Rapid mitochondrial genome sequencing based on Oxford Nanopore Sequencing and a proxy for vertebrate species identification

Abstract

Molecular information is crucial for species identification when facing challenging morphology‐based specimen identifications. The use of DNA barcodes partially solves this problem, but in some cases when PCR is not an option (i.e., primers are not available, problems in reaction standardization), amplification‐free approaches could be an optimal alternative. Recent advances in DNA sequencing, like the MinION device from Oxford Nanopore Technologies (ONT), allow to obtain genomic data with low laboratory and technical requirements, and at a relatively low cost. In this study, we explore ONT sequencing for molecular species identification from a total DNA sample obtained from a neotropical rodent and we also test the technology for complete mitochondrial genome reconstruction via genome skimming. We were able to obtain “de novo” the complete mitogenome of a specimen from the genus Melanomys (Cricetidae: Sigmodontinae) with average depth coverage of 78X using ONT‐only data and by combining multiple assembly routines. Our pipeline for an automated species identification was able to identify the sample using unassembled sequence data (raw) in a reasonable computing time, which was substantially reduced when a priori information related to the organism identity was known. Our findings suggest ONT sequencing as a suitable candidate to solve species identification problems in metazoan nonmodel organisms and generate complete mtDNA datasets.

Yes, we can use it: a formal test on the accuracy of low-pass nanopore long-read sequencing for mitophylogenomics and barcoding research using the Caribbean spiny lobster Panulirus argus

“…Still, the problem persists based on the results from this study. Previous studies using Canu have successfully assembled mitochondrial genomes [ 32 ] and larger circular chromosomes (i.e., chloroplasts - [ 40 ], bacteria - [ 22 ]). Importantly, in this study, Canu not only failed to assemble the mitochondrial genome of P. argus but also took 20–30 times longer to execute compared to the other pipelines that assembled and circularized the studied mitochondrial genome in < 2 h. The current version of Canu automatically detect available resources in a computer or cluster and configures itself to run ‘efficiently’ using those resources [ 39 ].…”

“…Triple, quadruple, and quintuple homopolymer insertions, and short deletions (≤ 3 bp) were rare. Unfortunately, the only previous study assembling a mitochondrial genome with nanopore long reads alone did not characterize errors [ 32 ]. For the MinION, independent error-assessments are rather scant [ 41 , 42 ].…”

“…In the case of animal mitochondrial genomes, hybrid assemblies have been successful in clawed lobsters ( Homarus gamarus - [ 24 ]) and land crabs ( Gecarcoidea natalis - [ 25 ]). To the best of the author’s knowledge, only a single study that employed a de novo assembly strategy using long reads alone produced a complete and fully accurate mitochondrial genome in a neotropical rodent ( Melanomys caliginosus - [ 32 ]). Importantly, the latter study benchmarked the long-read mitochondrial genome assembly using only two relatively short protein coding gene fragments obtained via Sanger sequencing [ 32 ].…”

“…To the best of the author’s knowledge, only a single study that employed a de novo assembly strategy using long reads alone produced a complete and fully accurate mitochondrial genome in a neotropical rodent ( Melanomys caliginosus - [ 32 ]). Importantly, the latter study benchmarked the long-read mitochondrial genome assembly using only two relatively short protein coding gene fragments obtained via Sanger sequencing [ 32 ]. Only after considerable manual curation, the authors (see [ 32 ]) claimed the assembly of a complete and fully accurate genome.…”

“…Importantly, the latter study benchmarked the long-read mitochondrial genome assembly using only two relatively short protein coding gene fragments obtained via Sanger sequencing [ 32 ]. Only after considerable manual curation, the authors (see [ 32 ]) claimed the assembly of a complete and fully accurate genome. However, the algorithm used for the final manual assembly curation was not explained in detail.…”