Utility of ForenSeq™ DNA Signature Prep Kit in the research of pairwise 2nd-degree kinship identification


The scope of forensic kinship analysis is being extended to more distant or complex relationships. However, current methods and standards in this field do not meet the needs of casework. The next-generation sequencing (NGS) technology may hold an advantage in this field to traditional methods due to its strong power to get much more genetic information. To evaluate the effectiveness of NGS to identify the 2nd-degree kinship pairs, DNA samples of 227 individuals from 49 Hebei Han pedigrees were tested by Goldeneye™ 20A kit using capillary electrophoresis (CE) to confirm the relationships within each pedigree, and those of 111 individuals within 97 confirmed grandparent-grandchild or avuncular pairs were analyzed by ForenSeq™ DNA Signature Prep Kit using MiSeq® FGx™ DNA sequencing platform. We calculated the likelihood ratio (LR) based on ITO method and the identical by state (IBS) score of 97 kinship pairs and compared with those of 97 unrelated pairs. According to the results summarized and analyzed by Fisher discriminant analysis and leave-one-out cross-validation (LOOCV) method, ITO method showed higher accuracy than IBS method, even with less information. Therefore, we proposed a recommendation of the thresholds for pairwise 2nd-degree kinship identification for Hebei Han population based on ITO method. When using ITO method based on 94 SNPs and the length information of 27 autosomal STRs, cumulative likelihood ratio (CLR) > 1 and CLR < 0.1 are recommended as the thresholds of confirming and excluding, respectively. The accuracy applying such thresholds is greater than 95%, indicating the promising application value of NGS in this field and providing a direction for further kinship identification strategy selection. Further studies are needed to get the population genetic data of loci contained in the kit based on all sequence information including flanking regions to make full use of the NGS data to improve the accuracy of kinship analysis.

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

Author information




SL and BC designed the study. MX, ZC, and QL recruited family samples and extracted DNA. MX and QD carried out CE test and NGS test. GM and LF performed data analysis. GM and SL wrote the first and last drafts of the manuscript, and all authors made critical revisions. SL and BC take responsibility for the integrity of the data and accuracy of the data analysis.

Corresponding authors

Correspondence to Bin Cong or Shujin Li.

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The ethical consent was obtained from the ethics committee of Hebei Medical University and statements on informed consent were signed by the adult volunteers and the parents of minors before the collection of blood samples

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Xu, M., Du, Q., Ma, G. et al. Utility of ForenSeq™ DNA Signature Prep Kit in the research of pairwise 2nd-degree kinship identification. Int J Legal Med 133, 1641–1650 (2019). https://doi.org/10.1007/s00414-019-02003-6

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  • 2nd-degree kinship identification
  • MiSeq® FGx™ sequencing platform
  • ForenSeq™ DNA signature prep kit
  • ITO method
  • IBS method