Computational protocol: Cervical small cell neuroendocrine tumor mutation profiles via whole exome sequencing

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Protocol publication

[…] After the FastQC step, trimmed FASTQ reads were aligned to human reference genome version 19 (hg19) using the TMAP mapping program with default parameters []. The results were sorted and compressed in BAM format using Picard SortSam. In accordance with manufacturer's instructions, duplicates were not removed from AmpliSeq data []. Local realignment around indels and recalibration were performed by GATK []. Recalibrated BAM files were used to call variants with MuTect software (version 1.1.4) using default parameters []. Nonsynonymous mutations within each sample were visualized by circos plot using the RCircos library ( The RCircos plot produced a human chromosome ideogram heatmap with five sample tracks for mutations. We used the chromosome ideogram tables from the UCSC genome browser ( The chromosome ideogram is shown in high-resolution to plot gene locations. Tools in the Database for Annotation, Visualization, and Integrated Discovery (DAVID) were used to assess gene ontology enrichment and to identify biological pathway categories associated with somatically mutated genes []. Sequence data for CSCNET samples used in this study were deposited in the European Nucleotide Archive under accession number, PRJEB12274. [...] The mutated genes in the NETs dataset are from a recent WES study of 55 SINETs [], 10 panNETs [], 42 SCLCs [], six NET cell lines [] and our five CSCNETs. The mutated CT dataset is from 188 samples from The Cancer Genome Atlas (TCGA). Publicly available somatic variant calls in mutation annotation format (MAF) files were used in this study. All MAF files were downloaded from TCGA cBioPortal or supplementary tables from each study. TCGA Pan-cancer analysis provided the distributions of mutation frequencies, types, and contexts across 12 tumor types []. In the TCGA study, only genes mutated in at least 5% of tumors were analyzed. With this cutoff percentage, mutated genes were selected with ≥5% recurrent rates from other NET and CT studies. We used ≥50% (≥3 samples) recurrent mutation as a cutoff for our CSCNETs. We also added significantly reported genes from each study. Selected gene mutations were nonsynonymous. Venn diagram analysis shows co-occurrence among NETs, and mutated gene overlap between CT, NETs, and CSCNETs. […]

Pipeline specifications

Software tools FastQC, Picard, GATK, MuTect, RCircos, DAVID, cBioPortal
Databases ENA TCGA Data Portal UCSC Genome Browser
Applications WES analysis, Genome data visualization
Diseases Neoplasms, Small Cell Lung Carcinoma, Desmoplastic Small Round Cell Tumor