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Multiplex amplicon sequencing for microbe identification in community-based culture collections

Microbiome analysis using metagenomic sequencing has revealed a vast microbial diversity associated with plants. Identifying the molecular functions associated with microbiome-plant interaction is a significant challenge concerning the development of microbiome-derived technologies applied to agricu...

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Autores principales: Armanhi, Jaderson Silveira Leite, de Souza, Rafael Soares Correa, de Araújo, Laura Migliorini, Okura, Vagner Katsumi, Mieczkowski, Piotr, Imperial, Juan, Arruda, Paulo
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4941570/
https://www.ncbi.nlm.nih.gov/pubmed/27404280
http://dx.doi.org/10.1038/srep29543
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author Armanhi, Jaderson Silveira Leite
de Souza, Rafael Soares Correa
de Araújo, Laura Migliorini
Okura, Vagner Katsumi
Mieczkowski, Piotr
Imperial, Juan
Arruda, Paulo
author_facet Armanhi, Jaderson Silveira Leite
de Souza, Rafael Soares Correa
de Araújo, Laura Migliorini
Okura, Vagner Katsumi
Mieczkowski, Piotr
Imperial, Juan
Arruda, Paulo
author_sort Armanhi, Jaderson Silveira Leite
collection PubMed
description Microbiome analysis using metagenomic sequencing has revealed a vast microbial diversity associated with plants. Identifying the molecular functions associated with microbiome-plant interaction is a significant challenge concerning the development of microbiome-derived technologies applied to agriculture. An alternative to accelerate the discovery of the microbiome benefits to plants is to construct microbial culture collections concomitant with accessing microbial community structure and abundance. However, traditional methods of isolation, cultivation, and identification of microbes are time-consuming and expensive. Here we describe a method for identification of microbes in culture collections constructed by picking colonies from primary platings that may contain single or multiple microorganisms, which we named community-based culture collections (CBC). A multiplexing 16S rRNA gene amplicon sequencing based on two-step PCR amplifications with tagged primers for plates, rows, and columns allowed the identification of the microbial composition regardless if the well contains single or multiple microorganisms. The multiplexing system enables pooling amplicons into a single tube. The sequencing performed on the PacBio platform led to recovery near-full-length 16S rRNA gene sequences allowing accurate identification of microorganism composition in each plate well. Cross-referencing with plant microbiome structure and abundance allowed the estimation of diversity and abundance representation of microorganism in the CBC.
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spelling pubmed-49415702016-07-20 Multiplex amplicon sequencing for microbe identification in community-based culture collections Armanhi, Jaderson Silveira Leite de Souza, Rafael Soares Correa de Araújo, Laura Migliorini Okura, Vagner Katsumi Mieczkowski, Piotr Imperial, Juan Arruda, Paulo Sci Rep Article Microbiome analysis using metagenomic sequencing has revealed a vast microbial diversity associated with plants. Identifying the molecular functions associated with microbiome-plant interaction is a significant challenge concerning the development of microbiome-derived technologies applied to agriculture. An alternative to accelerate the discovery of the microbiome benefits to plants is to construct microbial culture collections concomitant with accessing microbial community structure and abundance. However, traditional methods of isolation, cultivation, and identification of microbes are time-consuming and expensive. Here we describe a method for identification of microbes in culture collections constructed by picking colonies from primary platings that may contain single or multiple microorganisms, which we named community-based culture collections (CBC). A multiplexing 16S rRNA gene amplicon sequencing based on two-step PCR amplifications with tagged primers for plates, rows, and columns allowed the identification of the microbial composition regardless if the well contains single or multiple microorganisms. The multiplexing system enables pooling amplicons into a single tube. The sequencing performed on the PacBio platform led to recovery near-full-length 16S rRNA gene sequences allowing accurate identification of microorganism composition in each plate well. Cross-referencing with plant microbiome structure and abundance allowed the estimation of diversity and abundance representation of microorganism in the CBC. Nature Publishing Group 2016-07-12 /pmc/articles/PMC4941570/ /pubmed/27404280 http://dx.doi.org/10.1038/srep29543 Text en Copyright © 2016, Macmillan Publishers Limited http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
spellingShingle Article
Armanhi, Jaderson Silveira Leite
de Souza, Rafael Soares Correa
de Araújo, Laura Migliorini
Okura, Vagner Katsumi
Mieczkowski, Piotr
Imperial, Juan
Arruda, Paulo
Multiplex amplicon sequencing for microbe identification in community-based culture collections
title Multiplex amplicon sequencing for microbe identification in community-based culture collections
title_full Multiplex amplicon sequencing for microbe identification in community-based culture collections
title_fullStr Multiplex amplicon sequencing for microbe identification in community-based culture collections
title_full_unstemmed Multiplex amplicon sequencing for microbe identification in community-based culture collections
title_short Multiplex amplicon sequencing for microbe identification in community-based culture collections
title_sort multiplex amplicon sequencing for microbe identification in community-based culture collections
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4941570/
https://www.ncbi.nlm.nih.gov/pubmed/27404280
http://dx.doi.org/10.1038/srep29543
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