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Title: A pipeline for targeted metagenomics of environmental bacteria

Abstract

Background:Metagenomics and single cell genomics provide a window into the genetic repertoire of yet uncultivated microorganisms, but both methods are usually taxonomically untargeted. The combination of fluorescence in situ hybridization (FISH) and fluorescence activated cell sorting (FACS) has the potential to enrich taxonomically well-defined clades for genomic analyses. Methods:Cells hybridized with a taxon-specific FISH probe are enriched based on their fluorescence signal via flow cytometric cell sorting. A recently developed FISH procedure, the hybridization chain reaction (HCR)-FISH, provides the high signal intensities required for flow cytometric sorting while maintaining the integrity of the cellular DNA for subsequent genome sequencing. Sorted cells are subjected to shotgun sequencing, resulting in targeted metagenomes of low diversity. Results: Pure cultures of different taxonomic groups were used to (1) adapt and optimize the HCR-FISH protocol and (2) assess the effects of various cell fixation methods on both the signal intensity for cell sorting and the quality of subsequent genome amplification and sequencing. Best results were obtained for ethanol-fixed cells in terms of both HCR-FISH signal intensity and genome assembly quality. Our newly developed pipeline was successfully applied to a marine plankton sample from the North Sea yielding good quality metagenome assembled genomes from a yetmore » uncultivated flavobacterial clade. Conclusions: With the developed pipeline, targeted metagenomes at various taxonomic levels can be efficiently retrieved from environmental samples. The resulting metagenome assembled genomes allow for the description of yet uncharacterized microbial clades.« less

Authors:
 [1];  [2];  [1];  [2];  [2];  [2];  [2]; ORCiD logo [1]
+ Show Author Affiliations
  1. Max Planck Inst. for Marine Microbiology, Bremen (Germany)
  2. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). DOE Joint Genome Inst.
Publication Date:
Research Org.:
Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER); Max Planck Society
OSTI Identifier:
1615302
Grant/Contract Number:  
AC02-05CH11231
Resource Type:
Accepted Manuscript
Journal Name:
Microbiome
Additional Journal Information:
Journal Volume: 8; Journal Issue: 1; Journal ID: ISSN 2049-2618
Publisher:
BioMed Central
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; FACS; Cell fixation; HCR-FISH; Mini-metagenomics

Citation Formats

Grieb, Anissa, Bowers, Robert M., Oggerin, Monike, Goudeau, Danielle, Lee, Janey, Malmstrom, Rex R., Woyke, Tanja, and Fuchs, Bernhard M. A pipeline for targeted metagenomics of environmental bacteria. United States: N. p., 2020. Web. doi:10.1186/s40168-020-0790-7.
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Grieb, Anissa, Bowers, Robert M., Oggerin, Monike, Goudeau, Danielle, Lee, Janey, Malmstrom, Rex R., Woyke, Tanja, & Fuchs, Bernhard M. A pipeline for targeted metagenomics of environmental bacteria. United States. https://doi.org/10.1186/s40168-020-0790-7
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Grieb, Anissa, Bowers, Robert M., Oggerin, Monike, Goudeau, Danielle, Lee, Janey, Malmstrom, Rex R., Woyke, Tanja, and Fuchs, Bernhard M. Sat . "A pipeline for targeted metagenomics of environmental bacteria". United States. https://doi.org/10.1186/s40168-020-0790-7. https://www.osti.gov/servlets/purl/1615302.
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@article{osti_1615302,
title = {A pipeline for targeted metagenomics of environmental bacteria},
author = {Grieb, Anissa and Bowers, Robert M. and Oggerin, Monike and Goudeau, Danielle and Lee, Janey and Malmstrom, Rex R. and Woyke, Tanja and Fuchs, Bernhard M.},
abstractNote = {Background:Metagenomics and single cell genomics provide a window into the genetic repertoire of yet uncultivated microorganisms, but both methods are usually taxonomically untargeted. The combination of fluorescence in situ hybridization (FISH) and fluorescence activated cell sorting (FACS) has the potential to enrich taxonomically well-defined clades for genomic analyses. Methods:Cells hybridized with a taxon-specific FISH probe are enriched based on their fluorescence signal via flow cytometric cell sorting. A recently developed FISH procedure, the hybridization chain reaction (HCR)-FISH, provides the high signal intensities required for flow cytometric sorting while maintaining the integrity of the cellular DNA for subsequent genome sequencing. Sorted cells are subjected to shotgun sequencing, resulting in targeted metagenomes of low diversity. Results: Pure cultures of different taxonomic groups were used to (1) adapt and optimize the HCR-FISH protocol and (2) assess the effects of various cell fixation methods on both the signal intensity for cell sorting and the quality of subsequent genome amplification and sequencing. Best results were obtained for ethanol-fixed cells in terms of both HCR-FISH signal intensity and genome assembly quality. Our newly developed pipeline was successfully applied to a marine plankton sample from the North Sea yielding good quality metagenome assembled genomes from a yet uncultivated flavobacterial clade. Conclusions: With the developed pipeline, targeted metagenomes at various taxonomic levels can be efficiently retrieved from environmental samples. The resulting metagenome assembled genomes allow for the description of yet uncharacterized microbial clades.},
doi = {10.1186/s40168-020-0790-7},
journal = {Microbiome},
number = 1,
volume = 8,
place = {United States},
year = {Sat Feb 15 00:00:00 EST 2020},
month = {Sat Feb 15 00:00:00 EST 2020}
}
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Journal Article:
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Publisher's Version of Record
https://doi.org/10.1186/s40168-020-0790-7
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Figures / Tables:

Fig. 1 Fig. 1: HCR-FISH fluorescence (green fluorescence, 530/40 nm band-pass filter) of four isolates treated with different fixatives. The median signal of the population from flow cytometric analysis is shown. The dashed line indicates the level of background noise. NA = not analyzed
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Action of Hydrogen Peroxide on Degradation of DNA after Irradiation in Escherichia Coli
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QUAST: quality assessment tool for genome assemblies
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ARB: a software environment for sequence data
journal, February 2004

MEROPS: the peptidase database
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PHASTER: a better, faster version of the PHAST phage search tool
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Genomes OnLine database (GOLD) v.7: updates and new features
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A comparison of fixatives for the estimation of abundance and biovolume of marine planktonic ciliate populations
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Effects of fixatives on ciliates as related to cell size
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Direct‐geneFISH: a simplified protocol for the simultaneous detection and quantification of genes and rRNA in microorganisms
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Substrate-Controlled Succession of Marine Bacterioplankton Populations Induced by a Phytoplankton Bloom
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    Measuring the microbiome: Best practices for developing and benchmarking microbiomics methods
    journal, January 2020

    • Bokulich, Nicholas A.; Ziemski, Michal; Robeson, Michael S.
    • Computational and Structural Biotechnology Journal, Vol. 18
    • DOI: 10.1016/j.csbj.2020.11.049
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