# Enhancement of the Precision ID Mitochondrial DNA Whole Genome System for Challenging Unidentified Human Remains

## TL;DR

Adding BSA to a DNA sequencing system improves its performance when dealing with difficult human remains samples.

## Contribution

BSA enhances the Precision ID system's robustness against inhibitors and microbial DNA in challenging samples.

## Key findings

- BSA improves sequencing success without affecting uninhibited samples.
- BSA is effective against specific concentrations of inhibitors like humic acid and hematin.
- BSA helps overcome non-specific adaptor ligation caused by microbial DNA.

## 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.