Establishment and Application of a 42-plex Microhaplotype Assay in Forensic Genetics

doi:10.12116/j.issn.1004-5619.2023.530803

Abstract

Abstract:

Objective To establish and forensically verify a 42 microhaplotypes （mircohaps, MHs） multiplex assay system based on next-generation sequencing （NGS）, and to explore the application value of this system in the practice of forensic genetics. Methods A total of 42 highly polymorphic MHs were selected from previous studies, and sequenced by the MiSeq FGx^TM platform to verify the repeata-bility, sensitivity, specificity, stability, and mixture analysis ability of the detection system. Through population genetic investigation of 102 unrelated Chinese Han individuals in Liyang City, Jiangsu Province, China, the application value of this system in forensic genetics was evaluated. Results The sequencing repeatability of the 42-plex MHs assay was 100% and the sensitivity was as low as 0.062 5 ng. The system had the ability to withstand the interference of indigo （≤2 500 ng/μL）, humic acid （≤9 ng/μL）, hemoglobin（≤20 μmol）, and urea （≤200 ng/μL） and to detect mixtures of 2 people （1∶19）， 3 people （1∶1∶9） and 4 people （1∶1∶1∶9）. Based on 102 individual data, the combined power of discrimination and the combined power of exclusion were 1-3.45×10^-30 and 1-3.77×10^-11, respectively, and the average effect value of alleles was 2.899. Conclusion The 42-plex MHs assay was successfully established in this study and this system has high repeatability and sensitivity, good anti-jamming ability and mixture analysis ability. The 42 MHs are highly polymorphism and have good application value in individual identification and paternity testing.

Key words: forensic genetics, microhaplotype, next-generation sequencing, mixture, individual identification

CLC Number:

Peng YUN, An-qi CHEN, Li-qin CHEN, Cheng-tao LI. Establishment and Application of a 42-plex Microhaplotype Assay in Forensic Genetics[J]. Journal of Forensic Medicine, 2024, 40(1): 50-58.

Figures/Tables 5

References 21

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位点	位点组成	染色体	扩增长度/bp
MHEUR2-2	rs112073382/rs4970451/rs113727072/rs112357356/rs1623400	1	84
MHEUR9-1	rs12409047/rs12409060/rs2151940/s2171988	1	168
MHSAS2-1	rs1123130/rs114194062/rs35008184/rs857831	1	172
MHSAS4-1	rs17714228/rs77295887/rs875891/rs875890	3	156
MHSAS8-1	rs2107560/rs2107559/rs12492078	3	179
MHAFR4-1	rs16844737/rs4916615/rs1884412/rs1884411/rs58827274	4	125
MHAMR13-1	rs10461216/rs187032722/rs9995348/rs10017946	4	116
MHEUR6-1	rs4833021/rs4833022/rs4833023	4	146
MHSAS9-1	rs2114312/rs9985976/rs4437264/rs4640681	4	190
MHAMR3-1	rs200588122/rs67283146/rs540158574/rs202023291/rs9401441/rs6925945/rs141891023	6	75
MHAMR5-1	rs36182126/rs75391776/rs35548356/rs7743540	6	182
MHEAS2-1	rs12175290/rs9452163/rs73529991/rs2211027	6	129
MHEUR11-1	rs138007191/rs851988/rs567978840/rs851987	6	194
MHEUR7-1	rs535901401/rs72655638/rs58870591/rs7840076	8	161
MHSAS3-1	rs10956351/rs78607349/rs34375527/rs35719509	8	98
MHEAS5-1	rs73374371/rs117251178/rs10738378/rs139150924/rs7468912/rs12346031	9	112
MHEUR1-1	rs57542309/rs10810635/rs1096259/rs67920508	9	171
MHEUR5-1	rs10756388/rs35552759/rs34112289/rs201672851/rs12156438	9	74
MHSAS5-3	rs4836757/rs12347183/rs4836758/rs77808236	9	155
MHSAS7-1	rs10761217/rs71364374/rs4743915	9	123
MHEAS4-2	rs670712/rs561514/rs11220401/rs562361	11	161
MHEAS6-1	rs34896965/rs1508537/rs4604870/rs7482592	11	38
MHAMR7-1	rs1152971/rs4761274/rs1152970/rs10879055	12	165
MHAMR14-1	rs35748706/rs11170250/rs12829851/rs4761944/rs4761808	12	185
MHEUR8-1	rs2730952/rs2134395/rs2730951/rs2134396	12	169
MHAMR8-1	rs149236313/rs12875658/rs9599297/rs9571876	13	123
MHAFR1-1	rs10130834/rs10131007/rs11621409/rs12881752/rs74090738	14	157
MHAMR9-7	rs61981010/rs74368593/rs146292209/rs111272295/rs1756651/rs12590697/rs192833681	14	166
MHAMR11-1	rs4255712/rs7494541/rs10139593	14	110
MHAFR3-3	rs530176837/rs563921248/rs62020715/rs62020716/rs11856096	15	158
MHEUR4-1	rs10532258/rs28698741/rs34113852	15	112
MHAFR5-1	rs2318185/rs5818409/rs74029053/rs28526585	16	155
MHAMR1-1	rs1351151/rs6205084/rs1351152/rs1351153	16	195
MHAMR4-1	rs449842/rs17642520/rs17796342/rs7198164	16	140
MHSAS1-1	rs7190552/rs7196129/rs12447919	16	144
MHAMR10-1	rs12051807/rs72859113/rs192005315/rs12952969	17	145
MHEUR3-25	rs201685439/rs72639203/rs199861716/rs567424484/rs542946310/rs551770630/rs201245768/rs569956882/rs199736881	17	198
MHEUR10-2	rs35718812/rs58008957/rs17742582	18	155
MHSAS6-1	rs35843459/rs113586740/rs2247133/rs11668692/rs2247129	19	170
MHAMR2-17	rs28446958/rs147532699/rs28435537/rs530142559/rs574425142	22	185
MHAMR6-1	rs135975/rs135976/rs146297030/rs7287191/rs2040412/rs7292599	22	197
MHEAS1-7	rs111946844/rs138120872/rs4050488/rs2257292/rs462915	22	179

位点	位点组成	染色体	扩增长度/bp
MHEUR2-2	rs112073382/rs4970451/rs113727072/rs112357356/rs1623400	1	84
MHEUR9-1	rs12409047/rs12409060/rs2151940/s2171988	1	168
MHSAS2-1	rs1123130/rs114194062/rs35008184/rs857831	1	172
MHSAS4-1	rs17714228/rs77295887/rs875891/rs875890	3	156
MHSAS8-1	rs2107560/rs2107559/rs12492078	3	179
MHAFR4-1	rs16844737/rs4916615/rs1884412/rs1884411/rs58827274	4	125
MHAMR13-1	rs10461216/rs187032722/rs9995348/rs10017946	4	116
MHEUR6-1	rs4833021/rs4833022/rs4833023	4	146
MHSAS9-1	rs2114312/rs9985976/rs4437264/rs4640681	4	190
MHAMR3-1	rs200588122/rs67283146/rs540158574/rs202023291/rs9401441/rs6925945/rs141891023	6	75
MHAMR5-1	rs36182126/rs75391776/rs35548356/rs7743540	6	182
MHEAS2-1	rs12175290/rs9452163/rs73529991/rs2211027	6	129
MHEUR11-1	rs138007191/rs851988/rs567978840/rs851987	6	194
MHEUR7-1	rs535901401/rs72655638/rs58870591/rs7840076	8	161
MHSAS3-1	rs10956351/rs78607349/rs34375527/rs35719509	8	98
MHEAS5-1	rs73374371/rs117251178/rs10738378/rs139150924/rs7468912/rs12346031	9	112
MHEUR1-1	rs57542309/rs10810635/rs1096259/rs67920508	9	171
MHEUR5-1	rs10756388/rs35552759/rs34112289/rs201672851/rs12156438	9	74
MHSAS5-3	rs4836757/rs12347183/rs4836758/rs77808236	9	155
MHSAS7-1	rs10761217/rs71364374/rs4743915	9	123
MHEAS4-2	rs670712/rs561514/rs11220401/rs562361	11	161
MHEAS6-1	rs34896965/rs1508537/rs4604870/rs7482592	11	38
MHAMR7-1	rs1152971/rs4761274/rs1152970/rs10879055	12	165
MHAMR14-1	rs35748706/rs11170250/rs12829851/rs4761944/rs4761808	12	185
MHEUR8-1	rs2730952/rs2134395/rs2730951/rs2134396	12	169
MHAMR8-1	rs149236313/rs12875658/rs9599297/rs9571876	13	123
MHAFR1-1	rs10130834/rs10131007/rs11621409/rs12881752/rs74090738	14	157
MHAMR9-7	rs61981010/rs74368593/rs146292209/rs111272295/rs1756651/rs12590697/rs192833681	14	166
MHAMR11-1	rs4255712/rs7494541/rs10139593	14	110
MHAFR3-3	rs530176837/rs563921248/rs62020715/rs62020716/rs11856096	15	158
MHEUR4-1	rs10532258/rs28698741/rs34113852	15	112
MHAFR5-1	rs2318185/rs5818409/rs74029053/rs28526585	16	155
MHAMR1-1	rs1351151/rs6205084/rs1351152/rs1351153	16	195
MHAMR4-1	rs449842/rs17642520/rs17796342/rs7198164	16	140
MHSAS1-1	rs7190552/rs7196129/rs12447919	16	144
MHAMR10-1	rs12051807/rs72859113/rs192005315/rs12952969	17	145
MHEUR3-25	rs201685439/rs72639203/rs199861716/rs567424484/rs542946310/rs551770630/rs201245768/rs569956882/rs199736881	17	198
MHEUR10-2	rs35718812/rs58008957/rs17742582	18	155
MHSAS6-1	rs35843459/rs113586740/rs2247133/rs11668692/rs2247129	19	170
MHAMR2-17	rs28446958/rs147532699/rs28435537/rs530142559/rs574425142	22	185
MHAMR6-1	rs135975/rs135976/rs146297030/rs7287191/rs2040412/rs7292599	22	197
MHEAS1-7	rs111946844/rs138120872/rs4050488/rs2257292/rs462915	22	179

生物抑制剂	质量浓度（含量）	检出率/%	测序深度/×
靛蓝	0 ng/μL	100.00	3 636.03
	1 000 ng/μL	100.00	1 444.56
	1 500 ng/μL	100.00	804.23
	2 000 ng/μL	100.00	674.69
	2 500 ng/μL	100.00	493.41
腐殖酸	0 ng/μL	100.00	3 636.03
	3 ng/μL	100.00	1 622.34
	6 ng/μL	100.00	1 347.80
	9 ng/μL	100.00	1 236.11
	12 ng/μL	92.85	90.98
血红素	0 μmol	100.00	3 636.03
	5 μmol	100.00	1 063.31
	10 μmol	100.00	924.77
	15 μmol	100.00	326.28
	20 μmol	100.00	189.98
尿素	0 ng/μL	100.00	3 636.03
	200 ng/μL	100.00	496.85
	400 ng/μL	97.61	111.13
	600 ng/μL	95.23	63.61
	800 ng/μL	92.85	47.65
黑色素	0 ng/μL	100.00	3 636.03
	5 ng/μL	95.23	62.04
	10 ng/μL	90.47	24.70
	15 ng/μL	78.57	5.51
	20 ng/μL	73.80	3.60

生物抑制剂	质量浓度（含量）	检出率/%	测序深度/×
靛蓝	0 ng/μL	100.00	3 636.03
	1 000 ng/μL	100.00	1 444.56
	1 500 ng/μL	100.00	804.23
	2 000 ng/μL	100.00	674.69
	2 500 ng/μL	100.00	493.41
腐殖酸	0 ng/μL	100.00	3 636.03
	3 ng/μL	100.00	1 622.34
	6 ng/μL	100.00	1 347.80
	9 ng/μL	100.00	1 236.11
	12 ng/μL	92.85	90.98
血红素	0 μmol	100.00	3 636.03
	5 μmol	100.00	1 063.31
	10 μmol	100.00	924.77
	15 μmol	100.00	326.28
	20 μmol	100.00	189.98
尿素	0 ng/μL	100.00	3 636.03
	200 ng/μL	100.00	496.85
	400 ng/μL	97.61	111.13
	600 ng/μL	95.23	63.61
	800 ng/μL	92.85	47.65
黑色素	0 ng/μL	100.00	3 636.03
	5 ng/μL	95.23	62.04
	10 ng/μL	90.47	24.70
	15 ng/μL	78.57	5.51
	20 ng/μL	73.80	3.60

混合人数	混合比例	检出等位基因数/个	检测基因频率/%	主要贡献者		次要贡献者
混合人数	混合比例	检出等位基因数/个	检测基因频率/%	等位基因数/个	等位基因检出率/%	等位基因数/个	等位基因检出率/%
2人	1∶1	96	100.00	66	100.00	30	100.00
	1∶9	96	100.00	66	100.00	30	100.00
	1∶19	94	100.00	66	100.00	28	100.00
	1∶49	84	89.36	66	100.00	18	60.00
	1∶99	76	79.16	66	100.00	10	33.33
3人	1∶1∶1	119	100.00	66	100.00	53	100.00
	1∶1∶9	119	100.00	66	100.00	53	100.00
	1∶1∶19	117	98.31	66	100.00	51	96.62
	1∶1∶49	106	89.36	66	100.00	40	75.47
	1∶1∶99	103	86.55	66	100.00	37	69.81
4人	1∶1∶1∶1	124	100.00	66	100.00	58	100.00
	1∶1∶1∶9	124	100.00	66	100.00	58	100.00
	1∶1∶1∶19	120	96.77	66	100.00	54	93.31
	1∶1∶1∶49	112	89.36	66	100.00	46	79.31
	1∶1∶1∶99	107	86.55	66	100.00	41	70.68