Optimization and Developmental Validation of 38-plex InDel Panel for Ancestry Inference

doi:10.12116/j.issn.1004-5619.2021.510105

Abstract

Abstract:

Objective The previously established 38-plex InDel system was optimized and its performance was validated according to the Scientific Working Group on DNA Analysis Method （SWGDAM） application guidelines. The ancestry inference accuracy of individuals from East Asian， European， African and mixed populations was verified. Methods DNA standard sample 9947A was used as the template to establish the optimal amplification conditions by adjusting primer balance， Mg²⁺ final concentration and optimizing PCR thermal cycle parameters and amplification volume. The allelic dropout， nonspecific amplification and whether the origin of the inferred samples matched the known information were compared to evaluate the performance of this system. Results The optimal dosage of this system was 0.125-2 ng DNA template. The results of InDel typing were accurate， the amplification equilibrium was good， and the species specificity was good. This system showed certain tolerance to DNA samples including the inhibitor such as hemoglobin （≤80 μmol/L）， indigo （≤40 mmol/L）， calcium ion （≤1.0 mmol/L）， and humic acid （≤90 ng/μL）. The system enabled the direct amplification of DNA from saliva and blood on filter paper， and the results of ethnic inference were accurate. The system successfully detected the mixed DNA sample from two individuals. The test results of the system for common biological materials in practical cases were accurate. Conclusion The results of the 38-plex InDel system are accurate and reliable， and the performance of the system meets the requirement of the SWGDAM guidelines. This system can accurately differentiate the ancestry origins of individuals from African， European， East Asian， and Eurasian populations and can be implemented in forensic practice.

Key words: forensic genetics, ancestry inference, InDel polymorphism, multiplex amplification, developmental validation

CLC Number:

DF795.2

Qing-guo WANG, Lei ZHAO, Tang-song LI, Wang FU, He-xin XIE, Yuan MA, Wen-ping SUN, Jun-ping HAN. Optimization and Developmental Validation of 38-plex InDel Panel for Ancestry Inference[J]. Journal of Forensic Medicine, 2022, 38(5): 611-617.

Figures/Tables 9

Tab. 1 The reaction system of PCR （μL）

组分	体积
2.5×NiHi S9 PCR Mix	2	4	10	20
引物混合物	0.5	1	2.5	5
9947A DNA	0.5	1	2.5	5
去离子灭菌水	2	4	10	20
总体积	5	10	25	50

Fig. 1 CE maps from amplification of 9947A with different concentrations of Mg2+

Fig. 2 CE maps from amplification of 9947A with different cycle numbers

Fig. 3 CE maps from amplification of 9947A with different annealing temperatures

Fig. 4 CE maps from amplification of 9947A with different final extension times

Fig. 5 CE maps from amplification of 9947A with different reaction volumes

Fig. 6 The sensitivity study results of 9947A

Fig. 7 Comparison of test results of a sample from two laboratories using the 38-plex InDel assay

Tab. 2 The results of stability study using 4 concentration of inhibitors

抑制剂	体系终浓度	分型成功率/%
血红蛋白	60 μmol/L	100.00
	80 μmol/L	100.00
	100 μmol/L	97.36
	150 μmol/L	65.79
	200 μmol/L	39.47
靛蓝	30 mmol/L	100.00
	40 mmol/L	100.00
	52 mmol/L	97.36
	60 mmol/L	97.36
	70 mmol/L	92.10
钙离子	0.8 mmol/L	100.00
	1.0 mmol/L	100.00
	1.25 mmol/L	97.36
	1.5 mmol/L	89.47
	2.0 mmol/L	71.05
腐殖酸	80 ng/μL	100.00
	90 ng/μL	100.00
	100 ng/μL	97.36
	130 ng/μL	92.10
	160 ng/μL	92.10

References 19

1	DE LA PUENTE M， SANTOS C， FONDEVILA M， et al. The global AIMs nano set： A 31-plex SNaPshot assay of ancestry- informative SNPs[J]. Forensic Sci Int Genet，2016，22：81-88. doi：10.1016/j.fsigen.2016.01.015 .
2	FONDEVILA M， PHILLIPS C， SANTOS C， et al. Revision of the SNPforID 34-plex forensic ancestry test： Assay enhancements， standard reference sample genotypes and extended population studies[J]. Forensic Sci Int Genet，2013，7（1）：63-74. doi：10.1016/j.fsigen.2012.06.007 .
3	PHILLIPS C， SALAS A， SÁNCHEZ J J， et al. Inferring ancestral origin using a single multiplex assay of ancestry-informative marker SNPs[J]. Forensic Sci Int Genet，2007，1（3/4）：273-280. doi：10.1016/j.fsigen.2007.06.008 .
4	LI C X， PAKSTIS A J， JIANG L， et al. A panel of 74 AISNPs： Improved ancestry inference within Eastern Asia[J]. Forensic Sci Int Genet，2016，23：101-110. doi：10.1016/j.fsigen.2016.04.002 .
5	WEI Y L， WEI L， ZHAO L， et al. A single-tube 27-plex SNP assay for estimating individual ancestry and admixture from three continents[J]. Int J Leg Med，2016，130（1）：27-37. doi：10.1007/s00414-015-1183-5 .
6	PEREIRA R， PHILLIPS C， ALVES C， et al. A new multiplex for human identification using insertion/deletion polymorphisms[J]. Electrophoresis，2009，30（21）：3682-3690. doi：10.1002/elps.200900274 .
7	PEREIRA R， PHILLIPS C， PINTO N， et al. Straightforward inference of ancestry and admixture proportions through ancestry-informative insertion deletion multiplexing[J]. PLoS One，2012，7（1）： e29684. doi：10.1371/journal.pone.0029684 .
8	FONDEVILA M， PHILLIPS C， SANTOS C， et al. Forensic performance of two insertion-deletion marker assays[J]. Int J Leg Med，2012，126（5）：725-737. doi：10.1007/s00414-012-0721-7 .
9	SANTOS C， PHILLIPS C， OLDONI F， et al. Completion of a worldwide reference panel of samples for an ancestry informative Indel assay[J]. Forensic Sci Int Genet，2015，17：75-80. doi：10.1016/j.fsigen.2015.03.011 .
10	ZAUMSEGEL D， ROTHSCHILD M A， SCHNEIDER P M. A 21 marker insertion deletion polymorphism panel to study biogeographic ancestry[J]. Forensic Sci Int Genet，2013，7（2）：305-312. doi：10.1016/j.fsigen.2012.12.007 .
11	韩俊萍，赵蕾，王庆国，等. 单管直扩38重祖先信息InDels体系的构建及其在微流控芯片系统的应用[J].生物化学与生物物理进展，2020，47（9）：968-981. doi：10.16476/j.pibb.2020.0020 .
	HAN J P， ZHAO L， WANG Q G， et al. A single-tube direct amplification 38-plex ancestry informative InDels assay and its integration with a microfluidic system[J]. Shengwu Huaxue Yu Shengwu Wuli Jinzhan，2020，47（9）：968-981.
12	MILLS R E， LUTTIG C T， LARKINS C E， et al. An initial map of insertion and deletion （INDEL） variation in the human genome[J]. Genome Res，2006，16（9）：1182-1190. doi：10.1101/gr.4565806 .
13	BASTOS-RODRIGUES L， PIMENTA J R， PENA S D J. The genetic structure of human populations studied through short insertion-deletion polymorphisms[J]. Ann Hum Genet，2006，70（Pt 5）：658-665. doi：10.1111/j.1469-1809.2006.00287.x .
14	LAN Q， SHEN C M， JIN X Y， et al. Front Cover： Distinguishing three distinct biogeographic regions with an in-house developed 39-AIM-InDel panel and further admixture proportion estimation for Uyghurs[J]. Electrophoresis，2019，40（11）：elps. 201970101. doi：10.1002/elps.201970101 .
15	江丽，孙启凡，马泉，等. 27-plex SNP种族推断方法的优化及验证[J].遗传，2017，39（2）：166-173. doi：10.16288/j.yczz.16-346 .
	JIANG L， SUN Q F， MA Q， et al. Optimization and validation of analysis method based on 27-plex SNP panel for ancestry inference[J]. Yichuan，2017，39（2）：166-173.
16	PARK S J， KIM J Y， YANG Y G， et al. Direct STR amplification from whole blood and blood- or saliva-spotted FTA without DNA purification[J]. J Forensic Sci，2008，53（2）：335-341. doi：10.1111/j.1556-4029.2008.00666.x .
17	SANTOS C， FONDEVILA M， BALLARD D， et al. Forensic ancestry analysis with two capillary electrophoresis ancestry informative marker （AIM） panels： Results of a collaborative EDNAP exercise[J]. Forensic Sci Int Genet，2015，19：56-67. doi：10.1016/j.fsigen.2015.06.004 .
18	解通. 35个InDel位点荧光复合扩增检测体系的构建、验证及新疆三个少数民族遗传背景的探索研究[D].广州：南方医科大学，2019：76-86.
	XIE T. Development and validation of 35 InDels fluorescence multiplex system and genetic background explorative analysis of three ethnic minorities in Chinese Xinjiang area[D]. Guangzhou： Southern Medical University，2019：76-86.
19	ZHUANG B， HAN J P， XIANG G X， et al. A fully integrated and automated microsystem for rapid pharmacogenetic typing of multiple warfarin-related single-nucleotide polymorphisms[J]. Lab Chip，2016，16（1）：86-95. doi：10.1039/c5lc01094b .

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