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. 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.This research has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 824110 – EASI-Genomics (I.G.G.) and the ERC Synergy project BCLL@las under grant agreement No 810287 (I.G.G.). Institutional support was from the Spanish Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias and cofunded with ERDF funds (PI19/01772). We acknowledge the institutional support of the Spanish Ministry of Science and Innovation through the Instituto de Salud Carlos III and the 2014–2020 Smart Growth Operating Program, to the EMBL partnership and institutional co-financing with the European Regional Development Fund (MINECO/FEDER, BIO2015-71792-P). We also acknowledge the support of the Centro de Excelencia Severo Ochoa, and the Generalitat de Catalunya through the Departament de Salut, Departament d’Empresa i Coneixement and the CERCA Programme to the institute