Since skeletal remains recovered from missing persons' cases are often exposed to harsh environmental conditions that result in the DNA being damaged, degraded, and/or the samples containing PCR inhibitors, the current study evaluated the efficacy of common extraction methods in removing high levels of PCR inhibitors commonly encountered with human remains, as well as their downstream compatibility with the two leading sequencing chemistries and platforms for human identification purposes.
Blood, hair, and bone samples were spiked with high levels of inhibitors commonly identified in each substrate in order to test the efficiency of various DNA extraction methods prior to sequencing. Samples were extracted using three commercial extraction kits (DNA IQ, DNA Investigator, and PrepFiler BTA), organic (blood and hair only), and two total demineralization protocols (bone only)). Massively parallel sequencing (MPS) was performed using two different systems: Precision ID chemistry and a custom AmpliSeq STR and iiSNP panel on the Ion S5 System and the ForenSeq DNA Signature Prep Kit on the MiSeq FGx. The overall results showed that all DNA extraction methods were efficient and fully compatible with both MPS systems. Key performance indicators such as STR and SNP reportable alleles, read depth, and heterozygote balance were comparable for each extraction method. In samples where CE-based STRs yielded partial profiles (bone), MPS-based STRs generated more complete or full profiles. Moreover, MPS panels contain more STR loci than current CE-based STR kits and also include SNPs, which can further increase the power of discrimination obtained from these samples, making MPS a desirable choice for the forensic analysis of such challenging samples. (publisher abstract modified)