Accurate and comprehensive analysis of single nucleotide variants and large deletions of the human mitochondrial genome in DNA and single cells

Abstract

Massive parallel sequencing (MPS) can accurately quantify mitochondrial DNA (mtDNA) single nucleotide variants (SNVs), but no MPS methods are currently validated to simultaneously and accurately establish the breakpoints and frequency of large deletions at low heteroplasmic loads. Here we present the thorough validation of an MPS protocol to quantify the load of very low frequency, large mtDNA deletions in bulk DNA and single cells, along with SNV calling by standard methods. We used a set of well-characterized DNA samples, DNA mixes and single cells to thoroughly control the study. We developed a custom script for the detection of mtDNA rearrangements that proved to be more accurate in detecting and quantifying deletions than pre-existing tools. We also show that PCR conditions and primersets must be carefully chosen to avoid biases in the retrieved variants and an increase in background noise, and established a lower detection limit of 0.5% heteroplasmic load for large deletions, and 1.5 and 2% for SNVs, for bulk DNA and single cells, respectively. Finally, the analysis of different single cells provided novel insights into mtDNA cellular mosaicism.

Splice-Break: exploiting an RNA-seq splice junction algorithm to discover mitochondrial DNA deletion breakpoints and analyses of psychiatric disorders

“…The catalogue of 4489 mtDNA deletions we describe here only includes those with breakpoints between positions 357–15925 (NC_012920.1) of the mitochondrial genome as we filtered out the majority of the control region in this analysis. Future studies may include analysis of mtDNA deletion breakpoints in the D-loop and extended control region, which includes a 3′ breakpoint ‘hotspot’ at position 16071, perhaps using different primers (with different binding positions) for the LR PCR (7,14,32). Indeed any analysis of mtDNA deletions that utilizes LR PCR will be limited to only discover molecules that can be successfully amplified with the primers chosen.…”

“…Additional computational methods we have identified that may warrant future comparisons with our pipeline include a customized Perl script provided by Zambelli et al. (32), and the eKLIPse tool recently published by Goudenège et al. (33).…”

Random Mutagenesis, Clonal Events, and Embryonic or Somatic Origin Determine the mtDNA Variant Type and Load in Human Pluripotent Stem Cells

“…This method can reliably detect SNVs with loads ≥1.5% in DNA samples and oocytes, and ≥2% in single cells and ICMs (Zambelli et al., 2017). …”

Mitochondrial DNA Variations in Tumors: Drivers or Passengers?

“…In a comparative analysis, it has been demonstrated that Sanger sequencing is valid for quantification of heteroplasmies with more than 10% of cells/mitochondria carrying the mutation, whereas NGS is capable of reliably detecting and quantifying heteroplasmic variants down to the 1% level [113]. Recently, a massive parallel sequencing (MPS) protocol reliably quantified low frequency, large mtDNA deletions in single cells with a lower detection limit of 0.5% [114]. mtDNA NGS has been also suggested as a useful quality check of pluripotent stem cells for drug discovery and regenerative medicine purposes [115].…”