Computational protocol: Characterization of genetic intratumor heterogeneity in colorectal cancer and matching patient‐derived spheroid cultures

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Protocol publication

[…] WES was performed using the KAPA‐Hyper prep kit from Illumina (Roche, Basel, Switzerland) for library construction, followed by exome capture using NimbleGen SeqCap EZ Human Exome Library v3.0 (Roche). Sequencing was performed on an Illumina NextSeq500, with 10–50 ng of genomic DNA as input per sample (depending on material available). Reads were mapped using BWA MEM against the human reference genome HG19, and duplicates were marked with Picard MarkDuplicates. SNPs were called using GATK HaplotypeCaller. Somatic mutations were called using MuTect2, with matched germline WES data obtained from blood samples as reference. Mutation allele frequencies (AFs) were calculated using SAMtools mpileup. Copy number alterations (CNAs) and allelic imbalance were estimated using FACETS (Shen and Seshan, ). Tumor purity was estimated using PurBayes (Larson and Fridley, ) and Sequenza (Favero et al., ). Tumor drivers and potentially druggable targets were identified using the IntOGen catalog of cancer drivers, available for download at Intogen.org (Tamborero D, Rubio‐Perez C, Deu‐Pons J, Schroeder M, Vivancos A, Rovira A, Tusquets I, Albanell J, Rodon J, Tabernero J, Dienstmann R, Gonzalez‐Perez A and Lopez‐Bigas N, unpublished data). [...] RNA sequencing was performed as previously described using ScriptSeq RNA‐Seq Library preparation Kit from Illumina (Hedegaard et al., ). The paired raw sequence reads were processed using TopHat2 (Kim et al., ) and mapped to the human reference genome HG19. FPKM values were called using Cufflinks (Trapnell et al., ) and GenCode v19 transcript information. The FPKM gene expression values were used to assign a molecular subtype to each sample using our previously reported TUMOR subtype classifier (Bramsen et al., ). CMS were assigned using the nearest‐centroid Single Sample Predictor CMS classifier (Guinney et al., ), and CRIS types were assigned using the CRIS classifier (Isella et al., ). Tumor purity was estimated from the RNA sequencing data using ESTIMATE (Yoshihara et al., ). […]

Pipeline specifications

Software tools TopHat, Cufflinks
Databases GENCODE
Application RNA-seq analysis
Organisms Homo sapiens
Diseases Neoplasms, Colorectal Neoplasms, Genetic Diseases, Inborn