Computational protocol: The genomic landscape of malignant peripheral nerve sheath tumors: diverse drivers of Ras pathway activation

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Protocol publication

[…] Patients with a diagnosis of MPNST were identified by the treating physician. Samples utilized for sequencing were collected as part of routine clinical care. All subjects gave written institutional review board (IRB)-approved informed consent for their clinical samples to be utilized for research. All methods were carried out in accordance with relevant guidelines and regulations and all experimental protocols were approved by both the local scientific review committee and IRB at the Moffitt Cancer Center.Whole-exome sequencing (WES) was performed by the Molecular Genomics Core Facility at the Moffitt Cancer Center. The genomic DNA recovered from FFPE blocks was used to generate a sequencing library using the NuGEN Ovation Ultralow Library System (NuGEN, Inc., San Carlos, CA). Whole-exome enrichment utilized the Roche NimbleGen SeqCap EZ Exome Library v2.0 kit, which targets 64 Mb of genomic DNA sequence regions (Roche NimbleGen, Inc., Madison WI). Sequencing was performed on an Illumina HiScan SQ sequencer to generate 75 million 100-base paired-end reads for a target coverage depth of 30–50X. The raw sequence data was demultiplexed using the Illumina CASAVA 1.8.2 software (Illumina, Inc., San Diego, CA). Sequence reads were aligned to the reference human genome (hg19) with the Burrows-Wheeler Alignment Tool, and duplicate identification, insertion/deletion realignment, quality score recalibration, and variant identification were performed with the Picard toolkit and Genome Analysis ToolKit. Somatic mutations were identified by comparing matched tumor and normal samples using Strelka. Sequence variants were annotated to determine genic context using ANNOVAR. Additional contextual information was incorporated, including allele frequency in other studies such as 1000 Genomes, the NHLBI Exome Sequence Project, in silico functional impact predictions, and observed impacts from databases like ClinVar ( and the Collection Of Somatic Mutations In Cancer (COSMIC) ( (Tier 3) data from The Cancer Genome Atlas sarcoma project was obtained from the Genomic Data Commons Data Portal ( including annotated mutation calls from whole exome sequencing, segmented CNV from SNP arrays, and clinical data. Criteria for inclusion in our analysis included have a listed diagnosis of MPNST with accompanying clinical data to determine syndromic or sporadic and having available processed data from both exome sequencing and SNP array.Variant analysis from whole exome sequencing from both in-house and TCGA sequencing data was performed by filtering variants to include only those that were rare in population databases (VAF < 0.01) and had compelling evidence for oncogenicity either by presence in a hotspot position in COSMIC, have been previously reported to be oncogenic at the position level, or are a loss of function mutation type (nonsense, frameshift, splice site) in a putative tumor suppressor gene. To screen for mutations that involve the Ras-pathway, we utilized the DAVID tool ( for pathway mapping.Copy number analysis from in-house whole exome sequencing data was performed via paired tumor-normal analysis using EXCAVATOR. Results were further filtered to include only segments with ProbCall >0.9 for novel findings or >0.5 for previously described recurrent CNV in this disease. To further focus on likely pathogenic changes, copy number variation from both WES (in-house) and SNP arrays (TCGA) were further filtered to include for reporting to only segments of predicted high level amplification (CN ≥ 8) or complete deletion (CN = 0) in regions that included an oncogene or tumor suppressor gene, respectively. In addition, we evaluated the genomic regions of the five previously reported recurrently affected tumor suppressors in MPNST (NF1, SUZ12, EED, TP53, and CDKN2A) for heterozygous loss (CN = 1).For literature review, a pubmed search was conducted 05/01/2017 using terms “MPNST and sequencing,” “MPNST and genomics”, and “MPNST and PRC2” and the results were manually reviewed. Studies were included for review/analysis if next generation sequencing was performed on at least 5 MPNST. […]

Pipeline specifications

Software tools BaseSpace, Picard, GATK, Strelka, ANNOVAR, DAVID, EXCAVATOR
Databases ClinVar TCGA Data Portal GDC
Application WES analysis
Diseases Neoplasms, Neurilemmoma, Sarcoma