High-density universal 16S rRNA microarray analysis revealsbroader diversity than typical clone library when sampling theenvironment
Molecular approaches aimed at detection of a broad-range ofprokaryotes in the environment routinely rely upon classifyingheterogeneous 16S rRNA genes amplified by PCR using primers with broadspecificity. The general method of sampling and categorizing DNA has beento clone then sequence the PCR products. However, the number of clonesrequired to adequately catalogue the majority of taxa in a sample isunwieldy. Alternatively, hybridizing target sequences to a universal 16SrRNA gene microarray may provide a more rapid and comprehensive view ofprokaryotic community composition. This study investigated the breadthand accuracy of a microarray in detecting diverse 16S rRNA gene sequencetypes compared to clone-and-sequencing using three environmental samples:urban aerosol, subsurface soil and subsurface water. PCR productsgenerated from universal 16S rRNA gene-targeted primers were classifiedusing either the clone-and-sequence method or by hybridization to a novelhigh-density microarray of 297,851 probes complementary to 842prokaryotic sub-families. The three clone libraries comprised 1,391high-quality sequences. Approximately 8 percent of the clones could notbe placed into a known sub-family and were considered novel. Themicroarray results confirmed the majority of clone-detected sub-familiesand additionally demonstrated greater amplicon diversity extending intophyla not observed by the cloning method. Sequences matching OTUs withinthe phyla Nitrospira, Planctomycetes, and TM7, which were uniquelydetected by the array, were verified with specific primers and subsequentamplicon sequencing. Sub-family richness detected by the arraycorresponded well with non-parametric richness predictions extrapolatedfrom clone libraries except in the water community where clone-basedrichness predictions were greatly exceeded. It was concluded thatalthough the microarray is unreliable inidentifying novel prokaryotictaxa, it reveals greater diversity in environmental samples thansequencing a typically sized clone library. Furthermore, the microarrayallowed samples to be rapidly evaluated with replication, a significantadvantage in studies of microbial ecology.
- Research Organization:
- Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
- Sponsoring Organization:
- USDOE; US Department of Homeland Security (DHS)
- DOE Contract Number:
- DE-AC02-05CH11231
- OSTI ID:
- 926473
- Report Number(s):
- LBNL-59763; R&D Project: G4W064; BnR: 400904010
- Journal Information:
- Microbial Ecology, Vol. 53, Issue 3; Related Information: Journal Publication Date: 03/02/2007
- Country of Publication:
- United States
- Language:
- English
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