This study assessed the usefulness of DNA quantification to predict the success of historical samples when analyzing SNPs, mtDNA, and STR targets. Thirty burials from six historical contexts were utilized, ranging in age from 80 to 800 years postmortem. Samples underwent library preparation and hybridization capture with two bait panels (FORCE and mitogenome), and STR typing (autosomal STR and Y-STR). All 30 samples generated small (~80 bp) autosomal DNA target qPCR results, despite mean mappable fragments ranging from 55–125 bp. The qPCR results were positively correlated with DNA profiling success. Samples with human DNA inputs as low as 100 pg resulted in ≥80% FORCE SNPs at 10X coverage. All 30 samples resulted in mitogenome coverage ≥100X despite low human DNA input (as low as 1 pg). With PowerPlex Fusion, ≥30 pg human DNA input resulted in >40% of auSTR loci. At least 59% of Y-STR loci were recovered with Y-target qPCR-based inputs of ≥24 pg. The results also indicate that human DNA quantity is a better predictor of success than the ratio of human to exogenous DNA. Accurate quantification with qPCR is feasible for historical bone samples, allowing for the screening of extracts to predict the success of DNA profiling.

Background/Objectives: 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. Methods: 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. Results: 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. Conclusions: 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.