Second- and third-degree kinship analysis by NGS-based SNP genotyping and evaluation of 2045-SNP performance on...

We developed a novel next-generation sequencing-based single-nucleotide polymorphism (SNP) genotyping method for second- and third-degree kinship analysis, and designed 1144- and 2045-SNP panels using one (Set A) and two sets (Sets A and B) of primers. These SNP loci were analyzed in 120 Japanese individuals, and likelihood ratios (LRs) for kinship discrimination were calculated to evaluate the effect of number of SNP loci in simulated analysis. Likelihood evaluation was performed using DNA profiles from two individuals, namely, the unknown and one reference relative. Genotyping of the 1144 and 2045 SNPs was informative to discriminate aunt-nephew/niece as second-degree relatives and unrelated pairs. In third-degree relationship analysis, distributions of log₁₀LRs between the unknown and a first cousin (FC) were not separated from those of unrelated individuals even in 2045-SNP genotyping. To perform enhanced discrimination of third-degree relationships, we also evaluated the effectiveness of DNA profiles from three individuals, namely, the unknown and two first cousins (2FC). A likelihood evaluation from the 2045 SNPs using 2FC was more useful than the use of FC. Our method was applied to 12 kinship cases for second- and third-degree relationship analysis, and LRs from 1144- and 2045-SNP genotypes were markedly higher than those from conventional short tandem repeat profiles. Moreover, we evaluated the performance of the 2045 SNPs using limited or degraded DNA, compared with that with a larger amount of DNA. Using 0.1 ng of non-degraded DNA, the average concordance was higher than 97 %. In analysis with heavily degraded DNA (degradation index=32.2), we also detected high concordance (85.5 %) from 2045-SNP genotypes, compared with the lower rate (52.4 %) from 21 short tandem repeat profiles. Our method should be highly sensitive with discriminatory DNA profiles for analyzing second- and third-degree relationships.