适用于降解样本的201个遗传标记检测体系的构建和验证

doi:10.16288/j.yczz.23-253

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Hereditas(Beijing) ›› 2024, Vol. 46 ›› Issue (4): 306-318.doi: 10.16288/j.yczz.23-253

• Research Article • Previous Articles Next Articles

A DNA typing panel of 201 genetic markers for degraded samples: development and validation

Wei Han(), Qingzhen Zhang, Jing Yang(), Zhe Zhou()

Bioinformatics Center of Academy of Military Medical Sciences, Beijing 100850, China

Received:2023-12-05 Revised:2024-01-30 Online:2024-04-20 Published:2024-01-31
Contact: Jing Yang, Zhe Zhou E-mail:651430459@qq.com;jingyang0511@163.com;zhouzhe@bmi.ac.cn

Abstract

Abstract:

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.

Key words: next generation sequencing, multiple types, genetic markers, degraded samples

Wei Han, Qingzhen Zhang, Jing Yang, Zhe Zhou. A DNA typing panel of 201 genetic markers for degraded samples: development and validation[J]. Hereditas(Beijing), 2024, 46(4): 306-318.

Figures/Tables 8

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样本名称	大片段(pg/μL)	小片段(pg/μL)	Y片段(pg/μL)	降解指数 DI	STR分型成功率(%)	SNP+InDel分型成功率(%)	MH分型成功率(%)	全部遗传标记分型成功率(%)
D1	0.44	0.80	1.32	1.84	98.51	99.11	100	98.97
D2	0.24	0.71	1.18	2.93	98.51	100	100	99.66
D3	0.14	0.70	1.03	4.84	97.01	99.11	100	98.63
D4	0.31	0.86	1.29	2.72	97.01	99.11	100	98.63
D5	0.07	0.69	0.94	10.45	95.52	98.22	100	97.6
D6	0.04	0.65	0.99	15.78	92.54	97.33	100	96.23
D7	0.02	0.40	0.74	16.66	97.01	100	100	99.32
D8	0.03	0.51	0.80	20.35	97.01	99.11	100	98.63
D9	0.01	0.41	0.69	37.61	97.01	98.67	100	98.29
D10	0.01	0.54	0.66	82.78	91.04	98.22	100	96.58
D11	0.00	0.49	0.62	401.02	83.58	95.56	87.5	92.12

样品名	分型错误STR	分型错误SNP	分型错误InDel	分型错误MH
血红素-空白	/	/	/	/
血红素-5 µmol/L	CSF1PO	/	/	/
血红素-10 µmol/L	CSF1PO 、D7S820、D9S1122、DYS437、DYS392、DYS448、DYS438	/	rs3032356	/
血红素-20 µmol/L	/	/	/	/
血红素-40 µmol/L	D17S974、D19S433、D3S1358、D7S820、DYS481、DYS19、DYS643、Y_GATA_H4、DYS389II	/	/	/
血红素-80 µmol/L	×	×	×	×
靛蓝-空白	/	/	/	/
靛蓝-2 mmol/L	D18S51、Penta D、FGA、DYS458、DYS518、DYS389II	rs1413212、rs2399332、rs717302、rs159606、rs12174864、rs1336071、rs826472、rs3780962、rs2107612、rs1295330、rs2096458、rs914165、rs747718394	rs35771136、rs2307805、rs16660	mh13KK-213
靛蓝-4 mmol/L	×	×	×	×
腐殖酸-空白	/	/	/	/
腐殖酸-1 ng/µL	/	/	/	/
腐殖酸-5 ng/µL	CSF1PO、D7S820、DYS393、DYS389I	rs1295330	/	/
腐殖酸-10 ng/µL	DYS389II	rs1295330	/	/
腐殖酸-15 ng/µL	D18S51、D21S11、Penta D、DYS518、DYS389II	rs1295330、rs2096458	/	/
腐殖酸-50 ng/µL	×	×	×	×

A DNA typing panel of 201 genetic markers for degraded samples: development and validation

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