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Printing Microbial Dark Matter: Using Single Cell Dispensing and Genomics to Investigate the Patescibacteria/Candidate Phyla Radiation

As of today, the majority of environmental microorganisms remain uncultured. They are therefore referred to as “microbial dark matter.” In the recent past, cultivation-independent methods like single-cell genomics (SCG) enabled the discovery of many previously unknown microorganisms, among them the...

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Autores principales: Wiegand, Sandra, Dam, Hang T., Riba, Julian, Vollmers, John, Kaster, Anne-Kristin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8241940/
https://www.ncbi.nlm.nih.gov/pubmed/34220732
http://dx.doi.org/10.3389/fmicb.2021.635506
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author Wiegand, Sandra
Dam, Hang T.
Riba, Julian
Vollmers, John
Kaster, Anne-Kristin
author_facet Wiegand, Sandra
Dam, Hang T.
Riba, Julian
Vollmers, John
Kaster, Anne-Kristin
author_sort Wiegand, Sandra
collection PubMed
description As of today, the majority of environmental microorganisms remain uncultured. They are therefore referred to as “microbial dark matter.” In the recent past, cultivation-independent methods like single-cell genomics (SCG) enabled the discovery of many previously unknown microorganisms, among them the Patescibacteria/Candidate Phyla Radiation (CPR). This approach was shown to be complementary to metagenomics, however, the development of additional and refined sorting techniques beyond the most commonly used fluorescence-activated cell sorting (FACS) is still desirable to enable additional downstream applications. Adding image information on the number and morphology of sorted cells would be beneficial, as would be minimizing cell stress caused by sorting conditions such as staining or pressure. Recently, a novel cell sorting technique has been developed, a microfluidic single-cell dispenser, which assesses the number and morphology of the cell in each droplet by automated light microscopic processing. Here, we report for the first time the successful application of the newly developed single-cell dispensing system for label-free isolation of individual bacteria from a complex sample retrieved from a wastewater treatment plant, demonstrating the potential of this technique for single cell genomics and other alternative downstream applications. Genome recovery success rated above 80% with this technique—out of 880 sorted cells 717 were successfully amplified. For 50.1% of these, analysis of the 16S rRNA gene was feasible and led to the sequencing of 50 sorted cells identified as Patescibacteria/CPR members. Subsequentially, 27 single amplified genomes (SAGs) of 15 novel and distinct Patescibacteria/CPR members, representing yet unseen species, genera and families could be captured and reconstructed. This phylogenetic distinctness of the recovered SAGs from available metagenome-assembled genomes (MAGs) is accompanied by the finding that these lineages—in whole or in part—have not been accessed by genome-resolved metagenomics of the same sample, thereby emphasizing the importance and opportunities of SCGs.
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spelling pubmed-82419402021-07-01 Printing Microbial Dark Matter: Using Single Cell Dispensing and Genomics to Investigate the Patescibacteria/Candidate Phyla Radiation Wiegand, Sandra Dam, Hang T. Riba, Julian Vollmers, John Kaster, Anne-Kristin Front Microbiol Microbiology As of today, the majority of environmental microorganisms remain uncultured. They are therefore referred to as “microbial dark matter.” In the recent past, cultivation-independent methods like single-cell genomics (SCG) enabled the discovery of many previously unknown microorganisms, among them the Patescibacteria/Candidate Phyla Radiation (CPR). This approach was shown to be complementary to metagenomics, however, the development of additional and refined sorting techniques beyond the most commonly used fluorescence-activated cell sorting (FACS) is still desirable to enable additional downstream applications. Adding image information on the number and morphology of sorted cells would be beneficial, as would be minimizing cell stress caused by sorting conditions such as staining or pressure. Recently, a novel cell sorting technique has been developed, a microfluidic single-cell dispenser, which assesses the number and morphology of the cell in each droplet by automated light microscopic processing. Here, we report for the first time the successful application of the newly developed single-cell dispensing system for label-free isolation of individual bacteria from a complex sample retrieved from a wastewater treatment plant, demonstrating the potential of this technique for single cell genomics and other alternative downstream applications. Genome recovery success rated above 80% with this technique—out of 880 sorted cells 717 were successfully amplified. For 50.1% of these, analysis of the 16S rRNA gene was feasible and led to the sequencing of 50 sorted cells identified as Patescibacteria/CPR members. Subsequentially, 27 single amplified genomes (SAGs) of 15 novel and distinct Patescibacteria/CPR members, representing yet unseen species, genera and families could be captured and reconstructed. This phylogenetic distinctness of the recovered SAGs from available metagenome-assembled genomes (MAGs) is accompanied by the finding that these lineages—in whole or in part—have not been accessed by genome-resolved metagenomics of the same sample, thereby emphasizing the importance and opportunities of SCGs. Frontiers Media S.A. 2021-06-16 /pmc/articles/PMC8241940/ /pubmed/34220732 http://dx.doi.org/10.3389/fmicb.2021.635506 Text en Copyright © 2021 Wiegand, Dam, Riba, Vollmers and Kaster. 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
Wiegand, Sandra
Dam, Hang T.
Riba, Julian
Vollmers, John
Kaster, Anne-Kristin
Printing Microbial Dark Matter: Using Single Cell Dispensing and Genomics to Investigate the Patescibacteria/Candidate Phyla Radiation
title Printing Microbial Dark Matter: Using Single Cell Dispensing and Genomics to Investigate the Patescibacteria/Candidate Phyla Radiation
title_full Printing Microbial Dark Matter: Using Single Cell Dispensing and Genomics to Investigate the Patescibacteria/Candidate Phyla Radiation
title_fullStr Printing Microbial Dark Matter: Using Single Cell Dispensing and Genomics to Investigate the Patescibacteria/Candidate Phyla Radiation
title_full_unstemmed Printing Microbial Dark Matter: Using Single Cell Dispensing and Genomics to Investigate the Patescibacteria/Candidate Phyla Radiation
title_short Printing Microbial Dark Matter: Using Single Cell Dispensing and Genomics to Investigate the Patescibacteria/Candidate Phyla Radiation
title_sort printing microbial dark matter: using single cell dispensing and genomics to investigate the patescibacteria/candidate phyla radiation
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8241940/
https://www.ncbi.nlm.nih.gov/pubmed/34220732
http://dx.doi.org/10.3389/fmicb.2021.635506
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