Genomics

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Design and testing of genome-proxy microarrays to profile marine microbial communities


ABSTRACT: Microarrays are useful tools for detecting and quantifying specific functional and phylogenetic genes in natural microbial communities. In order to track uncultivated microbial genotypes and their close relatives in an environmental context, we designed and implemented a “genome proxy” microarray that targets microbial genome fragments recovered directly from the environment. Fragments consisted of sequenced clones from large-insert genomic libraries from microbial communities in Monterey Bay, the Hawaii Ocean Time-series station ALOHA, and Antarctic coastal waters. In a prototype array, we designed probe sets to thirteen of the sequenced genome fragments and to genomic regions of the cultivated cyanobacterium Prochlorococcus MED4. Each probe set consisted of multiple 70-mers, each targeting an individual ORF, and distributed along each ~40-160kbp contiguous genomic region. The targeted organisms or clones, and close relatives, were hybridized to the array both as pure DNA mixtures and as additions of cells to a background of coastal seawater. This prototype array correctly identified the presence or absence of the target organisms and their relatives in laboratory mixes, with negligible cross-hybridization to organisms having ≤~75% genomic identity. In addition, the array correctly identified target cells added to a background of environmental DNA, with a limit of detection of ~0.1% of the community, corresponding to ~10^3 cells/ml in these samples. Signal correlated to cell concentration with an R2 of 1.0 across six orders of magnitude. In addition the array could track a related strain (at 86% genomic identity to that targeted) with a linearity of R2=0.9999 and a limit of detection of ~1% of the community. Closely related genotypes were distinguishable by differing hybridization patterns across each probe set. This array’s multiple-probe, “genome-proxy” approach and consequent ability to track both target genotypes and their close relatives is important for the array’s environmental application given the recent discoveries of considerable intra-population diversity within marine microbial communities. Keywords: target addition experiment, proof-of-concept for GPL6012

ORGANISM(S): uncultured marine bacterium EB000_55B11 uncultured proteobacterium 60D04 uncultured marine bacterium EB0_41B09 uncultured proteobacterium 65D09 uncultured marine group II euryarchaeote 37F11 Prochlorococcus marinus subsp. pastoris str. CCMP1986 Prochlorococcus marinus str. MIT 9313 uncultured gamma proteobacterium eBACHOT4E07 uncultured marine bacterium 440 uncultured marine bacterium 583 marine metagenome uncultured crenarchaeote 74A4 uncultured marine gamma proteobacterium EBAC31A08 uncultured crenarchaeote 4B7 Prochlorococcus marinus str. MIT 9312 uncultured marine group II euryarchaeote EF100_57A08 uncultured proteobacterium uncultured marine bacterium 577 uncultured marine alpha proteobacterium Prochlorococcus marinus str. MIT 9515

PROVIDER: GSE9384 | GEO | 2007/10/31

SECONDARY ACCESSION(S): PRJNA103099

REPOSITORIES: GEO

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