Accurate mapping of mitochondrial DNA deletions and duplications using deep sequencing

Abstract

Deletions and duplications in mitochondrial DNA (mtDNA) cause mitochondrial disease and accumulate in conditions such as cancer and age-related disorders, but validated high-throughput methodology that can readily detect and discriminate between these two types of events is lacking. Here we establish a computational method, MitoSAlt, for accurate identification, quantification and visualization of mtDNA deletions and duplications from genomic sequencing data. Our method was tested on simulated sequencing reads and human patient samples with single deletions and duplications to verify its accuracy. Application to mouse models of mtDNA maintenance disease demonstrated the ability to detect deletions and duplications even at low levels of heteroplasmy.

Mitochondrial DNA variants in inclusion body myositis characterized by deep sequencing

“…To identify large mtDNA deletions and duplications, alignment to chrM (rCRS assembly, NC_012920.1) was performed (20,21). After realignment, a mean chrM coverage depth of 46,000x was observed.…”

“…The mitochondrial DNA coverage was normalized to account for shifts due to deletions and duplications by considering the coverage at positions m.4000‐6000 in the mitochondrial genome, a region with low amounts of deletions/duplications, for all the samples. Gapped alignments, indicative of deletions/duplications, were clustered and visualized as described (20–22). A deletion or duplication was considered only if it was supported by five sequencing reads with a minimum heteroplasmy level of 0.01%.…”

“…A deletion or duplication was considered only if it was supported by five sequencing reads with a minimum heteroplasmy level of 0.01%. Deletions were reclassified as likely duplications in case of overlap with replication origins, as previously described (20,21). Heteroplasmy levels for individual deletions and duplications were estimated by comparing the number of reads supporting the corresponding breakpoints to the total number of reads overlapping the breakpoints (including wild type).…”

Mitochondrial DNA Instability Is Common in HIV-Exposed Uninfected Newborns

“…It is noteworthy that our NGS approach uses information from short DNA fragments (median 180 bp) sequencing and does not provide information on the full structure of mtDNA. The observed deletions are likely to originate from an incomplete duplication of mtDNA resulting in a chimeric molecule, the full-length sequence conferring the replication origins lacking in the deleted sequence, as already hypothesized by others [ 37 ]. Such rearrangements have already been described in human tumors and the aging process [ 35 , 36 , 38 ].…”

Mitochondrial Heteroplasmy Shifting as a Potential Biomarker of Cancer Progression

“…To identify new diagnostic and prognostic biomarkers and even new potential molecular targets, current research focuses on the nuclear genome by detecting mutations and on gene expression and epigenomic signatures [ 2 , 3 , 4 , 5 ]; however, given the number of biological processes in which mitochondria participate (energy synthesis, metabolism, apoptosis and signaling pathways, among others), these organelles are gaining great relevance in addressing these questions [ 6 ]. More specifically, it has been shown that the mitochondrial DNA (mtDNA) of tumor cells accumulates many alterations, including punctual mutations, insertions, deletions and variations in mtDNA copy number (mtCNV), which trigger the metabolic reprogramming of transformed cells as a biological strategy that favors tumor cell proliferation and survival [ 7 , 8 , 9 , 10 , 11 , 12 , 13 ]. Even though the mutational spectrum of mtDNA has already been studied in different types of cancer, the mechanism by which mtDNA mutations contributes to tumor development is not yet well known [ 14 , 15 , 16 ].…”