For the adoption of massively parallel sequencing (MPS) systems by forensic laboratories, validation studies on specific workflows are needed to support the feasibility of implementation and the reliability of the data they produce. As such, the whole mitochondrial genome sequencing methodology—Precision ID mtDNA Whole Genome Panel, Ion Chef, Ion S5, and Converge—has been subjected to a variety of developmental validation studies. These validation studies were completed in accordance with the Scientific Working Group on DNA Analysis Methods (SWGDAM) validation guidelines and assessed reproducibility, repeatability, accuracy, sensitivity, specificity to human DNA, and ability to analyze challenging (e.g., mixed, degraded, or low quantity) samples. Intra- and inter-run replicates produced an average maximum pairwise difference in variant frequency of 1.2%. Concordance with data generated with traditional Sanger sequencing and an orthogonal MPS platform methodology was used to assess accuracy, and generation of complete and concordant haplotypes at DNA input levels as low as 37.5 pg of nuclear DNA or 187.5 mitochondrial genome copies illustrated the sensitivity of the system. Overall, data presented herein demonstrate that highly accurate and reproducible results were generated for a variety of sample qualities and quantities, supporting the reliability of this specific whole genome mitochondrial DNA MPS system for analysis of forensic biological evidence.

The efficient extraction of DNA from challenging samples, such as bones, is critical for the success of downstream genotyping analysis in molecular genetic disciplines. Even though the ancient DNA community has developed several protocols targeting small DNA fragments that are typically present in decomposed or old specimens, only recently forensic geneticists have started to adopt those protocols. Here, we compare an ancient DNA extraction protocol (Dabney) with a bone extraction method (Loreille) typically used in forensics. Real-time quantitative PCR and forensically representative typing methods including fragment size analysis and sequencing were used to assess protocol performance. We used four bone samples of different age in replicates to study the effects of both extraction methods. Our results confirm Loreille’s overall increased gain of DNA when enough tissue is available and Dabney’s improved efficiency for retrieving shorter DNA fragments that is beneficial when highly degraded DNA is present. The results suggest that the choice of extraction method needs to be based on available sample, degradation state, and targeted genotyping method. We modified the Dabney protocol by pooling parallel lysates prior to purification to study gain and performance in single tube typing assays and found that up to six parallel lysates lead to an almost linear gain of extracted DNA. These data are promising for further forensic investigations as the adapted Dabney protocol combines increased sensitivity for degraded DNA with necessary total DNA amount for forensic applications.

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.