title: Development of a fluorescence-activated cell sorting method coupled with whole genome amplification to analyze minority and trace Dehalococcoides genomes in microbial communities.
abstract: Dehalococcoides mccartyi are functionally important bacteria that catalyze the reductive dechlorination of chlorinated ethenes. However, these anaerobic bacteria are fastidious to isolate, making downstream genomic characterization challenging. In order to facilitate genomic analysis, a fluorescence-activated cell sorting (FACS) method was developed in this study to separate D. mccartyi cells from a microbial community, and the DNA of the isolated cells was processed by whole genome amplification (WGA) and hybridized onto a D. mccartyi microarray for comparative genomics against four sequenced strains. First, FACS was successfully applied to a D. mccartyi isolate as positive control, and then microarray results verified that WGA from 10(6) cells or ∼1 ng of genomic DNA yielded high-quality coverage detecting nearly all genes across the genome. As expected, some inter- and intrasample variability in WGA was observed, but these biases were minimized by performing multiple parallel amplifications. Subsequent application of the FACS and WGA protocols to two enrichment cultures containing ∼10% and ∼1% D. mccartyi cells successfully enabled genomic analysis. As proof of concept, this study demonstrates that coupling FACS with WGA and microarrays is a promising tool to expedite genomic characterization of target strains in environmental communities where the relative concentrations are low.
authors.affiliation: Department of Civil and Environmental Engineering, University of California , Berkeley, California 94720, United States.
authors.forename: Patrick K H
authors.initials: PK
authors.lastname: Lee
authors.forename: Yujie
authors.initials: Y
authors.lastname: Men
authors.forename: Shanquan
authors.initials: S
authors.lastname: Wang
authors.forename: Jianzhong
authors.initials: J
authors.lastname: He
authors.forename: Lisa
authors.initials: L
authors.lastname: Alvarez-Cohen
mesh_terms: D041862:Chloroflexi / Q000166:cytology / Q000235:genetics*; D005434:Flow Cytometry / Q000379:methods*; D016680:Genome, Bacterial; D023281:Genomics / Q000379:methods*; D017404:In Situ Hybridization, Fluorescence; D059013:Microbial Consortia / Q000235:genetics; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods