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Molecular Ecology Resources
Article . 2023 . Peer-reviewed
License: CC BY NC ND
Data sources: Crossref
https://doi.org/10.22541/au.16...
Article . 2022 . Peer-reviewed
Data sources: Crossref
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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; Krista M. Nichols;

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

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.

Country
Spain
Keywords

Long fragment, High-Throughput Nucleotide Sequencing, Cas9 targeted sequencing, Sequence Analysis, DNA, DNA, Mitochondrial, Mitochondria, Mitogenome, Fish, Mitochondrial enrichment, Genome, Mitochondrial, Animals, eDNA

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selected citations
These citations are derived from selected sources.
This is an alternative to the "Influence" indicator, which also reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Citations provided by BIP!
popularity
This indicator reflects the "current" impact/attention (the "hype") of an article in the research community at large, based on the underlying citation network.
BIP!Popularity provided by BIP!
influence
This indicator reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Influence provided by BIP!
impulse
This indicator reflects the initial momentum of an article directly after its publication, based on the underlying citation network.
BIP!Impulse provided by BIP!
views
OpenAIRE UsageCountsViews provided by UsageCounts
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12
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