1. Accurate and comprehensive analysis of single nucleotide variants and large deletions of the human mitochondrial genome in DNA and single cells
- Author
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Ben Caljon, Claudia Spits, Luca Gianaroli, Joke Mertens, Sara Seneca, Kim Vancampenhout, Karen Sermon, Filippo Zambelli, Thierry Voet, Daniel Brown, Dorien Daneels, Sonia Van Dooren, Basic (bio-) Medical Sciences, Reproduction and Genetics, Faculty of Medicine and Pharmacy, and Clinical sciences
- Subjects
0301 basic medicine ,Mitochondrial DNA ,Polymerase Chain Reaction/methods ,030105 genetics & heredity ,Biology ,Human mitochondrial genetics ,Polymerase Chain Reaction ,Polymorphism, Single Nucleotide ,Sensitivity and Specificity ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Journal Article ,Humans ,Nucleotide ,Genetics(clinical) ,genetics ,Genetics (clinical) ,Cells, Cultured ,Genetics ,chemistry.chemical_classification ,Fibroblasts/metabolism ,Massive parallel sequencing ,Breakpoint ,Single-Cell Analysis/methods ,Sequence Analysis, DNA ,Standard methods ,Fibroblasts ,Heteroplasmy ,030104 developmental biology ,chemistry ,Sequence Analysis, DNA/methods ,Genome, Mitochondrial ,Genome-Wide Association Study/methods ,Single-Cell Analysis ,DNA ,Gene Deletion ,Genome-Wide Association Study - 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.European Journal of Human Genetics advance online publication, 23 August 2017; doi:10.1038/ejhg.2017.129.
- Published
- 2017