Highlights
- •Miscoding lesions accumulate as storage condition and targeted DNA damage worsens.
- •The majority of miscoding lesions have a 1–2% minor variant frequency.
- •Miscoding lesions are typically not replicated from the same DNA extract.
- •Miscoding lesions reduce the mtDNA heteroplasmy transition:transversion ratio.
- •Sequences with DNA damage may require modified heteroplasmy reporting thresholds.
Abstract
Resolution of mitochondrial (mt) DNA heteroplasmy is now possible when applying a
massively parallel sequencing (MPS) approach, including minor components down to 1%.
However, reporting thresholds and interpretation criteria will need to be established
for calling heteroplasmic variants that address a number of important topics, one
of which is DNA damage. We assessed the impact of increasing amounts of DNA damage
on the interpretation of minor component sequence variants in the mtDNA control region,
including low-level mixed sites. A passive approach was used to evaluate the impact
of storage conditions, and an active approach was employed to accelerate the process
of hydrolytic damage (for example, replication errors associated with depurination
events). The patterns of damage were compared and assessed in relation to damage typically
encountered in poor quality samples. As expected, the number of miscoding lesions
increased as conditions worsened. Single nucleotide polymorphisms (SNPs) associated
with miscoding lesions were indistinguishable from innate heteroplasmy and were most
often observed as 1–2% of the total sequencing reads. Numerous examples of miscoding
lesions above 2% were identified, including two complete changes in the nucleotide
sequence, presenting a challenge when assessing the placement of reporting thresholds
for heteroplasmy. To mitigate the impact, replication of miscoding lesions was not
observed in stored samples, and was rarely seen in data associated with accelerated
hydrolysis. In addition, a significant decrease in the expected transition:transversion
ratio was observed, providing a useful tool for predicting the presence of damage-induced
lesions. The results of this study directly impact MPS analysis of minor sequence
variants from poorly preserved DNA extracts, and when biological samples have been
exposed to agents that induce DNA damage. These findings are particularly relevant
to clinical and forensic investigations.
Keywords
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Article info
Publication history
Published online: September 28, 2016
Accepted:
September 27,
2016
Received in revised form:
September 15,
2016
Received:
May 31,
2016
Identification
Copyright
© 2016 Elsevier Ireland Ltd. All rights reserved.
