Abstract
We present an in silico approach for the reconstruction of complete mitochondrial genomes of non-model organisms directly from next-generation sequencing (NGS) data—mitochondrial baiting and iterative mapping (MITObim). The method is straightforward even if only (i) distantly related mitochondrial genomes or (ii) mitochondrial barcode sequences are available as starting-reference sequences or seeds, respectively. We demonstrate the efficiency of the approach in case studies using real NGS data sets of the two monogenean ectoparasites species Gyrodactylus thymalli and Gyrodactylus derjavinoides including their respective teleost hosts European grayling (Thymallus thymallus) and Rainbow trout (Oncorhynchus mykiss). MITObim appeared superior to existing tools in terms of accuracy, runtime and memory requirements and fully automatically recovered mitochondrial genomes exceeding 99.5% accuracy from total genomic DNA derived NGS data sets in <24 h using a standard desktop computer. The approach overcomes the limitations of traditional strategies for obtaining mitochondrial genomes for species with little or no mitochondrial sequence information at hand and represents a fast and highly efficient in silico alternative to laborious conventional strategies relying on initial long-range PCR. We furthermore demonstrate the applicability of MITObim for metagenomic/pooled data sets using simulated data. MITObim is an easy to use tool even for biologists with modest bioinformatics experience. The software is made available as open source pipeline under the MIT license at https://github.com/chrishah/MITObim.
Highlights
Mitochondrial DNA is a useful and popular marker in molecular ecology, population genetics, evolutionary biology, as well as in phylogeographic and phylogenetic studies of animals [1,2,3,4]
We demonstrate the novel approach of reconstructing complete mitochondrial genomes using exclusively cytochrome-oxidase subunit 1 (COI) sequences, i.e. the commonly used mitochondrial DNA barcode for animals [33], as seed references
mitochondrial baiting and iterative mapping (MITObim) required 15 and 12 iterations to complete the mitochondrial genomes of G. thymalli and G. derjavinoides into a single contig, respectively
Summary
Mitochondrial DNA is a useful and popular marker in molecular ecology, population genetics, evolutionary biology, as well as in phylogeographic and phylogenetic studies of animals [1,2,3,4]. Only relatively short mitochondrial regions were targeted, but with improving methodology, sequencing of complete mitochondrial genomes became more common, even when exploring difficult templates such as ancient DNA. Until recently, sequencing of mitochondrial genomes was a somewhat challenging and resource demanding task. It has been approached using the conventional strategy of combining long-range PCR with subsequent primer walking [e.g. Approaches using exclusively e129 Nucleic Acids Research, 2013, Vol 41, No 13
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