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Microbial dark matter sequences verification in amplicon sequencing and environmental metagenomics data

Although microorganisms constitute the most diverse and abundant life form on Earth, in many environments, the vast majority of them remain uncultured. As it is based on information gleaned mainly from cultivated microorganisms, our current body of knowledge regarding microbial life is partial and d...

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Autores principales: Barak, Hana, Fuchs, Naomi, Liddor-Naim, Michal, Nir, Irit, Sivan, Alex, Kushmaro, Ariel
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10656735/
https://www.ncbi.nlm.nih.gov/pubmed/38029171
http://dx.doi.org/10.3389/fmicb.2023.1247119
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author Barak, Hana
Fuchs, Naomi
Liddor-Naim, Michal
Nir, Irit
Sivan, Alex
Kushmaro, Ariel
author_facet Barak, Hana
Fuchs, Naomi
Liddor-Naim, Michal
Nir, Irit
Sivan, Alex
Kushmaro, Ariel
author_sort Barak, Hana
collection PubMed
description Although microorganisms constitute the most diverse and abundant life form on Earth, in many environments, the vast majority of them remain uncultured. As it is based on information gleaned mainly from cultivated microorganisms, our current body of knowledge regarding microbial life is partial and does not reflect actual microbial diversity. That diversity is hidden in the uncultured microbial majority, termed by microbiologists as “microbial dark matter” (MDM), a term borrowed from astrophysics. Metagenomic sequencing analysis techniques (both 16S rRNA gene and shotgun sequencing) compare gene sequences to reference databases, each of which represents only a small fraction of the existing microorganisms. Unaligned sequences lead to groups of “unknown microorganisms” that are usually ignored and rarefied from diversity analysis. To address this knowledge gap, we analyzed the 16S rRNA gene sequences of microbial communities from four different environments—a living organism, a desert environment, a natural aquatic environment, and a membrane bioreactor for wastewater treatment. From those datasets, we chose representative sequences of potentially unknown bacteria for additional examination as “microbial dark matter sequences” (MDMS). Sequence existence was validated by specific amplification and re-sequencing. These sequences were screened against databases and aligned to the Genome Taxonomy Database to build a comprehensive phylogenetic tree for additional sequence classification, revealing potentially new candidate phyla and other lineages. These putative MDMS were also screened against metagenome-assembled genomes from the explored environments for additional validation and for taxonomic and metabolic characterizations. This study shows the immense importance of MDMS in environmental metataxonomic analyses of 16S rRNA gene sequences and provides a simple and readily available methodology for the examination of MDM hidden behind amplicon sequencing results.
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spelling pubmed-106567352023-11-02 Microbial dark matter sequences verification in amplicon sequencing and environmental metagenomics data Barak, Hana Fuchs, Naomi Liddor-Naim, Michal Nir, Irit Sivan, Alex Kushmaro, Ariel Front Microbiol Microbiology Although microorganisms constitute the most diverse and abundant life form on Earth, in many environments, the vast majority of them remain uncultured. As it is based on information gleaned mainly from cultivated microorganisms, our current body of knowledge regarding microbial life is partial and does not reflect actual microbial diversity. That diversity is hidden in the uncultured microbial majority, termed by microbiologists as “microbial dark matter” (MDM), a term borrowed from astrophysics. Metagenomic sequencing analysis techniques (both 16S rRNA gene and shotgun sequencing) compare gene sequences to reference databases, each of which represents only a small fraction of the existing microorganisms. Unaligned sequences lead to groups of “unknown microorganisms” that are usually ignored and rarefied from diversity analysis. To address this knowledge gap, we analyzed the 16S rRNA gene sequences of microbial communities from four different environments—a living organism, a desert environment, a natural aquatic environment, and a membrane bioreactor for wastewater treatment. From those datasets, we chose representative sequences of potentially unknown bacteria for additional examination as “microbial dark matter sequences” (MDMS). Sequence existence was validated by specific amplification and re-sequencing. These sequences were screened against databases and aligned to the Genome Taxonomy Database to build a comprehensive phylogenetic tree for additional sequence classification, revealing potentially new candidate phyla and other lineages. These putative MDMS were also screened against metagenome-assembled genomes from the explored environments for additional validation and for taxonomic and metabolic characterizations. This study shows the immense importance of MDMS in environmental metataxonomic analyses of 16S rRNA gene sequences and provides a simple and readily available methodology for the examination of MDM hidden behind amplicon sequencing results. Frontiers Media S.A. 2023-11-02 /pmc/articles/PMC10656735/ /pubmed/38029171 http://dx.doi.org/10.3389/fmicb.2023.1247119 Text en Copyright © 2023 Barak, Fuchs, Liddor-Naim, Nir, Sivan and Kushmaro. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Microbiology
Barak, Hana
Fuchs, Naomi
Liddor-Naim, Michal
Nir, Irit
Sivan, Alex
Kushmaro, Ariel
Microbial dark matter sequences verification in amplicon sequencing and environmental metagenomics data
title Microbial dark matter sequences verification in amplicon sequencing and environmental metagenomics data
title_full Microbial dark matter sequences verification in amplicon sequencing and environmental metagenomics data
title_fullStr Microbial dark matter sequences verification in amplicon sequencing and environmental metagenomics data
title_full_unstemmed Microbial dark matter sequences verification in amplicon sequencing and environmental metagenomics data
title_short Microbial dark matter sequences verification in amplicon sequencing and environmental metagenomics data
title_sort microbial dark matter sequences verification in amplicon sequencing and environmental metagenomics data
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10656735/
https://www.ncbi.nlm.nih.gov/pubmed/38029171
http://dx.doi.org/10.3389/fmicb.2023.1247119
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