A new method for long-read sequencing of animal mitochondrial genomes: application to the identification of equine mitoc
- PDF / 2,004,298 Bytes
- 15 Pages / 595.276 x 790.866 pts Page_size
- 7 Downloads / 137 Views
METHODOLOGY ARTICLE
Open Access
A new method for long-read sequencing of animal mitochondrial genomes: application to the identification of equine mitochondrial DNA variants Sophie Dhorne-Pollet1, Eric Barrey1† and Nicolas Pollet2*†
Abstract Background: Mitochondrial DNA is remarkably polymorphic. This is why animal geneticists survey mitochondrial genomes variations for fundamental and applied purposes. We present here an approach to sequence whole mitochondrial genomes using nanopore long-read sequencing. Our method relies on the selective elimination of nuclear DNA using an exonuclease treatment and on the amplification of circular mitochondrial DNA using a multiple displacement amplification step. Results: We optimized each preparative step to obtain a 100 million-fold enrichment of horse mitochondrial DNA relative to nuclear DNA. We sequenced these amplified mitochondrial DNA using nanopore sequencing technology and obtained mitochondrial DNA reads that represented up to half of the sequencing output. The sequence reads were 2.3 kb of mean length and provided an even coverage of the mitochondrial genome. Long-reads spanning half or more of the whole mtDNA provided a coverage that varied between 118X and 488X. We evaluated SNPs identified using these long-reads by Sanger sequencing as ground truth and found a precision of 100.0%; a recall of 93.1% and a F1-score of 0.964 using the Twilight horse mtDNA reference. The choice of the mtDNA reference impacted variant calling efficiency with F1-scores varying between 0.947 and 0.964. Conclusions: Our method to amplify mtDNA and to sequence it using the nanopore technology is usable for mitochondrial DNA variant analysis. With minor modifications, this approach could easily be applied to other large circular DNA molecules. Keywords: Mitochondria, mtDNA, Horse, Nanopore sequencing, MinION device, Single nucleotide polymorphism
* Correspondence: [email protected] † Eric Barrey and Nicolas Pollet should be considered senior co-authors. 2 Université Paris-Saclay, CNRS, IRD, UMR Évolution, Génomes, Comportement et Écologie, 91198 Gif-sur-Yvette, France Full list of author information is available at the end of the article © The Author(s). 2020 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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommon
Data Loading...
