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Effectiveness of blocking primers and a peptide nucleic acid (PNA) clamp for 18S metabarcoding dietary analysis of herbivorous fish

The structure of food webs and carbon flow in aquatic ecosystems can be better understood by studying contributing factors such as the diets of herbivorous fish. Metabarcoding using a high-throughput sequencer has recently been used to clarify prey organisms of various fish except herbivorous fish....

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Autores principales: Homma, Chiho, Inokuchi, Daiki, Nakamura, Yohei, Uy, Wilfredo H., Ohnishi, Kouhei, Yamaguchi, Haruo, Adachi, Masao
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9020718/
https://www.ncbi.nlm.nih.gov/pubmed/35442965
http://dx.doi.org/10.1371/journal.pone.0266268
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author Homma, Chiho
Inokuchi, Daiki
Nakamura, Yohei
Uy, Wilfredo H.
Ohnishi, Kouhei
Yamaguchi, Haruo
Adachi, Masao
author_facet Homma, Chiho
Inokuchi, Daiki
Nakamura, Yohei
Uy, Wilfredo H.
Ohnishi, Kouhei
Yamaguchi, Haruo
Adachi, Masao
author_sort Homma, Chiho
collection PubMed
description The structure of food webs and carbon flow in aquatic ecosystems can be better understood by studying contributing factors such as the diets of herbivorous fish. Metabarcoding using a high-throughput sequencer has recently been used to clarify prey organisms of various fish except herbivorous fish. Since sequences of predator fish have dominated in sequences obtained by metabarcoding, we investigated a method for suppressing the amplification of fish DNA by using a blocking primer or peptide nucleic acid (PNA) clamp to determine the prey organisms of herbivorous fish. We designed three blocking primers and one PNA clamp that anneal to fish-specific sequences and examined how efficient they were in suppressing DNA amplification in various herbivorous fish. The results showed that the PNA clamp completely suppressed fish DNA amplification, and one of the blocking primers suppressed fish DNA amplification but less efficiently than the PNA clamp. Finally, we conducted metabarcoding using mock community samples as templates to determine whether the blocking primer or the PNA clamp was effective in suppressing fish DNA amplification. The results showed that the PNA clamp suppressed 99.3%–99.9% of fish DNA amplification, whereas the blocking primer suppressed 3.3%–32.9%. Therefore, we propose the application of the PNA clamp for clarifying the prey organisms and food preferences of various herbivorous fish.
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spelling pubmed-90207182022-04-21 Effectiveness of blocking primers and a peptide nucleic acid (PNA) clamp for 18S metabarcoding dietary analysis of herbivorous fish Homma, Chiho Inokuchi, Daiki Nakamura, Yohei Uy, Wilfredo H. Ohnishi, Kouhei Yamaguchi, Haruo Adachi, Masao PLoS One Research Article The structure of food webs and carbon flow in aquatic ecosystems can be better understood by studying contributing factors such as the diets of herbivorous fish. Metabarcoding using a high-throughput sequencer has recently been used to clarify prey organisms of various fish except herbivorous fish. Since sequences of predator fish have dominated in sequences obtained by metabarcoding, we investigated a method for suppressing the amplification of fish DNA by using a blocking primer or peptide nucleic acid (PNA) clamp to determine the prey organisms of herbivorous fish. We designed three blocking primers and one PNA clamp that anneal to fish-specific sequences and examined how efficient they were in suppressing DNA amplification in various herbivorous fish. The results showed that the PNA clamp completely suppressed fish DNA amplification, and one of the blocking primers suppressed fish DNA amplification but less efficiently than the PNA clamp. Finally, we conducted metabarcoding using mock community samples as templates to determine whether the blocking primer or the PNA clamp was effective in suppressing fish DNA amplification. The results showed that the PNA clamp suppressed 99.3%–99.9% of fish DNA amplification, whereas the blocking primer suppressed 3.3%–32.9%. Therefore, we propose the application of the PNA clamp for clarifying the prey organisms and food preferences of various herbivorous fish. Public Library of Science 2022-04-20 /pmc/articles/PMC9020718/ /pubmed/35442965 http://dx.doi.org/10.1371/journal.pone.0266268 Text en © 2022 Homma et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://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
Homma, Chiho
Inokuchi, Daiki
Nakamura, Yohei
Uy, Wilfredo H.
Ohnishi, Kouhei
Yamaguchi, Haruo
Adachi, Masao
Effectiveness of blocking primers and a peptide nucleic acid (PNA) clamp for 18S metabarcoding dietary analysis of herbivorous fish
title Effectiveness of blocking primers and a peptide nucleic acid (PNA) clamp for 18S metabarcoding dietary analysis of herbivorous fish
title_full Effectiveness of blocking primers and a peptide nucleic acid (PNA) clamp for 18S metabarcoding dietary analysis of herbivorous fish
title_fullStr Effectiveness of blocking primers and a peptide nucleic acid (PNA) clamp for 18S metabarcoding dietary analysis of herbivorous fish
title_full_unstemmed Effectiveness of blocking primers and a peptide nucleic acid (PNA) clamp for 18S metabarcoding dietary analysis of herbivorous fish
title_short Effectiveness of blocking primers and a peptide nucleic acid (PNA) clamp for 18S metabarcoding dietary analysis of herbivorous fish
title_sort effectiveness of blocking primers and a peptide nucleic acid (pna) clamp for 18s metabarcoding dietary analysis of herbivorous fish
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9020718/
https://www.ncbi.nlm.nih.gov/pubmed/35442965
http://dx.doi.org/10.1371/journal.pone.0266268
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