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Selecting 16S rRNA Primers for Microbiome Analysis in a Host–Microbe System: The Case of the Jellyfish Rhopilema nomadica

Amplicon sequencing of the 16S rRNA gene is extensively used to characterize bacterial communities, including those living in association with eukaryotic hosts. Deciding which region of the 16S rRNA gene to analyze and selecting the appropriate PCR primers remains a major decision when initiating an...

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Autores principales: Barak, Noga, Fadeev, Eduard, Brekhman, Vera, Aharonovich, Dikla, Lotan, Tamar, Sher, Daniel
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10144005/
https://www.ncbi.nlm.nih.gov/pubmed/37110378
http://dx.doi.org/10.3390/microorganisms11040955
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author Barak, Noga
Fadeev, Eduard
Brekhman, Vera
Aharonovich, Dikla
Lotan, Tamar
Sher, Daniel
author_facet Barak, Noga
Fadeev, Eduard
Brekhman, Vera
Aharonovich, Dikla
Lotan, Tamar
Sher, Daniel
author_sort Barak, Noga
collection PubMed
description Amplicon sequencing of the 16S rRNA gene is extensively used to characterize bacterial communities, including those living in association with eukaryotic hosts. Deciding which region of the 16S rRNA gene to analyze and selecting the appropriate PCR primers remains a major decision when initiating any new microbiome study. Based on a detailed literature survey of studies focusing on cnidarian microbiomes, we compared three commonly used primers targeting different hypervariable regions of the 16S rRNA gene, V1V2, V3V4, and V4V5, using the jellyfish Rhopilema nomadica as a model. Although all primers exhibit a similar pattern in bacterial community composition, the performance of the V3V4 primer set was superior to V1V2 and V4V5. The V1V2 primers misclassified bacteria from the Bacilli class and exhibited low classification resolution for Rickettsiales, which represent the second most abundant 16S rRNA gene sequence in all the primers. The V4V5 primer set detected almost the same community composition as the V3V4, but the ability of these primers to also amplify the eukaryotic 18S rRNA gene may hinder bacterial community observations. However, after overcoming the challenges possessed by each one of those primers, we found that all three of them show very similar bacterial community dynamics and compositions. Nevertheless, based on our results, we propose that the V3V4 primer set is potentially the most suitable for studying jellyfish-associated bacterial communities. Our results suggest that, at least for jellyfish samples, it may be feasible to directly compare microbial community estimates from different studies, each using different primers but otherwise similar experimental protocols. More generally, we recommend specifically testing different primers for each new organism or system as a prelude to large-scale 16S rRNA gene amplicon analyses, especially of previously unstudied host–microbe associations.
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spelling pubmed-101440052023-04-29 Selecting 16S rRNA Primers for Microbiome Analysis in a Host–Microbe System: The Case of the Jellyfish Rhopilema nomadica Barak, Noga Fadeev, Eduard Brekhman, Vera Aharonovich, Dikla Lotan, Tamar Sher, Daniel Microorganisms Article Amplicon sequencing of the 16S rRNA gene is extensively used to characterize bacterial communities, including those living in association with eukaryotic hosts. Deciding which region of the 16S rRNA gene to analyze and selecting the appropriate PCR primers remains a major decision when initiating any new microbiome study. Based on a detailed literature survey of studies focusing on cnidarian microbiomes, we compared three commonly used primers targeting different hypervariable regions of the 16S rRNA gene, V1V2, V3V4, and V4V5, using the jellyfish Rhopilema nomadica as a model. Although all primers exhibit a similar pattern in bacterial community composition, the performance of the V3V4 primer set was superior to V1V2 and V4V5. The V1V2 primers misclassified bacteria from the Bacilli class and exhibited low classification resolution for Rickettsiales, which represent the second most abundant 16S rRNA gene sequence in all the primers. The V4V5 primer set detected almost the same community composition as the V3V4, but the ability of these primers to also amplify the eukaryotic 18S rRNA gene may hinder bacterial community observations. However, after overcoming the challenges possessed by each one of those primers, we found that all three of them show very similar bacterial community dynamics and compositions. Nevertheless, based on our results, we propose that the V3V4 primer set is potentially the most suitable for studying jellyfish-associated bacterial communities. Our results suggest that, at least for jellyfish samples, it may be feasible to directly compare microbial community estimates from different studies, each using different primers but otherwise similar experimental protocols. More generally, we recommend specifically testing different primers for each new organism or system as a prelude to large-scale 16S rRNA gene amplicon analyses, especially of previously unstudied host–microbe associations. MDPI 2023-04-06 /pmc/articles/PMC10144005/ /pubmed/37110378 http://dx.doi.org/10.3390/microorganisms11040955 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Barak, Noga
Fadeev, Eduard
Brekhman, Vera
Aharonovich, Dikla
Lotan, Tamar
Sher, Daniel
Selecting 16S rRNA Primers for Microbiome Analysis in a Host–Microbe System: The Case of the Jellyfish Rhopilema nomadica
title Selecting 16S rRNA Primers for Microbiome Analysis in a Host–Microbe System: The Case of the Jellyfish Rhopilema nomadica
title_full Selecting 16S rRNA Primers for Microbiome Analysis in a Host–Microbe System: The Case of the Jellyfish Rhopilema nomadica
title_fullStr Selecting 16S rRNA Primers for Microbiome Analysis in a Host–Microbe System: The Case of the Jellyfish Rhopilema nomadica
title_full_unstemmed Selecting 16S rRNA Primers for Microbiome Analysis in a Host–Microbe System: The Case of the Jellyfish Rhopilema nomadica
title_short Selecting 16S rRNA Primers for Microbiome Analysis in a Host–Microbe System: The Case of the Jellyfish Rhopilema nomadica
title_sort selecting 16s rrna primers for microbiome analysis in a host–microbe system: the case of the jellyfish rhopilema nomadica
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10144005/
https://www.ncbi.nlm.nih.gov/pubmed/37110378
http://dx.doi.org/10.3390/microorganisms11040955
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