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GoFish: A versatile nested PCR strategy for environmental DNA assays for marine vertebrates

Here we describe GoFish, a strategy for single-species environmental DNA (eDNA) presence/absence assays using nested PCR. The assays amplify a mitochondrial 12S rDNA segment with vertebrate metabarcoding primers, followed by nested PCR with M13-tailed, species-specific primers. Sanger sequencing con...

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Autores principales: Stoeckle, Mark Y., Das Mishu, Mithun, Charlop-Powers, Zachary
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6289459/
https://www.ncbi.nlm.nih.gov/pubmed/30533051
http://dx.doi.org/10.1371/journal.pone.0198717
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author Stoeckle, Mark Y.
Das Mishu, Mithun
Charlop-Powers, Zachary
author_facet Stoeckle, Mark Y.
Das Mishu, Mithun
Charlop-Powers, Zachary
author_sort Stoeckle, Mark Y.
collection PubMed
description Here we describe GoFish, a strategy for single-species environmental DNA (eDNA) presence/absence assays using nested PCR. The assays amplify a mitochondrial 12S rDNA segment with vertebrate metabarcoding primers, followed by nested PCR with M13-tailed, species-specific primers. Sanger sequencing confirms positives detected by gel electrophoresis. We first obtained 12S sequences from 77 fish specimens for 36 northwestern Atlantic taxa not well documented in GenBank. Using these and existing 12S records, we designed GoFish assays for 11 bony fish species common in the lower Hudson River estuary and tested seasonal abundance and habitat preference at two sites. Additional assays detected nine cartilaginous fish species and a marine mammal, bottlenose dolphin, in southern New York Bight. GoFish sensitivity was equivalent to Illumina MiSeq metabarcoding. Unlike quantitative PCR (qPCR), GoFish does not require tissues of target and related species for assay development and a basic thermal cycler is sufficient. Unlike Illumina metabarcoding, indexing and batching samples are unnecessary and advanced bioinformatics expertise is not needed. From water collection to Sanger sequencing results, the assay can be carried out in three days. The main limitations to this approach, which employs metabarcoding primers, are the same as for metabarcoding, namely, inability to distinguish species with shared target sequences and inconsistent amplification of rarer eDNA. In addition, the performance of the 20 assays reported here as compared to other single-species eDNA assays is not known. This approach will be a useful addition to current eDNA methods when analyzing presence/absence of known species, when turnaround time is important, and in educational settings.
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spelling pubmed-62894592018-12-28 GoFish: A versatile nested PCR strategy for environmental DNA assays for marine vertebrates Stoeckle, Mark Y. Das Mishu, Mithun Charlop-Powers, Zachary PLoS One Research Article Here we describe GoFish, a strategy for single-species environmental DNA (eDNA) presence/absence assays using nested PCR. The assays amplify a mitochondrial 12S rDNA segment with vertebrate metabarcoding primers, followed by nested PCR with M13-tailed, species-specific primers. Sanger sequencing confirms positives detected by gel electrophoresis. We first obtained 12S sequences from 77 fish specimens for 36 northwestern Atlantic taxa not well documented in GenBank. Using these and existing 12S records, we designed GoFish assays for 11 bony fish species common in the lower Hudson River estuary and tested seasonal abundance and habitat preference at two sites. Additional assays detected nine cartilaginous fish species and a marine mammal, bottlenose dolphin, in southern New York Bight. GoFish sensitivity was equivalent to Illumina MiSeq metabarcoding. Unlike quantitative PCR (qPCR), GoFish does not require tissues of target and related species for assay development and a basic thermal cycler is sufficient. Unlike Illumina metabarcoding, indexing and batching samples are unnecessary and advanced bioinformatics expertise is not needed. From water collection to Sanger sequencing results, the assay can be carried out in three days. The main limitations to this approach, which employs metabarcoding primers, are the same as for metabarcoding, namely, inability to distinguish species with shared target sequences and inconsistent amplification of rarer eDNA. In addition, the performance of the 20 assays reported here as compared to other single-species eDNA assays is not known. This approach will be a useful addition to current eDNA methods when analyzing presence/absence of known species, when turnaround time is important, and in educational settings. Public Library of Science 2018-12-11 /pmc/articles/PMC6289459/ /pubmed/30533051 http://dx.doi.org/10.1371/journal.pone.0198717 Text en © 2018 Stoeckle et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Stoeckle, Mark Y.
Das Mishu, Mithun
Charlop-Powers, Zachary
GoFish: A versatile nested PCR strategy for environmental DNA assays for marine vertebrates
title GoFish: A versatile nested PCR strategy for environmental DNA assays for marine vertebrates
title_full GoFish: A versatile nested PCR strategy for environmental DNA assays for marine vertebrates
title_fullStr GoFish: A versatile nested PCR strategy for environmental DNA assays for marine vertebrates
title_full_unstemmed GoFish: A versatile nested PCR strategy for environmental DNA assays for marine vertebrates
title_short GoFish: A versatile nested PCR strategy for environmental DNA assays for marine vertebrates
title_sort gofish: a versatile nested pcr strategy for environmental dna assays for marine vertebrates
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6289459/
https://www.ncbi.nlm.nih.gov/pubmed/30533051
http://dx.doi.org/10.1371/journal.pone.0198717
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