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Data from: Affordable de novo generation of fish mitogenomes using amplification-free enrichment of mitochondrial DNA and deep sequencing of long fragments

Cite this dataset

Ramon-Laca, Ana (2024). Data from: Affordable de novo generation of fish mitogenomes using amplification-free enrichment of mitochondrial DNA and deep sequencing of long fragments [Dataset]. Dryad. https://doi.org/10.5061/dryad.jm63xsjdj

Abstract

Biomonitoring surveys from environmental DNA make use of metabarcoding tools to describe the community composition. These studies match their sequencing results against public genomic databases to identify the species. However, mitochondrial genomic reference data are yet incomplete, only a few genes may be available, or the suitability of existing sequence data is suboptimal for species-level resolution. Here we present a dedicated and cost-effective workflow with no DNA amplification for generating complete fish mitogenomes for the purpose of strengthening fish mitochondrial databases. Two different long-fragment sequencing approaches using Oxford Nanopore sequencing coupled with mitochondrial DNA enrichment were used. One where the enrichment is achieved by preferential isolation of mitochondria followed by DNA extraction and nuclear DNA depletion (‘mitoenrichment’).  A second enrichment approach takes advantage of the CRISPR-Cas9 targeted scission on previously dephosphorylated DNA (‘targeted mitosequencing’). The sequencing results varied between tissue, species, and integrity of the DNA. The mitoenrichment method yielded 0.17-12.33 % of sequences on target and a mean coverage ranging from 74.9 to 805-fold. The targeted mitosequencing experiment from native genomic DNA yielded 1.83-55 % of sequences on target and a 38 to 2123-fold mean coverage. This produced complete the mitogenome of species with homopolymeric regions, tandem repeats, and gene rearrangements. We demonstrate that deep sequencing of long fragments of native fish DNA is possible and can be achieved with low computational resources in a cost-effective manner, opening the discovery of mitogenomes of non-model or understudied fish taxa to a broad range of laboratories worldwide.

README: Data from: Affordable de novo generation of fish mitogenomes using amplification-free enrichment of mitochondrial DNA and deep sequencing of long fragments

https://doi.org/10.5061/dryad.jm63xsjdj

Description of the data and file structure

Article: Affordable de novo generation of fish mitogenomes using amplification-free enrichment of mitochondrial DNA and deep sequencing of long fragments

Authors: Ana Ramón-Laca, Ramón Gallego and Krista M. Nichols

These are the raw data fast5 files that passed filter (Q>9) from fish mitogenome sequencing MinION runs using R9.4 and R10.3 flow cells for the following species:

  • Oncorhynchus tshawytscha
  • Merluccius productus
  • Thaleichthys pacificus
  • Tarletonbeania crenularis
  • Diaphus theta
  • Stenobrachius leucopsarus

The .fast5 files can be basecalled with Guppy or latest Dorado versions of https://github.com/nanoporetech

Funding

NOAA National Marine Fisheries Service