Dataset Information

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.

SUBMITTER: Franco-Sierra ND

PROVIDER: S-EPMC7141017 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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

Franco-Sierra Nicolás D ND Díaz-Nieto Juan F JF

Ecology and evolution 20200311 7

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 l ...[more]

PMID: 32274008

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Project description:BackgroundHepatitis C (HCV) and many other RNA viruses exist as rapidly mutating quasi-species populations in a single infected host. High throughput characterization of full genome, within-host variants is still not possible despite advances in next generation sequencing. This limitation constrains viral genomic studies that depend on accurate identification of hemi-genome or whole genome, within-host variants, especially those occurring at low frequencies. With the advent of third generation long read sequencing technologies, including Oxford Nanopore Technology (ONT) and PacBio platforms, this problem is potentially surmountable. ONT is particularly attractive in this regard due to the portable nature of the MinION sequencer, which makes real-time sequencing in remote and resource-limited locations possible. However, this technology (termed here 'nanopore sequencing') has a comparatively high technical error rate. The present study aimed to assess the utility, accuracy and cost-effectiveness of nanopore sequencing for HCV genomes. We also introduce a new bioinformatics tool (Nano-Q) to differentiate within-host variants from nanopore sequencing.ResultsThe Nanopore platform, when the coverage exceeded 300 reads, generated comparable consensus sequences to Illumina sequencing. Using HCV Envelope plasmids (~ 1800 nt) mixed in known proportions, the capacity of nanopore sequencing to reliably identify variants with an abundance as low as 0.1% was demonstrated, provided the autologous reference sequence was available to identify the matching reads. Successful pooling and nanopore sequencing of 52 samples from patients with HCV infection demonstrated its cost effectiveness (AUD$ 43 per sample with nanopore sequencing versus $100 with paired-end short read technology). The Nano-Q tool successfully separated between-host sequences, including those from the same subtype, by bulk sorting and phylogenetic clustering without an autologous reference sequence (using only a subtype-specific generic reference). The pipeline also identified within-host viral variants and their abundance when the parameters were appropriately adjusted.ConclusionCost effective HCV whole genome sequencing and within-host variant identification without haplotype reconstruction are potential advantages of nanopore sequencing.

Dataset Information

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

Publications

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

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