Computational protocol: Investigation of Radiosensitivity Gene Signatures in Cancer Cell Lines

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Protocol publication

[…] Microarray data were normalised using RMA . The R/BioConductor package annmap and the annmap database were used to remove non-exonic and multi-targeting probesets. Array performance was measured as the percentage of probesets flagged as “present” with a conservative cut-off (%Detection Above BackGround [%DABG] P<0.01) and only those probesets flagged “present” in at least three samples were retained. This filtering reduced the number of probesets considered from 1,411,399 to 353,981 exonic probesets, of which 243,301 passed DABG filtering. Gene level summaries were calculated by taking the median signal of filtered probesets that mapped to unique gene symbols. When summarised this resulted in 31,345 genes considered. Unsupervised hierarchical clustering was performed on the 1000 most variant genes (ranked on coefficient of variation) to show the separation of samples based on the most variable genes in the data, while minimising computational requirements. Signature Generation: A gene signature was determined to be the set of genes or probesets that were significantly differentially expressed between two groups of cell lines according to either LIMMA or Rank Product Analysis. The cut-off for significance was a false discovery corrected p value of 0.01. Packages: R: 3.0.2, Annmap: v1.2.1 using human database build 66, LIMMA: 3.17.26, RankProd: 2.32.0, Pheatmap: 0.7.7. [...] Head and neck cell line Affymetrix U133A Plus2 array data were RMA normalised using the affy package in R. Affymetrix control probesets (‘AFFX’ annotated) were removed. For variance analysis, _x_, _a_ and _s_ annotated probesets were also removed. NCI-60 - Affymetrix Plus2 cel files were downloaded from CellMiner ( and RMA normalised as before. After normalisation, replicate arrays for each cell line were averaged. For comparison to the gene-level summarised exon array data, Plus2 probesets were mapped to gene symbols using annmap. […]

Pipeline specifications

Software tools Annmap, limma, RankProd, PHeatmaps, affy
Databases CellMiner
Applications Gene expression microarray analysis, Transcriptome data visualization
Organisms Homo sapiens
Diseases Head and Neck Neoplasms, Neoplasms