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CaptureSeq: Hybridization-Based Enrichment of cpn60 Gene Fragments Reveals the Community Structures of Synthetic and Natural Microbial Ecosystems

Background. The molecular profiling of complex microbial communities has become the basis for examining the relationship between the microbiome composition, structure and metabolic functions of those communities. Microbial community structure can be partially assessed with “universal” PCR targeting...

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Autores principales: Links, Matthew G., Dumonceaux, Tim J., McCarthy, E. Luke, Hemmingsen, Sean M., Topp, Edward, Town, Jennifer R.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8069376/
https://www.ncbi.nlm.nih.gov/pubmed/33924343
http://dx.doi.org/10.3390/microorganisms9040816
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author Links, Matthew G.
Dumonceaux, Tim J.
McCarthy, E. Luke
Hemmingsen, Sean M.
Topp, Edward
Town, Jennifer R.
author_facet Links, Matthew G.
Dumonceaux, Tim J.
McCarthy, E. Luke
Hemmingsen, Sean M.
Topp, Edward
Town, Jennifer R.
author_sort Links, Matthew G.
collection PubMed
description Background. The molecular profiling of complex microbial communities has become the basis for examining the relationship between the microbiome composition, structure and metabolic functions of those communities. Microbial community structure can be partially assessed with “universal” PCR targeting taxonomic or functional gene markers. Increasingly, shotgun metagenomic DNA sequencing is providing more quantitative insight into microbiomes. However, both amplicon-based and shotgun sequencing approaches have shortcomings that limit the ability to study microbiome dynamics. Methods. We present a novel, amplicon-free, hybridization-based method (CaptureSeq) for profiling complex microbial communities using probes based on the chaperonin-60 gene. Molecular profiles of a commercially available synthetic microbial community standard were compared using CaptureSeq, whole metagenome sequencing, and 16S universal target amplification. Profiles were also generated for natural ecosystems including antibiotic-amended soils, manure storage tanks, and an agricultural reservoir. Results. The CaptureSeq method generated a microbial profile that encompassed all of the bacteria and eukaryotes in the panel with greater reproducibility and more accurate representation of high G/C content microorganisms compared to 16S amplification. In the natural ecosystems, CaptureSeq provided a much greater depth of coverage and sensitivity of detection compared to shotgun sequencing without prior selection. The resulting community profiles provided quantitatively reliable information about all three domains of life (Bacteria, Archaea, and Eukarya) in the different ecosystems. The applications of CaptureSeq will facilitate accurate studies of host-microbiome interactions for environmental, crop, animal and human health. Conclusions: cpn60-based hybridization enriched for taxonomically informative DNA sequences from complex mixtures. In synthetic and natural microbial ecosystems, CaptureSeq provided sequences from prokaryotes and eukaryotes simultaneously, with quantitatively reliable read abundances. CaptureSeq provides an alternative to PCR amplification of taxonomic markers with deep community coverage while minimizing amplification biases.
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spelling pubmed-80693762021-04-26 CaptureSeq: Hybridization-Based Enrichment of cpn60 Gene Fragments Reveals the Community Structures of Synthetic and Natural Microbial Ecosystems Links, Matthew G. Dumonceaux, Tim J. McCarthy, E. Luke Hemmingsen, Sean M. Topp, Edward Town, Jennifer R. Microorganisms Article Background. The molecular profiling of complex microbial communities has become the basis for examining the relationship between the microbiome composition, structure and metabolic functions of those communities. Microbial community structure can be partially assessed with “universal” PCR targeting taxonomic or functional gene markers. Increasingly, shotgun metagenomic DNA sequencing is providing more quantitative insight into microbiomes. However, both amplicon-based and shotgun sequencing approaches have shortcomings that limit the ability to study microbiome dynamics. Methods. We present a novel, amplicon-free, hybridization-based method (CaptureSeq) for profiling complex microbial communities using probes based on the chaperonin-60 gene. Molecular profiles of a commercially available synthetic microbial community standard were compared using CaptureSeq, whole metagenome sequencing, and 16S universal target amplification. Profiles were also generated for natural ecosystems including antibiotic-amended soils, manure storage tanks, and an agricultural reservoir. Results. The CaptureSeq method generated a microbial profile that encompassed all of the bacteria and eukaryotes in the panel with greater reproducibility and more accurate representation of high G/C content microorganisms compared to 16S amplification. In the natural ecosystems, CaptureSeq provided a much greater depth of coverage and sensitivity of detection compared to shotgun sequencing without prior selection. The resulting community profiles provided quantitatively reliable information about all three domains of life (Bacteria, Archaea, and Eukarya) in the different ecosystems. The applications of CaptureSeq will facilitate accurate studies of host-microbiome interactions for environmental, crop, animal and human health. Conclusions: cpn60-based hybridization enriched for taxonomically informative DNA sequences from complex mixtures. In synthetic and natural microbial ecosystems, CaptureSeq provided sequences from prokaryotes and eukaryotes simultaneously, with quantitatively reliable read abundances. CaptureSeq provides an alternative to PCR amplification of taxonomic markers with deep community coverage while minimizing amplification biases. MDPI 2021-04-13 /pmc/articles/PMC8069376/ /pubmed/33924343 http://dx.doi.org/10.3390/microorganisms9040816 Text en © 2021 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
Links, Matthew G.
Dumonceaux, Tim J.
McCarthy, E. Luke
Hemmingsen, Sean M.
Topp, Edward
Town, Jennifer R.
CaptureSeq: Hybridization-Based Enrichment of cpn60 Gene Fragments Reveals the Community Structures of Synthetic and Natural Microbial Ecosystems
title CaptureSeq: Hybridization-Based Enrichment of cpn60 Gene Fragments Reveals the Community Structures of Synthetic and Natural Microbial Ecosystems
title_full CaptureSeq: Hybridization-Based Enrichment of cpn60 Gene Fragments Reveals the Community Structures of Synthetic and Natural Microbial Ecosystems
title_fullStr CaptureSeq: Hybridization-Based Enrichment of cpn60 Gene Fragments Reveals the Community Structures of Synthetic and Natural Microbial Ecosystems
title_full_unstemmed CaptureSeq: Hybridization-Based Enrichment of cpn60 Gene Fragments Reveals the Community Structures of Synthetic and Natural Microbial Ecosystems
title_short CaptureSeq: Hybridization-Based Enrichment of cpn60 Gene Fragments Reveals the Community Structures of Synthetic and Natural Microbial Ecosystems
title_sort captureseq: hybridization-based enrichment of cpn60 gene fragments reveals the community structures of synthetic and natural microbial ecosystems
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8069376/
https://www.ncbi.nlm.nih.gov/pubmed/33924343
http://dx.doi.org/10.3390/microorganisms9040816
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