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Old 11-26-2014, 07:12 AM   #1
Location: Wisconsin

Join Date: Jun 2011
Posts: 87
Default Extract exon-intron reads from RNA-Seq data


We are trying to look at potential DNA contamination in our RNA/cDNA libraries using the RNA-Seq data (single-end Ion Torrent). One method of doing this seems to be looking at reads that start in the exon and "run into" the intron or vice versa. I thought of may be creating a custom annotation which will potentially have these "junctions" as the start and end and then use HTSeq or bedtools (intersect or coverage) to quantify/extract these reads. However, I am not quite sure of how to configure my intesect (customize the -r and -f parameters) or HTSeq command and also the annotation file.

Any suggestions on this and also if anybody has a different bioinformatics way of looking at DNA contamination will be greatly appreciated.

Thank you,
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Old 11-26-2014, 08:17 AM   #2
Richard Finney
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Location: bethesda

Join Date: Feb 2009
Posts: 700

Though procedures may be supposed to only corral mature RNA transcripts, immature transcripts often remain. This is not "contamination". Additionally there may be functional intronic transcripts. Additionally, very short "spillover" into the intron maybe inability of aligner to identify the "next" exon. People have gotten burned thinking these are evidence for mutations.
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bioinformatics, ion torrent

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