Forensic Validation of the 95K SNP Panel and the Parabon Fx Forensic Analysis Platform for Identification of US Military Unknowns Using Extended Kinship Inference.

To identify US military unknowns, the Armed Forces Medical Examiner System's Armed Forces DNA Identification Laboratory has historically relied upon mitochondrial DNA and Y-chromosomal short tandem repeat testing. Where no appropriate family reference sample (FRS) is available or skeletal samples are degraded, autosomal single nucleotide polymorphism (SNP) testing with next-generation sequencing could assist.A method utilizing hybridization capture enrichment of a 95,000 (95K) SNP panel, amenable to FRS and extremely challenging samples, was validated. The Parabon Fx Forensic Analysis Platform was used for analysis and extended kinship inference. Skeletal samples (n = 65) and associated FRS (n = 64) were selected for a performance evaluation and case-type sample study.Considering FRS with ≥7 ng DNA input into library preparation, 94% yielded ≥66,320 SNPs at ≥5X coverage. SNP recovery for skeletal samples at ≥1X coverage ranged from 5 to 94,197 SNPs, averaging 40,770 SNPs. When skeletal samples resulted in ≥13,000 SNPs, the most likely relationship category was consistent with the expected relationship. A log10 likelihood ratio of ≥4 and a posterior probability of ≥99.99% were established as thresholds for strong statistical support, and 87% of inferences met these thresholds while 13% were considered inconclusive. Pairwise kinship inference between unrelated individuals yielded an unrelated result in 85% of comparisons, 66% with strong statistical support. There were 170 instances of false positive 4th degree relationship inferences with strong statistical support. All false positives involved skeletal samples from individuals of admixed ancestry.With this approach, autosomal SNP testing can result in reliable kinship inferences between related individuals out to 3rd, and in some cases 4th, degree relationships, increasing the scope of eligible FRS to aid in identifications.