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. 2018 Dec 11;33(52):e337.
doi: 10.3346/jkms.2018.33.e337. eCollection 2018 Dec 24.

Detection of Innate and Artificial Mitochondrial DNA Heteroplasmy by Massively Parallel Sequencing: Considerations for Analysis

Moon-Young Kim  1 , Sohee Cho  2 , Ji Hyun Lee  1 , Hee Jin Seo  1 , Soong Deok Lee  1   2
Affiliations

Detection of Innate and Artificial Mitochondrial DNA Heteroplasmy by Massively Parallel Sequencing: Considerations for Analysis

Moon-Young Kim et al. J Korean Med Sci. .

Abstract

Background: Mitochondrial heteroplasmy, the co-existence of different mitochondrial polymorphisms within an individual, has various forensic and clinical implications. But there is still no guideline on the application of massively parallel sequencing (MPS) in heteroplasmy detection. We present here some critical issues that should be considered in heteroplasmy studies using MPS.

Methods: Among five samples with known innate heteroplasmies, two pairs of mixture were generated for artificial heteroplasmies with target minor allele frequencies (MAFs) ranging from 50% to 1%. Each sample was amplified by two-amplicon method and sequenced by Ion Torrent system. The outcomes of two different analysis tools, Torrent Suite Variant Caller (TVC) and mtDNA-Server (mDS), were compared.

Results: All the innate heteroplasmies were detected correctly by both analysis tools. Average MAFs of artificial heteroplasmies correlated well to the target values. The detection rates were almost 90% for high-level heteroplasmies, but decreased for low-level heteroplasmies. TVC generally showed lower detection rates than mDS, which seems to be due to their own computation algorithms which drop out some reference-dominant heteroplasmies. Meanwhile, mDS reported several unintended low-level heteroplasmies which were suggested as nuclear mitochondrial DNA sequences. The average coverage depth of each sample placed on the same chip showed considerable variation. The increase of coverage depth had no effect on the detection rates.

Conclusion: In addition to the general accuracy of the MPS application on detecting heteroplasmy, our study indicates that the understanding of the nature of mitochondrial DNA and analysis algorithm would be crucial for appropriate interpretation of MPS results.

Keywords: Computational Biology; DNA Sequence Analysis; Heteroplasmy; Massively Parallel Sequencing; Mitochondrial DNA.

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Conflict of interest statement

Disclosure: The authors have no potential conflicts of interest to disclose.

Figures

Fig. 1
Fig. 1. Observed MAFs of each mixed pair. For A-B pair (A) and C-D pair (B), the MAF distribution of TVC is represented as gray boxes and mDS as white boxes. Both analysis tools showed generally lower MAFs than expected without significant differences.
MAF = minor allele frequency, TVC = Torrent Suite Variant Caller, mDS = mtDNA-Server.
Fig. 2
Fig. 2. Detection rates of artificial heteroplasmies according to the analysis tools. TVC with reference modification shows the highest detection rates for high-level heteroplasmies, while mDS shows better results for low-level heteroplasmies.
TVC = Torrent Suite Variant Caller, mDS = mtDNA-Server.
See this image and copyright information in PMC

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