Computational protocol: Substantial inter-individual and limited intra-individual genomic diversity among tumors from men with metastatic prostate cancer

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Protocol publication

[…] Unsupervised clustering of expression and CN profiles was performed in R on the 5000 most variable genes for each dataset using Pearson correlation as the distance metric and average-linkage hierarchical clustering. Clusters were visualized using the ape R package. Circular plots of expression and CN profiles by chromosome location were created with the OmicCircos R package version 1.8.0. [...] A total of 176 tumors from 63 patients were collected for whole exome sequencing. For 35 tumors, DNA was of insufficient quality or quantity leaving 141 tumors from 56 patients that were successfully sequenced using the Nimblegen V2 or V3 platforms as previously described with the following modification: in a subset of tumors, individually barcoded libraries were pooled in pairs prior to capture. Genomic DNA was isolated as described above. Sequencing was per formed using the Illumina Hiseq 2000 with either 50 bp or 100 bp paired end sequences. Reads were mapped to the human reference genome sequence (hg19) with bwa v0.7.1. After remov al of PCR duplicate pairs we performed local realignment around indels using the Genome Analysis Toolkit (GATK). We subsequently called mutations using the Mutect software package with the following parameters: “--minimum_normal_allele_fraction 0.02 -- max_alt_alleles_in_normal_count 12 --intervals poscont.list --fraction_contamination 0.02”. To remove common polymorphisms and enrich for likely somatic mutations, we imposed a number of additional requirements, including requiring variants to be observed in at least 10% of reads at a position and removing variants present within a modified database of SNPs (dbSNP v137) that had first been stripped of all COSMIC variants. We investigated mutations for significance with MutSigCV using standard parameters, and inspected mutations in the top 50 significant genes manually using the Integrated Genomics Viewer (IGV) to remove sequence artifacts. The data are available for visualization and analysis in the cBioPortal for Cancer Genomics at: http://www.cbioportal.org/study.do?cancer_study_id=prad_fhcrc”. […]

Pipeline specifications

Software tools OmicCircos, BWA, GATK, MuTect, MutSig, cBioPortal
Databases dbSNP
Applications WES analysis, Genome data visualization
Organisms Homo sapiens
Diseases Fanconi Anemia, Neoplasms, Prostatic Neoplasms, Retinoblastoma
Chemicals Carboplatin