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

Accurate analysis of mitochondrial DNA is important for mitochondrial disease clinical research and diagnostics. Here, authors present a method using Cas9 cleavage, nanopore sequencing and a custom pipeline to identify pathogenic variants, deletions and accurately quantify heteroplasmy to below 1%. 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. 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 custom-developed software, baldur, efficiently detects single nucleotide heteroplasmy to below 1%, physically determines phase and can accurately disentangle complex deletions. Our workflow is a tool for studying mtDNA variation and will accelerate mitochondrial research.

Automated summary

Accurate analysis of mitochondrial DNA is crucial for clinical research and diagnostics of mitochondrial diseases. This study presents a method that utilizes Cas9 cleavage, nanopore sequencing, and a custom pipeline to identify pathogenic variants and accurately quantify heteroplasmy. The authors demonstrate that their method is capable of detecting complex deletions, determining the phase of variants, and achieving high coverage of the mitochondrial genome, overcoming the limitations of short-read sequencing. The developed workflow provides a powerful tool for studying mtDNA variation and will greatly accelerate mitochondrial research.