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

Ieva Keraite, Philipp Becker, Davide Canevazzi, Cristina Frias-López, Marc Dabad, Raúl Tonda-Hernandez, Ida Paramonov, Matthew John Ingham, Isabelle Brun-Heath, Jordi Leno, Anna Abulí, Elena Garcia-Arumí, Simon Charles Heath, Marta Gut () and Ivo Glynne Gut ()
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Ieva Keraite: The Barcelona Institute of Science and Technology (BIST)
Philipp Becker: The Barcelona Institute of Science and Technology (BIST)
Davide Canevazzi: The Barcelona Institute of Science and Technology (BIST)
Cristina Frias-López: The Barcelona Institute of Science and Technology (BIST)
Marc Dabad: The Barcelona Institute of Science and Technology (BIST)
Raúl Tonda-Hernandez: The Barcelona Institute of Science and Technology (BIST)
Ida Paramonov: The Barcelona Institute of Science and Technology (BIST)
Matthew John Ingham: The Barcelona Institute of Science and Technology (BIST)
Isabelle Brun-Heath: Institute for Research in Biomedicine (IRB Barcelona) - The Barcelona Institute of Science and Technology (BIST)
Jordi Leno: Hospital Universitari Vall d’Hebron
Anna Abulí: Hospital Universitari Vall d’Hebron
Elena Garcia-Arumí: Hospital Universitari Vall d’Hebron
Simon Charles Heath: The Barcelona Institute of Science and Technology (BIST)
Marta Gut: The Barcelona Institute of Science and Technology (BIST)
Ivo Glynne Gut: The Barcelona Institute of Science and Technology (BIST)

Nature Communications, 2022, vol. 13, issue 1, 1-12

Abstract: Abstract 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.

Date: 2022
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DOI: 10.1038/s41467-022-33530-3

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