Computational protocol: TIAM1 variants improve clinical outcome in neuroblastoma

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Protocol publication

[…] Sequencing analysis was performed on the Genomic Unit of La Fe Hospital Research Institute with the Ion PGM™ / Ion PROTON™ system (Life Technologies). Amplicon library was prepared using the Ion Ampliseq Library kit 2.0 (Life Technologies). Briefly, multiplex primer pool was added to 10 ng of genomic DNA and amplified with the following PCR cycles: 99°C for 2 min, followed by 99°C for 15 s and at 60°C for 4 min (17 cycles), and holding on at 10°C. Primers are partially digested using a FuPa reagent, and then sequencing adapters were ligated to the amplicons. The final libraries were purified with Agentcourt AMPure XP (Beckman Coulter) and quantified with Qubit DS HS assay Kit (Life Technologies). DNA template preparation was performed with the semiautomated Ion OneTouch™ 2 instrument and Ion One Touch enrichment system (ES) (Life Technologies). Finally DNA high-throughput sequencing was performed on PGM or Proton instrument. Ion PGM™ HiQ Sequencing kit on the Ion 318 sequencing chip (Life Technologies) was used for PGM instrument. In case of PROTON instrument, we used Ion PI™ HiQ Sequencing Kit on the Ion PI sequencing chip (Life Technologies).Data from the Ion Torrent runs were analysed using the platform-specific pipeline software Torrent Suite v5.0 for base calling, trim adapter and primer sequences, filtering out poor quality reads and no call reads according to the barcode sequences. The sequence variants in each sample were identified using the Torrent Suite Variant Caller (TSVC; v4.0-r76860) plug-in and browser extensible data (BED) files (chromosome coordinates) that specify the coding regions of the target genes within the human reference genome (hg19) retrieved from the NCBI database (build 37) as a reference. Finally, Ion Reporter software (https://ionreporter.thermofisher.com) was used to further annotate variant caller format (VCF) files and Integrated Genomic Viewer (IGV) software [] was then used to complete the visualization and to discriminate false-positive variants. We considered only variants with balanced numbers of forward and reverse reads. Based on the American College of Medical Genetics and Genomics and the Association for Molecular Pathology [], variants with minor allele frequency (MAF) of > 1% in 1000G were considered polymorphisms and were excluded from further analyses. Investigation about the potential pathogenic role was done using databases (COSMIC, dbSNP, ClinVar (NCBI)), prediction algorithms (SIFT, PolyPhen-2 or Mutation Taster) or splice prediction tools (SpliceView, NetGene2, Human Splicing Finder, NNSplice). […]

Pipeline specifications

Software tools IGV, PolyPhen, SpliceView, NetGene2, NNSplice
Application WGS analysis