High-density microarray of small-subunit ribosomal DNA probes - PubMed (original) (raw)

High-density microarray of small-subunit ribosomal DNA probes

Kenneth H Wilson et al. Appl Environ Microbiol. 2002 May.

Abstract

Ribosomal DNA sequence analysis, originally conceived as a way to provide a universal phylogeny for life forms, has proven useful in many areas of biological research. Some of the most promising applications of this approach are presently limited by the rate at which sequences can be analyzed. As a step toward overcoming this limitation, we have investigated the use of photolithography chip technology to perform sequence analyses on amplified small-subunit rRNA genes. The GeneChip (Affymetrix Corporation) contained 31,179 20-mer oligonucleotides that were complementary to a subalignment of sequences in the Ribosomal Database Project (RDP) (B. L. Maidak et al., Nucleic Acids Res. 29:173-174, 2001). The chip and standard Affymetrix software were able to correctly match small-subunit ribosomal DNA amplicons with the corresponding sequences in the RDP database for 15 of 17 bacterial species grown in pure culture. When bacteria collected from an air sample were tested, the method compared favorably with cloning and sequencing amplicons in determining the presence of phylogenetic groups. However, the method could not resolve the individual sequences comprising a complex mixed sample. Given these results and the potential for future enhancement of this technology, it may become widely useful.

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Figures

FIG. 1.

FIG. 1.

Example of phylogenetic placement of 14 SSU rDNA sequences included in RDP's Escherichia Subgroup. These sequences are contained in phylogenetic clusters at five hierarchical levels, each designated by a name and number as shown. Not all of these sequences are unique in the region represented on the chip.

FIG. 2.

FIG. 2.

Data from hybridization of amplified rDNA from F. nucleatum to probe sets for F. simiae and F. nucleatum. Probe sequences are shown 3′ to 5′, the order of synthesis on the chip. Probe pair numbers 1 through 15 and 30 are shared (identical) between the two probe sets. Underlined bases highlight the site of a sequence discrepancy between the RDP database used in the software analysis and the amplicon from F. nucleatum and the corresponding site in the F. simiae probe set. Signal intensity is measured as fluorescent intensity of the perfect match (PM) probe minus fluorescent intensity of the mismatch (MM) probe (PM − MM). Considering probe pairs 16 through 29, the amplicon from F. nucleatum hybridized better to the F. simiae probe set, which was actually complementary to its sequence. At position 22, the MM probe for F. nucleatum, incorporating a guanosine at the site of the discrepancy, hybridized much better than the PM probe. This finding led to the prediction that the amplicon actually contained a cytosine in this position. Sequencing confirmed this assessment.

FIG. 3.

FIG. 3.

Average difference in intensity of fluorescence between probe cells and control cells for the phylogenetic groups identified by GeneChip software in experiments using amplicons derived from pure cultures. For each sample, the largest average difference was set at 100, and other peaks were adjusted relative to it.

FIG. 4.

FIG. 4.

Relative abundance of microbes detected in an air sample as determined by rDNA amplification followed both by cloning with sequencing and by GeneChip analysis. rDNA sequences as determined by each method were sorted into taxa. Vertical bars represent mean difference of replicate hybridizations on three chips from different manufacturers' production lots. Bar height is proportional to the greatest mean difference in fluorescence found among the sequences in that group. Abbreviations for taxa and RDP phylogenetic numerical codes: Sphingobacterium group, Sphingo (2.15.2); α-proteobacteria subdivision, α-proteo (2.28.1); β-proteobacteria subdivision, β-proteo (2.28.2); γ-proteobacteria subdivision, γ-proteo (2.28.3); gram-positive high G+C bacteria, high G+C (2.30.1); gram-positive Eubacterium and relatives, Eubact (2.30.4); gram-positive Bacillus-Lactobacillus-Streptococcus subdivision, Bacil (2.30.7); gram-positive Clostridium and relatives, Clostr (2.30.9); Ascomycota fungi, Asco (3.9.1); and Basidiomycota fungi, Basidio (3.9.2). The 28 novel sequences did not correspond well to any phylogenetic group in the RDP database.

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