Novel method for multiplexed, full-length single-molecule sequencing of human mitochondrial DNA using Cas9-mediated enrichment

Abstract

Traditional methods to reconstruct the mitochondrial DNA (mtDNA) sequence are challenged. They fail to determine variant phase, 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, utilising 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. Data analysis is done with a newly developed pipeline with our software baldur, which efficiently detects single nucleotide heteroplasmy to below 1%, physically determines phase, and accurately disentangles complex deletions. Our workflow is a unique tool for studying mtDNA variation in health and disease, and will accelerate mitochondrial research.