Evaluating the Usefulness of Human DNA Quantification to Predict DNA Profiling Success of Historical Bone Samples

Abstract

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.

Unearthing who and Y at Harewood Cemetery and inference of George Washington’s Y-chromosomal haplotype

“…Replicate amplifications were carried out for extracts B-7Ac, B-7A1, and B-28Ac due to suspected inhibition based on previous research ( Table S2 ). 13 Burials 3 and 4 failed to generate reportable profiles (<4 loci), as shown in Table S5 . Burial 3 had the lowest quantification result of the four DNA extracts that generated a value for the Y-DNA target ( Table 2 ); therefore, a lower recovery was expected.…”

“…A reduced total dsDNA input was used for extract B-7Ac due to signs of inhibition observed in previous research. 13 At adapter ligation the stock concentration (15 μM) of UDI adapter was used for all samples and RBs. NCs and PCs received a 1.5 μM dilution due to a dsDNA input ≤1ng.…”

“…Hybridization capture and post capture purification were performed according to Thomas et al. 13 Purified capture libraries were analyzed on the 2100 Bioanalyzer instrument using the DNA 7500 or DNA High Sensitivity assay (both Agilent Technologies).…”

“… 30 A range of DNA inputs were amplified for samples B-7 and B-28 with suspected inhibition based on previous testing. 13 An NC and 2800M PC were included with each amplification event.…”

Assessment of DNA quality for whole genome library preparation

“…While the use of microarrays has long been the state of the art for SNP genotyping, the requirement for high amounts of pure and intact DNA limits its application for several types of challenging samples [10,18]. The recent progress in sequencing techniques has enabled the analysis of low quantities of degraded DNA [19][20][21][22] and the successful generation of genotypes from aged samples [1, 10,14,[23][24][25][26][27]. The possibility of re-analyzing difficult DNA samples with more sophisticated methods has also spurred the continuation and reopening of unsolved investigations of "cold cases" and unidentified human remains [14,24,26].…”