Keraite, IevaBecker, PhilippCanevazzi, DavideFrias-López, CristinaDabad, MarcTonda, RaúlParamonov, IdaIngham, MatthewBrun-Heath, IsabelleLeno, JordiAbulí, AnnaGarcía-Arumí, ElenaHeath, SimonGut, MartaGut, Ivo Glynne2023-01-182023-01-182022Keraite I, Becker P, Canevazzi D, Frias-López C, Dabad M, Tonda-Hernandez R, Paramonov I, Ingham MJ, Brun-Heath I, Leno J, Abulí A, Garcia-Arumí E, Heath SC, Gut M, Gut IG. A method for multiplexed full-length single-molecule sequencing of the human mitochondrial genome. Nat Commun. 2022 Oct 6;13(1):5902. DOI: 10.1038/s41467-022-33530-32041-1723http://hdl.handle.net/10230/55326Methods 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.application/pdfeng© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.info:eu-repo/semantics/articlehttp://dx.doi.org/10.1038/s41467-022-33530-3CRISPR-Cas systemsData processingDNA sequencingGenomic analysisMitochondrial genomeinfo:eu-repo/semantics/openAccess