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.
Highlights
Species are the fundamental study unit in biology so that accurate species identification is a vital process in the biological and medical sciences (Mayr, 1982; de Queiroz, 2005)
Species identification efforts have drastically being influenced by the use of molecular data acting as a complement to taxonomic information obtained from morphology alone (Camargo & Sites, 2013; Carstens et al, 2013; Luo, Ling, Ho, & Zhu, 2018); the availability of molecular datasets is still restricted to particular biotic groups, while many others—in which high diversity occurs—lack proper molecular information, hindering the ability of addressing evolutionary questions, biogeographic hypothesis, and accurate species delimitation and identification (Helmy, Awad, & Mosa, 2016; Noreña, González Muñoz, MosqueraRendón, Botero, & Cristancho, 2018)
Traditional barcoding approaches have proven useful for species identification (Barco, Raupach, Laakmann, Neumann, & Knebelsberger, 2016; Hebert, Ratnasingham, et al, 2003), but they are not suitable for certain taxa lacking reference genomes or specific primers for PCR amplification when universal primers do not work as expected (Ford et al, 2009; Moulton, Song, & Whiting, 2010; Pino-Bodas, Martín, Burgaz, & Lumbsch, 2013)
Summary
Species are the fundamental study unit in biology so that accurate species identification is a vital process in the biological and medical sciences (Mayr, 1982; de Queiroz, 2005). Recent developments in DNA sequencing techniques, such as single-molecule sequencing detection like the nanopore-based method from Oxford Nanopore Technologies (ONT), are a breakthrough in molecular biology due to its multiple advantages such as long sequencing reads, portability (pocket-sized device called MinION), reduced cost, and relative simplicity for its setup and operation compared with the traditional sequencing platforms (Jain, Olsen, Paten, & Akeson, 2016; Laver et al, 2015) Given those benefits, a new range of applications can be explored in fields such as microbiology, human genetics, basic genome research, microbiome studies, and clinical and animal research (Norris, Workman, Fan, Eshleman, & Timp, 2016; Schmidt et al, 2017). As a case of study, we present the first complete mitochondrial genome assembled using ONT-only data of the South American sigmodontine rodent of the genus Melanomys and evaluate an assembly-free method for its molecular identification
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