Accurately quantifying low-abundant targets amid similar sequences by revealing hidden correlations in oligonucleotide microarray data

Abstract

Microarrays have enabled the determination of how thousands of genes are expressed to coordinate function within single organisms. Yet applications to natural or engineered communities where different organisms interact to produce complex properties are hampered by theoretical and technological limitations. Here we describe a general method to accurately identify low-abundant targets in systems containing complex mixtures of homologous targets. We combined an analytical predictor of nonspecific probe–target interactions (cross-hybridization) with an optimization algorithm that iteratively deconvolutes true probe–target signal from raw signal affected by spurious contributions (cross-hybridization, noise, background, and unequal specific hybridization response). The method was capable of quantifying, with unprecedented specificity and accuracy, ribosomal RNA (rRNA) sequences in artificial and natural communities. Controlled experiments with spiked rRNA into artificial and natural communities demonstrated the accuracy of identification and quantitative behavior over different concentration ranges. Finally, we illustrated the power of this methodology for accurate detection of low-abundant targets in natural communities. We accurately identified Vibrio taxa in coastal marine samples at their natural concentrations (<0.05% of total bacteria), despite the high potential for cross-hybridization by hundreds of different coexisting rRNAs, suggesting this methodology should be expandable to any microarray platform and system requiring accurate identification of low-abundant targets amid pools of similar sequences.

Development of a phylogenetic microarray for comprehensive analysis of ruminal bacterial communities

“…Consistent with previous studies (Marcelino et al . ; Pozhitkov et al . ), the RumenBactArray can provide relatively accurate quantification of bacteria, even bacteria present at low abundance.…”

Exon arrays provide accurate assessments of gene expression

“…However, other approaches to estimating gene-level expression are possible, including model-based approaches [8,12-14], and methods based on physical models [15,16]. Various methods for estimating probe background intensities have also been proposed, incorporating probe content [17,18] or using physical models [19]. With so many probes interrogating background signal, Exon arrays present the opportunity to fit improved background models.…”

Detection of Bacterial Pathogens in River Water Using Multiplex-PCR

“…Improved detection of pathogenic E. coli (Ogunjimi and Choudary, 1999) by immuno-capture PCR, and the sensitive detection of Salmonella (Hoorfar et al, 2000) and Campylobacter (Nogva et al, 2000) by real-time PCR have also been developed; but these procedures are all monospecific and are either laborious or very expensive for routine use in water testing laboratories. More recent improvements have allowed simultaneous detection of several microorganisms in a single assay (Maynard et al, 2005;Straub et al, 2005;Marcelino et al, 2006). The use of such multiplex polymerase chain reaction (m-PCR) has provided rapid and highly sensitive methods for the specific detection of pathogenic microbes in the aquatic environment (Kong et al, 2002).…”