Computational protocol: Read-through transcripts in normal human lung parenchyma are down-regulated in lung adenocarcinoma

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Protocol publication

[…] RNA-Seq was carried out for 64 samples of non-involved lung tissue as previously described []. Qseq files, containing the raw sequencing data, were de-indexed and converted to the Sanger FastQ file format. FastQ sequences were aligned to the human genome assembly (GRCh37/hg19 version) using TopHat v.1.2.0 software []. The resulting Sequence Alignment/Map (SAM) files were quality-tested using SAM-Profiler software [], and only those with high mean read quality (> 30 Phred) were analyzed with FusionAnalyser software []. Candidate chimeric events were considered all those detected by ≥ 10 independent reads with a mean read quality ≥ 25 and with at least 1 read mapping across the predicted exonic breakpoint. These events were further filtered according to their reciprocal mapping and strandness: only adjacent genes mapping to the same gDNA strand were considered as correct read-through predictions and subsequently tested by Sanger sequencing. […]

Pipeline specifications

Software tools TopHat, FusionAnalyser
Application RNA-seq analysis
Organisms Homo sapiens