Abstract
BACKGROUND: The Precision ID mitochondrial (mt) DNA Whole Genome system is a fully automated massively parallel sequencing (MPS) solution for the whole mitochondrial genome. While extremely sensitive, the Precision ID system is susceptible to inhibitors and microbial DNA that are often co-extracted from human remains. METHODS: DNA templates spiked with varying amounts of hematin, humic acid, and calcium, along with bones containing degraded and non-human DNA, were sequenced using the Precision ID system with and without the addition of bovine serum albumin (BSA). RESULTS: BSA added to the initial PCR reaction successfully improved the robustness of the Precision ID system while not negatively impacting the sequencing success of uninhibited samples. The success of BSA is inhibitor-concentration dependent and is effective for templates containing at least 50 ng/μL humic acid, 50 μM hematin, and 1500 μM calcium ions. Furthermore, the presence of microbial DNA in addition to an inhibitor, results in non-specific adaptor ligation to the non-human DNA; BSA can alleviate the inhibition, allowing the human mtDNA to be amplified and sequenced. CONCLUSIONS: The addition of BSA to the Precision ID mtDNA system can yield successful sequencing results from challenging case samples that would otherwise fail.