Computational protocol: Whole-exome analysis in osteosarcoma to identify a personalized therapy

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Protocol publication

[…] We sequenced the whole exomes of eight high-grade osteosarcoma tissues using NextSeq550 Illumina platform. The instrument generated approximately an average read length of 120.6 bases. After mapping to the human reference genome GRCh37 (hg19) using Burrows-Wheeler Alignment tool (BWA), we obtained the average depth of each base in the target regions as 17.7x. The average coverage of target regions was 121.7x.The raw data generated from Illumina NextSeq550 were converted using Bcl2toFastq tools provided by Illumina. The data analysis of exomes was performed by using the SeqMule pipeline1. The final Variant Call Format files (VCFs) were uploaded on the VariantStudio Illumina (Illumina San Diego, CA, USA) software to perform annotation and filtration steps. We filtered genomic data by quality score of 30, by mean read depth of 5, and by GMAF ≤ 1 (Global Minor Allele Frequency: the frequency of the second most frequent allele in the population). Next, the detected mutations were filtered for the variants absent in database of Single Nucleotide Polymorphisms (dbSNP) to distinguish known somatic mutations from germline mutations. We further selected the somatic variations in the coding sequence by excluding those variations in flanking sequences like splicing sites, 5ʹ-UTR, 3ʹUTR, introns and intergenic regions. Moreover, we performed bioinformatics analysis using SIFT (v4.0.3) and PolyPhen-2 (v2.1.0) tools, that predict whether an amino acid substitution affects protein function based on sequence homology. To be sure that the variations to study were only those that caused a protein alteration, we selected only those that were at the same time deleterious for SIFT and probably damaging for PolyPhen-2. […]

Pipeline specifications

Software tools BWA, SeqMule, VariantStudio, PolyPhen
Application WES analysis
Diseases Bone Neoplasms, Neoplasms, Osteosarcoma, Genetic Diseases, Inborn