Application of a dense SNP PCR multiplex for high throughput kinship determination using Next Generation Sequencing (NGS)

Abstract

The need to identify large numbers of missing persons from mass disasters, armed conflicts and human rights abuses has become increasingly prevalent. The ForenSeq® Kintelligence Library Prep Kit together with NGS (sometimes called massively parallel sequencing), has been designed to interrogate 10,230 SNPs for the purpose of solving cold cases, including missing persons identification. The kit allows up to 3 samples per sequencing run in order to call sufficient SNPs for detecting relationships up to 5th degree when searching direct to consumer DNA databases, such as GEDmatch PRO™. For missing persons cases with high numbers of post-mortem (P.M.) and ante-mortem (A.M.) samples from the deceased and their family members, respectively, a higher throughput solution is required. In this report, we present the feasibility of increasing sequencing plexity for PM to 12 and AM to 32 samples using the Kintelligence kit and workflow. This generates sufficient SNP calls for kinship determination up to 3rd order. DNA was extracted from bones and dental remains, as well as artificially degraded and low input DNA to simulate P.M. samples. DNA from diverse populations was typed to simulate A.M. samples. We determined the number of called SNPs overlapping between A.M. and P.M samples from these high-plexity sequencing runs and showed the impact on two methods of estimating kinship: kinship coefficients and likelihood ratios (LR). LR and kinship coefficient decrease as fewer SNPs are called (i.e., in more challenging samples) or for more distant relationships. However, filtering on both values achieved high sensitivity and specificity out to 3rd degree. Furthermore, relationship identification out to 3rd degree was robust to loss of heterozygosity in the samples. Overall, increasing plexity can facilitate higher throughput DNA analysis of missing persons cases, without severe loss in kinship estimation.