In-solution fluorescence in situ hybridization and fluorescence-activated cell sorting for single cell and population genome recovery.
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.
Australian Centre for Ecogenomics, School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, Queensland, Australia.
Mohamed F
MF
Haroon
Connor T
CT
Skennerton
Jason A
JA
Steen
Nancy
N
Lachner
Philip
P
Hugenholtz
Gene W
GW
Tyson
D001105:Archaea / Q000235:genetics; D005434:Flow Cytometry; D005828:Genetics, Population; D016678:Genome; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D056186:Metagenomics / Q000379:methods*; D044085:Microfluidics / Q000379:methods; D012336:RNA, Ribosomal, 16S; D017422:Sequence Analysis, DNA; D059010:Single-Cell Analysis
D012336:RNA, Ribosomal, 16S
Anaerobic methanotrophic archaea; Anaerobic oxidation of methane; FISH–FACS; Fixation-free FISH; Fluorescence in situ hybridization; Fluorescence-activated cell sorting; In-solution FISH; Metagenomics; Single-cell genomics; Single-population genomics