Design of a 200-SNP panel for individual identification and evaluation of genotyping performance using isolated cells.

We developed a novel next-generation sequencing-based genotyping panel for 200 single nucleotide polymorphism (SNP) loci, and evaluated the genotyping performance when using small amounts of DNA. We analyzed 1144 previously characterized autosomal SNPs, and selected 200 informative SNPs, based on the allele coverage ratio (ACR) and amplicon size, to minimize inter-locus depth of coverage imbalances. Analysis of the 200 SNPs was performed using 1 ng of DNA from 66 Japanese individuals. The average ACR of most of the SNP loci ranged from 0.85 to 0.95. We also detected 200-SNP genotypes using diluted DNA samples (500, 250, 125, 60, 30, and 15 pg/reaction), and found high ACRs and concordance from less than 100 pg of DNA: the concordances were as high as 95.7 % and 88.0 %, respectively, when 60 and 30 pg of DNA were used. Our method also showed higher inhibitory tolerance than other methods when humic acid was added to the initial amplification reaction for SNP genotyping. When genotyping in the presence of 10 ng/µL humic acid, no SNP profiles were detected, but profiles with more than 99 % concordance were recovered on addition of bovine serum albumin. The genotyping performance was also evaluated using small groups of cells (10, 5, 4, 3, and 2 cells/reaction) isolated by micromanipulation with micro-tweezers. High concordance was identified on analysis of three cells (96.1 %). The use of isolated cells allowed accurate determination of the sensitivity, indicating that our method is highly sensitive and can provide informative SNP profiles for individual identification.