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

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.