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.

Mitochondrial DNA (mtDNA) can help in the identification of biological evidence recovered from crime scenes and human remains. Typically the hypervariable regions are targeted for sequencing; however, more discriminating profiles are obtained if the whole genome is sequenced. Different approaches exist as to how best amplify and sequence whole mtDNA from forensic specimens. Here, we describe a method based on two-round PCR, combining multiplex and simplex PCRs. This method has been used in the analysis of mitochondrial genomes from archival saliva samples applied to FTA® cards after 10 years of transportation and preservation, without special protection. It is expected that this technique can be also used for the analysis of other old biological specimens directly or with modifications related to the level of DNA degradation.