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Methodology for Accurate Detection of Mitochondrial DNA Methylation
Published on: May 20, 2018
Enhancement of the Precision ID Mitochondrial DNA Whole Genome System for Challenging Unidentified Human Remains
Lauren C Canale1, Mavis Date-Chong1, Jeanette Wallin1
1California Department of Justice, Jan Bashinski DNA Laboratory, Richmond, CA 94804, USA.
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.
Insights
Bovine serum albumin (BSA) addition enhances the Precision ID mitochondrial DNA (mtDNA) Whole Genome system's robustness against inhibitors and microbial DNA. This improves sequencing success in challenging forensic samples.
Area of Science:
- Forensic Science
- Molecular Biology
- Genetics
Background:
- The Precision ID mitochondrial (mt) DNA Whole Genome system offers automated massively parallel sequencing (MPS) for the entire mitochondrial genome.
- This system is highly sensitive but vulnerable to co-extracted inhibitors (e.g., hematin, humic acid, calcium) and microbial DNA from human remains.
Purpose of the Study:
- To evaluate the efficacy of bovine serum albumin (BSA) in mitigating inhibitor and microbial DNA interference in the Precision ID mtDNA Whole Genome system.
- To determine the optimal conditions for BSA application to enhance sequencing success in challenging samples.
Main Methods:
- DNA templates were spiked with varying concentrations of hematin, humic acid, and calcium.
- Samples included bones with degraded and non-human DNA.
- Sequencing was performed using the Precision ID system with and without BSA in the initial PCR reaction.
Main Results:
- BSA addition significantly improved the robustness of the Precision ID system against inhibitors without compromising sequencing of uninhibited samples.
- BSA was effective at inhibitor concentrations up to 50 ng/μL humic acid, 50 μM hematin, and 1500 μM calcium.
- BSA prevented non-specific adaptor ligation to microbial DNA in the presence of inhibitors, enabling human mtDNA amplification and sequencing.
Conclusions:
- Incorporating BSA into the Precision ID mtDNA workflow enhances its reliability for analyzing challenging forensic samples.
- BSA addition allows for successful mitochondrial genome sequencing from samples that would typically fail due to inhibitors or microbial contamination.
