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Development of an NGS panel containing 42 autosomal STR loci and the evaluation focusing on secondary kinship analysis

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Abstract

High-throughput next-generation sequencing (NGS) is a feasible technique to detect considerably more markers and simultaneously obtain length and sequence information in a single reaction. In this study, we developed an NGS panel including 42 commonly used autosomal short tandem repeats (STRs) and amelogenin on the Illumina MiSeq FGx™. Sequencing accuracy was validated by the consistency of 2800M Control DNA detected using the ForenSeq™ DNA Signature Prep Kit and Sanger sequencing. Nomenclature incompatibility was found between NGS-STR and CE-STR typing at 9 loci (D3S3045, D6S477, D7S3048, D9S925, D14S608, D17S1290, D18S535, D21S1270, GATA198B05), despite the correct sequence. The difference was caused by the two different methods of identifying motif sequence and a one-to-one correspondence can be found. We evaluated the panel by investigating consistency, sequencing sensitivity and the effectiveness of the 2nd-degree relationship identification. Herein, we present sequencing results from 58 unrelated individuals of the Hebei Han population. The total discrimination power (TDP) and cumulative probability of exclusion for trio paternity testing (CPEtrio) of the 42 NGS-STR panels reached 1–2.84 × 10−57 and 1–9.87 × 10−21, respectively. By family simulation and likelihood ratio (LR) calculation, this panel was shown to have effectiveness for the 2nd-degree kinship identification similar to the ForenSeq™ DNA Signature Prep Kit and certain advantages compared with it due to the relatively small number of loci. As expected, it provides new data for the development of NGS-STR typing technology.

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Funding

This work was supported by the National Natural Science Foundation of China (81671875) and Hebei Province Science Fund (H2017206351).

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Author notes

  1. Qingxia Liu and Guanju Ma contributed equally to this work.

Authors and Affiliations

  1. College of Forensic Medicine, Hebei Key Laboratory of Forensic Medicine, Hebei Medical University, Shijiazhuang, 050017, People’s Republic of China

    Qingxia Liu, Guanju Ma, Qingqing Du, Chaolong Lu, Lihong Fu, Qian Wang, Guangping Fu, Shujin Li & Bin Cong

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  1. Qingxia Liu
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Contributions

SL and BC designed the study. QL, QD, QW and GF extracted DNA and operated CE test. QL and QD performed NGS test. QL, QD, GM and LF carried out the statistical analysis. GM performed the simulation and evaluation on secondary kinship analysis. All authors contributed to interpretation of the data. QL, GM and SL wrote the first and last drafts of the manuscript and all authors made critical revisions. SL takes responsibility for the integrity of the data and accuracy of the data analysis.

Corresponding authors

Correspondence to Shujin Li or Bin Cong.

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The authors declare that they have no conflict of interest.

Ethical standards

Ethical consent was obtained from the ethics committee of Hebei Medical University and statements on informed consent from the adult volunteers and the parents of minors before the collection of blood samples.

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ESM 1

Comparisons of log10(CGI) distribution curves from 3 aspects: (A) _SEQ vs. _LEN groups, (B) _P vs. _R groups and (C) between different panels. The u values and P-values of Mann-Whitney u tests are listed in the figures. All figures share common legends according to the following rules: (i) _42, _SIG and _COM groups are coloured red, blue and green, respectively; (ii) the colour of _R groups are darker than the corresponding _P groups; (iii) the _LEN groups are dotted; (iv) UR groups are labelled the same as, and just slightly thinner than, the corresponding KS groups. Number of Alleles. (DOCX 615 kb).

ESM 2

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Liu, Q., Ma, G., Du, Q. et al. Development of an NGS panel containing 42 autosomal STR loci and the evaluation focusing on secondary kinship analysis. Int J Legal Med 134, 2005–2014 (2020). https://doi.org/10.1007/s00414-020-02295-z

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  • DOI: https://doi.org/10.1007/s00414-020-02295-z

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