Computational protocol: Comparative Developmental Expression Profiling of Two C. elegans Isolates

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Protocol publication

[…] The array data was uploaded to the Princeton University Microarray Database (PUMAdb) for processing. The data was collapsed by SUID, using the average value of each probe. Spots were considered good data if intensity was well above background and the feature was not a nonuniformity outlier Only genes with greater than 80% good data were kept for further analysis. Missing values were imputed using KNN-impute in the MultiExperiment Viewer , . All array data will be made publicly available through puma.princeton.edu. For the analysis using only a single stage, 100% good data was required across all arrays for that time point. Data was hierarchically clustered by centroid linkage and a centered Pearson correlation using the average value from the four replicates . Clusters were visualized using JavaTreeView . An analysis of variance (ANOVA) test was used to see which genes changed significantly over developmental time, using the strain and stage of each array as the parameters. qvalue, an R-package, was used to obtain false-discovery rates (FDR) . Significant genes have an FDR of less than .05. Enrichment values for functional groups were calculated from lists of genes provided by WormBase , which uses the PFAM and Interpro databases. The list of innate immunity genes was hand-curated from the literature. Significance was assessed through the hypergeometric distribution. Short-Time series expression miner (STEM) was used to cluster the data and provide GO-term enrichments . FIRE was used for motif analysis . The best motif for each gene was defined as that motif with the highest pa+po+pd value. This value had to be greater than 4 in order for a motif to be considered real. The most recent C. elegans gene ontology was downloaded from the Gene Ontology Consortium on April 20th, 2008 . The assignment of function was done subjectively, using GO-enrichments of the clusters, the motifs, and previous knowledge about the named motifs. […]

Pipeline specifications

Software tools TreeView, STEM
Application Immune system analysis
Organisms Caenorhabditis elegans