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Pipeline publication

[…] 2 if have been associated with recessive cancer syndromes. All variants were manually checked with IGV., RNA was isolated using TRIzol reagent (Thermo Fisher Scientific, MA, USA). RNA with a RIN of 8 was used to construct a library using Illumina´s TruSeq RNA kit, following the manufacturer´s instructions. The paired-end library was sequenced using a GAIIx equipment (Illumina, CA) in a 72 bp configuration. After quality control and trimming, the reads were aligned with the STAR aligner and the resulting SAM was further processed with the PICARD tool to recalibrate reads. Finally, we called and filtered variants using the Haplotype Caller from GATK. Fusion transcripts were obtained with the TopHat-fusion pipeline. To quantify differentially-expressed transcripts, we realigned and processed., RNASeq data from 10 normal human fibroblasts samples (GEO dataset GSE51518 from the NCBI) together with the ovarian fibrosarcoma data and used RSEM and the R package EBSeq to normalize, quantitate and compare the expression data. microRNA analysis was performed using a miRNA 4.0 array (Affymetrix, Santa Clara, CA). A normal ovary cell line cel file was obtained from the GEO information system (GSE76449) for comparison. Quality control, background subtracted, quantile normalized and log2- transformed using robust multi-array analysis (RMA) and differential expression analysis of the three cel files were done with Partek v 6.6. Candidate miRNAs were considered to be differentially-regulated if they presented a Fold Change >  = 2, p-values < 0.05 and FDR less than 0.05., Copy-number variants (CNV) were predicted using Control-Freec ver 8.0, calculatin […]

Pipeline specifications

Software tools TopHat-Fusion, RSEM, EBSeq