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Mitochondrial genomes long read sequencing methodology applied to multispecies for the identification of genetic variants
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International audience. In animals, mitochondrial genome variability is poorly studied outside of the MT-CO1, MT-CYB and D-Loop used in phylogenetic studies. However, in genetics there is a real interest in better understanding the variability of the whole mtDNA and its functional impact and the potential association with particular traits of interest. This study aimed to develop an optimized workflow for routine high-throughput sequencing of mtDNA based on long read sequencing and applicable whatever the species.Four vertebrate species were selected to carry out this methodological project: Equus caballus, Bos taurus, Oncorhynchus mykiss and Xenopus tropicalis.The sequencing libraries and long read sequencing protocol using a MinION device followed the methods described in the horse [1, 2] and were generalized to the other species. We used the GATK pipeline to detect and compare SNPs on each mtDNA genome. Whatever the species the quality of long read sequencing was good: average length of reads equal to 2.3kb; coverage of 188X to 488X; 100% of SNPs validated by Sanger sequencing. The enrichment ratio of mtDNA reads varied between samples within each species, but the coverage was deep enough for variant calling. All the SNP obtained by long reads versus short reads were identical.References1. Dhorne-Pollet S., Barrey E., Pollet N.. A new method for long-read sequencing of animal mitochondrial genomes: application to the identification of equine mitochondrial DNA variants. BMC Genomics, BioMed Central, 2020, 21 (1).2. Dhorne-Pollet S, Pollet N, Barrey E. A New Method for Sequencing the Mitochondrial Genome by Using Long Read Technology. Methods Mol Biol. 2021;2277:331-343.
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