色体STR(Y-STR)、110个A-SNP、24个Y-SNP、9个A-InDel、1个Y-InDel、8个MH和Amelogenin共201个遗传标记，建立二代测序检测体系HID_AM Panel v1.0。根据DNA 分析方法科学工作组(Scientific Working Group on DNA Analysis Methods，SWGDAM)的验证指南，对该体系的重复性、准确性、灵敏度、对降解样本的适用性、物种特异性、抗抑制性等指标进行评估。本体系分型结果与基于毛细管电泳方法检测的48个STR和Amelogenin结果完全一致，与ForenSeq™ DNA Signature Prep Kit重合的79个SNP分型结果完全一致；当DNA加入量不低于200 pg时可获得全部等位基因分型结果；当检测降解指数大于15.87的模拟降解样本时，检出成功率明显高于GlobalFilerTM PCR扩增试剂盒；当体系内血红素浓度不高于40 µmol/L、靛蓝浓度不高于2 mmol/L、或腐殖酸浓度不高于15 ng/µL时，扩增无明显抑制；鸭、鼠、牛、兔、鸡的DNA特异性扩增较少；常规样本中STR检出率达99.74%，SNP、InDel、MH全部检出。综上所述，本研究建立的检测体系具有较高的准确性、灵敏度、物种特异性和抗抑制性，适用于降解样本的个体识别。.
Keywords: degraded samples; genetic markers; multiple types; next generation sequencing.