In-solution fluorescence in situ hybridization and fluorescence-activated cell sorting for single cell and population genome recovery

Haroon, Mohamed F., Skennerton, Connor T., Steen, Jason A., Lachner, Nancy, Hugenholtz, Philip and Tyson, Gene W. (2013) In-solution fluorescence in situ hybridization and fluorescence-activated cell sorting for single cell and population genome recovery. Methods in Enzymology, 531 3-19. doi:10.1016/B978-0-12-407863-5.00001-0


Author Haroon, Mohamed F.
Skennerton, Connor T.
Steen, Jason A.
Lachner, Nancy
Hugenholtz, Philip
Tyson, Gene W.
Title In-solution fluorescence in situ hybridization and fluorescence-activated cell sorting for single cell and population genome recovery
Formatted title
In-solution fluorescence in situ hybridization and fluorescence-activated cell sorting for single cell and population genome recovery
Journal name Methods in Enzymology   Check publisher's open access policy
ISSN 0076-6879
1557-7988
1079-2376
ISBN 9780124078635
Publication date 2013-01-01
Year available 2013
Sub-type Article (original research)
DOI 10.1016/B978-0-12-407863-5.00001-0
Open Access Status Not yet assessed
Volume 531
Start page 3
End page 19
Total pages 17
Editor Edward F. DeLong
Place of publication Maryland Heights, MO, United States
Publisher Academic Press
Language eng
Subject 1303 Biochemistry
1312 Molecular Biology
Abstract Over the past decade, technological advances in whole genome amplification, microfluidics, flow sorting, and high-throughput sequencing have led to the development of single-cell genomics. Single-cell genomic approaches are typically applied to anonymous microbial cells with only morphology providing clues to their identity. However, targeted separation of microorganisms based on phylogenetic markers, such as the 16S rRNA gene, is beginning to emerge in the single-cell genomics field. Here, we describe an in-solution fluorescence in situ hybridization (FISH) protocol which can be combined with fluorescence-activated cell sorting (FACS) for separation of single cells or populations of interest from environmental samples. Sequencing of DNA obtained from sorted cells can be used for the recovery of draft quality genomes, and when performed in parallel with deep metagenomics, can be used to validate and further scaffold metagenomic assemblies. We illustrate in this chapter the feasibility of this FISH-FACS approach by describing the targeted recovery of a novel anaerobic methanotrophic archaeon.
Formatted abstract
Over the past decade, technological advances in whole genome amplification, microfluidics, flow sorting, and high-throughput sequencing have led to the development of single-cell genomics. Single-cell genomic approaches are typically applied to anonymous microbial cells with only morphology providing clues to their identity. However, targeted separation of microorganisms based on phylogenetic markers, such as the 16S rRNA gene, is beginning to emerge in the single-cell genomics field. Here, we describe an in-solution fluorescence in situ hybridization (FISH) protocol which can be combined with fluorescence-activated cell sorting (FACS) for separation of single cells or populations of interest from environmental samples. Sequencing of DNA obtained from sorted cells can be used for the recovery of draft quality genomes, and when performed in parallel with deep metagenomics, can be used to validate and further scaffold metagenomic assemblies. We illustrate in this chapter the feasibility of this FISH–FACS approach by describing the targeted recovery of a novel anaerobic methanotrophic archaeon.
Keyword Fluorescence in situ hybridization
In-solution FISH
Fixation-free FISH
Fluorescence-activated cell sorting
FISH–FACS
Anaerobic methanotrophic archaea
Single-cell genomics
Single-population genomics
Metagenomics
Anaerobic oxidation of methane
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ
Additional Notes Special issue: "Microbial Metagenomics, Metatranscriptomics, and Metaproteomics".

 
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Created: Fri, 04 Oct 2013, 21:09:06 EST by Mrs Louise Nimwegen on behalf of School of Chemistry & Molecular Biosciences