Computational protocol: Chronic sun exposure-related fusion oncogenes EGFR-PPARGC1A in cutaneous squamous cell carcinoma

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Protocol publication

[…] The transcriptome analysis was performed using Illumina TruSeq RNA Sample Preparation Kit (San Diego, CA) according to the protocol provided by Riken Genesis (Kanagawa, Japan). In brief, poly-A-containing mRNA was purified from high quality total RNA using poly-T oligo-conjugated magnetic beads. The mRNA was fragmented by divalent cations, and the cleaved RNA fragments were transcribed into first-strand cDNA using random primers and SuperScriptII Reverse Transcriptase (Invitrogen, Carlsbad, CA), followed by second strand cDNA synthesis using DNA polymerase I and RNase H. The cDNA fragments were then subjected to an end repair process, A-base addition, and the ligation of adapters, to convert them into a library of template molecules suitable for the subsequent cluster generation. The product was purified and enriched by PCR to create final cDNA library. The library was sequenced in a paired-end 100-bp configuration on Illumina HiSeq2000 platform. Adapter sequences and low-quality sequences were eliminated using Cutadapt (v1.0) software. After performing quality control, the poly-A/T sequences were also eliminated using PRINSEQ (v0.16). [...] The cleaned and trimmed reads were aligned to the reference human sequence (GRCh37/hg19) using TopHat (v1.4.0), a fast splice junction mapping software that uses Bowtie alignment to align reads–. The mapped reads were assembled using Cufflinks (v2.0.0) software, and the transcripts across all samples were merged using Cuffmerge program. The reference GTF annotation file used in Cufflinks was downloaded from iGenomes database.The differential expression between samples was analyzed using Cuffdiff software by calculating the fragments per kilobase per million map reads (FPKM) and by testing the statistical significance of the differences. A bioinformatic analysis was performed to detect mutations using Samtools (v1.0). Potential gene fusion transcripts were identified and filtered using deFuse (v0.61) and Fusion Hunter (v1.4). […]

Pipeline specifications

Software tools TopHat, Bowtie, Cufflinks, SAMtools, deFuse
Application RNA-seq analysis
Organisms Homo sapiens
Chemicals Tyrosine