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Efficient oligonucleotide probe selection for pan-genomic tiling arrays.

ABSTRACT: Array comparative genomic hybridization is a fast and cost-effective method for detecting, genotyping, and comparing the genomic sequence of unknown bacterial isolates. This method, as with all microarray applications, requires adequate coverage of probes targeting the regions of interest. An unbiased tiling of probes across the entire length of the genome is the most flexible design approach. However, such a whole-genome tiling requires that the genome sequence is known in advance. For the accurate analysis of uncharacterized bacteria, an array must query a fully representative set of sequences from the species' pan-genome. Prior microarrays have included only a single strain per array or the conserved sequences of gene families. These arrays omit potentially important genes and sequence variants from the pan-genome.This paper presents a new probe selection algorithm (PanArray) that can tile multiple whole genomes using a minimal number of probes. Unlike arrays built on clustered gene families, PanArray uses an unbiased, probe-centric approach that does not rely on annotations, gene clustering, or multi-alignments. Instead, probes are evenly tiled across all sequences of the pan-genome at a consistent level of coverage. To minimize the required number of probes, probes conserved across multiple strains in the pan-genome are selected first, and additional probes are used only where necessary to span polymorphic regions of the genome. The viability of the algorithm is demonstrated by array designs for seven different bacterial pan-genomes and, in particular, the design of a 385,000 probe array that fully tiles the genomes of 20 different Listeria monocytogenes strains with overlapping probes at greater than twofold coverage.PanArray is an oligonucleotide probe selection algorithm for tiling multiple genome sequences using a minimal number of probes. It is capable of fully tiling all genomes of a species on a single microarray chip. These unique pan-genome tiling arrays provide maximum flexibility for the analysis of both known and uncharacterized strains.

SUBMITTER: Phillippy AM

PROVIDER: S-EPMC2753849 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Efficient oligonucleotide probe selection for pan-genomic tiling arrays.

Phillippy Adam M AM Deng Xiangyu X Zhang Wei W Salzberg Steven L SL

BMC bioinformatics 20090916

<h4>Background</h4>Array comparative genomic hybridization is a fast and cost-effective method for detecting, genotyping, and comparing the genomic sequence of unknown bacterial isolates. This method, as with all microarray applications, requires adequate coverage of probes targeting the regions of interest. An unbiased tiling of probes across the entire length of the genome is the most flexible design approach. However, such a whole-genome tiling requires that the genome sequence is known in ad ...[more]

PMID: 19758451

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Project description:High-density oligonucleotide (oligo) arrays are a powerful tool for transcript profiling. Arrays based on GeneChip technology are amongst the most widely used, although GeneChip arrays are currently available for only a small number of plant and animal species. Thus, we have developed a method to improve the sensitivity of high-density oligonucleotide arrays when applied to heterologous species and tested the method by analysing the transcriptome of Brassica oleracea L., a species for which no GeneChip array is available, using a GeneChip array designed for Arabidopsis thaliana (L.) Heynh. Genomic DNA from B. oleracea was labelled and hybridised to the ATH1-121501 GeneChip array. Arabidopsis thaliana probe-pairs that hybridised to the B. oleracea genomic DNA on the basis of the perfect-match (PM) probe signal were then selected for subsequent B. oleracea transcriptome analysis using a .cel file parser script to generate probe mask files. The transcriptional response of B. oleracea to a mineral nutrient (phosphorus; P) stress was quantified using probe mask files generated for a wide range of gDNA hybridisation intensity thresholds. An example probe mask file generated with a gDNA hybridisation intensity threshold of 400 removed > 68 % of the available PM probes from the analysis but retained >96 % of available A. thaliana probe-sets. Ninety-nine of these genes were then identified as significantly regulated under P stress in B. oleracea, including the homologues of P stress responsive genes in A. thaliana. Increasing the gDNA hybridisation intensity thresholds up to 500 for probe-selection increased the sensitivity of the GeneChip array to detect regulation of gene expression in B. oleracea under P stress by up to 13-fold. Our open-source software to create probe mask files is freely available http://affymetrix.arabidopsis.info/xspecies/ and may be used to facilitate transcriptomic analyses of a wide range of plant and animal species in the absence of custom arrays.

Dataset Information

Efficient oligonucleotide probe selection for pan-genomic tiling arrays.

Publications

Efficient oligonucleotide probe selection for pan-genomic tiling arrays.

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