A method for multiplexed full-length single-molecule sequencing of the human mitochondrial genome

Methods to reconstruct the mitochondrial DNA (mtDNA) sequence using short-read sequencing come with an inherent bias due to amplification and mapping. They can fail to determine the phase of variants, to capture multiple deletions and to cover the mitochondrial genome evenly. Long-read whole genome sequencing is prohibitively expensive for the purpose of mtDNA heteroplasmy detection and often does not represent the full mtDNA length. Here we describe a method to target, multiplex and sequence at high coverage full-length human mitochondrial genomes as native single-molecules, utilizing the RNA-guided DNA endonuclease Cas9. Combining Cas9 induced breaks, that define the mtDNA beginning and end of the sequencing reads, as barcodes, we achieve high demultiplexing specificity and delineation of the full-length of the mtDNA, regardless of the structural variant pattern. The long-read sequencing data is analysed with a pipeline where our newly developed software baldur efficiently detects single nucleotide heteroplasmy to below 1%, physically determines phase and can accurately disentangle complex deletions. Our workflow is a unique tool for studying mtDNA variation in health and disease, and will accelerate mitochondrial research.EGA study EGAS00001006280