Considering DNA damage when interpreting mtDNA heteroplasmy in deep sequencing data

Area of Science:

• Genetics
• Molecular Biology
• Forensic Science

Background:

• Mitochondrial DNA (mtDNA) heteroplasmy detection is improved by massively parallel sequencing (MPS), capable of identifying variants down to 1%.
• Establishing reporting thresholds for heteroplasmic variants is crucial, especially considering the impact of DNA damage.

Purpose of the Study:

• To assess how DNA damage affects the interpretation of low-level mtDNA heteroplasmy.
• To evaluate the influence of storage conditions and accelerated hydrolysis on DNA damage patterns and their impact on variant calling.

Main Methods:

• Assessed mtDNA control region variants under passive (storage) and active (hydrolysis) DNA damage conditions.
• Compared damage patterns to those in poor-quality samples.
• Analyzed the frequency and nature of miscoding lesions and their indistinguishability from true heteroplasmy.

Main Results:

• Increased DNA damage led to more miscoding lesions, often appearing as 1-2% heteroplasmy.
• Some lesions were indistinguishable from innate heteroplasmy, with two cases showing complete nucleotide sequence changes.
• Replication of miscoding lesions was minimal in stored and hydrolyzed samples.
• A decreased transition:transversion ratio was observed, aiding in the prediction of damage-induced lesions.

Conclusions:

• DNA damage poses a significant challenge for MPS analysis of low-level mtDNA variants in degraded samples.
• The findings are critical for accurate clinical and forensic investigations involving compromised DNA.
• The transition:transversion ratio can serve as a biomarker for DNA damage in sequencing data.