A new method for long-read sequencing of animal mitochondrial genomes: application to the identification of equine mitochondrial DNA variants

Abstract

14Background: We present here an approach to sequence whole mitochondrial genomes using 15 nanopore long-read sequencing. Our method relies on the selective elimination of nuclear DNA 16 using an exonuclease treatment and on the amplification of circular mitochondrial DNA using 17 a multiple displacement amplification step. 18Results: We optimized each preparative step to obtain a 100 million-fold enrichment of horse 19 mitochondrial DNA relative to nuclear DNA. We sequenced these amplified mitochondrial 20 DNA using nanopore sequencing technology and obtained mitochondrial DNA reads that 21 represented up to half of the sequencing output. The sequence reads were 2.3 kb of mean length 22 and provided an even coverage of the mitochondrial genome. Long-reads spanning half or more 23 of the whole mtDNA provided a coverage that varied between 118X and 488X. Finally, we 24 identified SNPs with a precision of 98.1%; recall of 85.2% and a F1-score of 0.912. 25 2 Conclusions : Our analyses show that our method to amplify mtDNA and to sequence it using 26 the nanopore technology is usable for mitochondrial DNA variant analysis. With minor 27 modifications, this approach could easily be applied to other large circular DNA molecules. 28 29 Nucleotide Polymorphism 31 32 33 Background 34Mitochondria have become crucial organelles of eukaryote cells since an ancestral bacterial 35 endosymbiosis event gave rise to the eukaryote branch of the tree of life, about 1.5 billion years 36 ago (1,2). The discovery that mitochondria contains DNA in the early 1960s attracted the 37 attention of many scientists and more recently the word of mitogenomics has been coined to 38 unite this field of study (3). Today, thousands of mitochondrial genomes (mtDNA) from many 39 species have been sequenced, and ongoing studies require robust, rapid and cheap method to 40 decipher novel genomes and identify genetic variants (4). In human alone, more than 48,882 41 full length mtDNA sequences are known, but much less sequences and variants are available 42 for other mammals (5). Numerous and diverse applications take advantage of mtDNA 43 sequencing, such as diagnostic of mitochondrial diseases in human and animals, breeding 44 selection, forensic studies, biodiversity surveys, conservation genetics and evolutionary 45 analysis. 46Animal mitochondrial genomes are usually encoded by a single circular DNA of about 10 47 to 20 kbp, but weird cases are known such as linear or very large molecules (6). The 48 mitochondrial genomes are characterized by a maternal transmission, a lack of recombination 49 and higher frequency of mutations than in the nuclear genome (7). In vertebrates, the ratio of 50 3 mtDNA over nuclear DNA mutation rate is usually above 20 (8). This means that mtDNA 51 polymorphisms may occur relatively often in animal populations with the potential for 52 associated disorders, and this is why sequencing these organelle genomes is an important 53 component of contemporaneous animal genetics. Knowing the whole mtDNA sequence enables 54 the i...

Extension of Mitogenome Enrichment Based on Single Long-Range PCR: mtDNAs and Putative Mitochondrial-Derived Peptides of Five Rodent Hibernators

“…The so-called ‘hybrid assembly’ helps to cope with the widespread presence of sequence duplications and repeats occurring in vertebrates mitogenomes, namely the (prevalent) short repeats of the NCR, but especially the (rarer) relatively large repetitive regions ( Gibb et al, 2007 ; Satoh et al, 2016 ; Rhie et al, 2021 ). Third-generation approaches for long-read sequencing such as Oxford Nanopore technology or PacBio sequencing are options of choice to overcome this challenge ( Kingan et al, 2019 ; Dhorne-Pollet et al, 2020 ). Moreover, the homology between NUMTs and mtDNA can confound the outcome of next generation sequencing (NGS) pipelines that align short reads of DNA to the reference genome based on sequence similarity ( Maude et al, 2019 ).…”

“…The use of plasma DNA as natural source of nuclear depletion is hampered by the fact that direct NGS analysis would produce unnecessary nuDNA reads since extrachromosomal circular DNA was found to be released from tissues into the circulation ( Kumar et al, 2017 ). This phenomenon impairs sequence analysis of circular mtDNA enriched by random-hexamer primed isothermal rolling circle amplification ( Simison et al, 2006 ) and the enrichment of circular mtDNA by exonuclease V-based depletion of nuDNA ( Gould et al, 2015 ; Zhu et al, 2017 ; Dhorne-Pollet et al, 2020 ). The latter likely explains the admixture of 70 to 41% of nuDNA reads following exonuclease depletion of linear DNA and phi29 polymerase-mediated isothermal amplification ( Dhorne-Pollet et al, 2020 ).…”

“…This phenomenon impairs sequence analysis of circular mtDNA enriched by random-hexamer primed isothermal rolling circle amplification ( Simison et al, 2006 ) and the enrichment of circular mtDNA by exonuclease V-based depletion of nuDNA ( Gould et al, 2015 ; Zhu et al, 2017 ; Dhorne-Pollet et al, 2020 ). The latter likely explains the admixture of 70 to 41% of nuDNA reads following exonuclease depletion of linear DNA and phi29 polymerase-mediated isothermal amplification ( Dhorne-Pollet et al, 2020 ). Another PCR-free pipeline for mtDNA enrichment and reduction of sequencing volume used a gene-capture chip that produced an up to 100-fold increase of the mtDNA fraction ( Liu et al, 2016 ).…”

Use of mitochondrial sequencing to detect gene doping in horses via gene editing and somatic cell nuclear transfer

“…Newer product innovations should increase base calling, however, and the loss of accuracy can be compensated for if read depth is sufficiently high enough. In addition to amplicon sequencing by long read sequencing, 46 an alternative method of sequencing mitochondrial genomes in horses using selective elimination of nuclear DNA followed by multiple displacement amplification has recently been described 47 …”

“…In addition to amplicon sequencing by long read sequencing, 46 an alternative method of sequencing mitochondrial genomes in horses using selective elimination of nuclear DNA followed by multiple displacement amplification has recently been described. 47…”