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Reconstructed ancestral sequences improve pathogen identification using resequencing DNA microarrays.

ABSTRACT: We describe the benefit of using reconstructed ancestral sequences (RAS) on resequencing microarrays for rapid pathogen identification, with Enterobacteriaceae rpoB sequences as a model. Our results demonstrate a sharp improvement of call rate and accuracy when using RASs as compared to extant sequences. This improvement was attributed to the lower sequence divergence of RASs, which also expanded the sequence space covered by the microarray. Extension of this novel microarray design strategy to viruses, antimicrobial resistance elements or toxins is straightforward.

SUBMITTER: Berthet N

PROVIDER: S-EPMC3004854 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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Reconstructed ancestral sequences improve pathogen identification using resequencing DNA microarrays.

Berthet Nicolas N Deletoile Alexis A Passet Virginie V Kennedy Giulia C GC Manuguerra Jean-Claude JC Cole Stewart T ST Brisse Sylvain S

PloS one 20101220 12

We describe the benefit of using reconstructed ancestral sequences (RAS) on resequencing microarrays for rapid pathogen identification, with Enterobacteriaceae rpoB sequences as a model. Our results demonstrate a sharp improvement of call rate and accuracy when using RASs as compared to extant sequences. This improvement was attributed to the lower sequence divergence of RASs, which also expanded the sequence space covered by the microarray. Extension of this novel microarray design strategy to ...[more]

PMID: 21187950

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Project description:BACKGROUND: Genome-wide detection of single feature polymorphisms (SFP) in swine using transcriptome profiling of day 25 placental RNA by contrasting probe intensities from either Meishan or an occidental composite breed with Affymetrix porcine microarrays is presented. A linear mixed model analysis was used to identify significant breed-by-probe interactions. RESULTS: Gene specific linear mixed models were fit to each of the log2 transformed probe intensities on these arrays, using fixed effects for breed, probe, breed-by-probe interaction, and a random effect for array. After surveying the day 25 placental transcriptome, 857 probes with a q-value < or = 0.05 and |fold change| > or = 2 for the breed-by-probe interaction were identified as candidates containing SFP. To address the quality of the bioinformatics approach, universal pyrosequencing assays were designed from Affymetrix exemplar sequences to independently assess polymorphisms within a subset of probes for validation. Additionally probes were randomly selected for sequencing to determine an unbiased confirmation rate. In most cases, the 25-mer probe sequence printed on the microarray diverged from Meishan, not occidental crosses. This analysis was used to define a set of highly reliable predicted SFPs according to their probability scores. CONCLUSION: By applying a SFP detection method to two mammalian breeds for the first time, we detected transition and transversion single nucleotide polymorphisms, as well as insertions/deletions which can be used to rapidly develop markers for genetic mapping and association analysis in species where high density genotyping platforms are otherwise unavailable.SNPs and INDELS discovered by this approach have been publicly deposited in NCBI's SNP repository dbSNP. This method is an attractive bioinformatics tool for uncovering breed-by-probe interactions, for rapidly identifying expressed SNPs, for investigating potential functional correlations between gene expression and breed polymorphisms, and is robust enough to be used on any Affymetrix gene expression platform.

Dataset Information

Reconstructed ancestral sequences improve pathogen identification using resequencing DNA microarrays.

Publications

Reconstructed ancestral sequences improve pathogen identification using resequencing DNA microarrays.

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