Present address: The Department of Civil and Environmental Engineering, Georgia Institute of Technology, 790 Atlantic Dr., Atlanta, GA 30332-0355, USA.
Design and testing of ‘genome-proxy’ microarrays to profile marine microbial communities
Article first published online: 19 NOV 2007
© 2007 The Authors
Volume 10, Issue 2, pages 506–521, February 2008
How to Cite
Rich, V. I., Konstantinidis, K. and DeLong, E. F. (2008), Design and testing of ‘genome-proxy’ microarrays to profile marine microbial communities. Environmental Microbiology, 10: 506–521. doi: 10.1111/j.1462-2920.2007.01471.x
- Issue published online: 17 DEC 2007
- Article first published online: 19 NOV 2007
- Received 16 June, 2007; accepted 21 September, 2007.
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 13 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 open reading frame, and distributed along each ∼40–160 kbp 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 ∼103 cells ml−1 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 intrapopulation diversity within marine microbial communities.