Transcriptomics

Dataset Information

37

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 106 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. The genomic DNA (gDNA) of each culture or sorted and amplifed DNA was analyzed in triplicate.

ORGANISM(S): Dehalococcoides mccartyi 195  

SUBMITTER: Yujie Men  Shanquan Wang   Patrick K Lee   Patrick Lee   Lisa Alvarez-Cohen   Jianzhong He    

PROVIDER: E-GEOD-54040 | ArrayExpress | 2015-02-08

SECONDARY ACCESSION(S): GSE54040PRJNA234435

REPOSITORIES: GEO, ArrayExpress

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Development of a fluorescence-activated cell sorting method coupled with whole genome amplification to analyze minority and trace Dehalococcoides genomes in microbial communities.

Lee Patrick K H PK   Men Yujie Y   Wang Shanquan S   He Jianzhong J   Alvarez-Cohen Lisa L  

Environmental science & technology 20150121 3


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 amplificatio  ...[more]

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