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Affordable de novo generation of fish mitogenomes using amplification-free enrichment of mitochondrial DNA and deep sequencing of long fragments
  • Ana Ramon-Laca,
  • Ramon Gallego,
  • Krista Nichols
Ana Ramon-Laca
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Corresponding Author:analaca@gmail.com

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Ramon Gallego
NOAA Fisheries Northwest Fisheries Science Center
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Krista Nichols
NOAA Fisheries Northwest Fisheries Science Center
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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 74.9-805-fold. The targeted mitosequencing experiment from native genomic DNA yielded 1.83-55 % of sequences on target and a 38-2123-fold mean coverage. This helped 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, can be achieved with low computational resources in a cost-effective manner, exceeding the widespread genome skimming approach, and allowing the discovery of mitogenomes of non-model or understudied fish taxa to a broad range of laboratories worldwide.
25 May 2022Submitted to Molecular Ecology Resources
09 Jun 2022Submission Checks Completed
09 Jun 2022Assigned to Editor
14 Jun 2022Reviewer(s) Assigned
07 Dec 2022Review(s) Completed, Editorial Evaluation Pending
19 Dec 2022Editorial Decision: Revise Minor
11 Jan 2023Review(s) Completed, Editorial Evaluation Pending
11 Jan 20231st Revision Received
20 Jan 2023Editorial Decision: Accept