Dear forum,
I have been using RNA-seq for some time now and have a rather basic question:
When doing paired-end data, I have to provide TopHat with the inner distance of the mate pairs/paired ends (parameter: -r). But when I align them to a reference (which is obviously DNA) and my RNA-seq data is cDNA (so introns left out), then there should be a bias regarding the inner distance. The question came up when I tested the best value for the -r option by setting different (meaningful) values and checking them with samtools flagstat. On one value the statistics told me that there were around 30% properly mapped (seems bad) and the other value had 70% (rather good). When I assume that there are also introns in the DNA that are left out, which value fits better and is it even useful to give this parameter? Ok, TopHat forces me to do so ;-) and actually it cares about splice junctions. But the procedure is not clear to me and if this properly mapped statistic is a useful one in this context.
I'm happy about any advice!
Best,
Oliver
I have been using RNA-seq for some time now and have a rather basic question:
When doing paired-end data, I have to provide TopHat with the inner distance of the mate pairs/paired ends (parameter: -r). But when I align them to a reference (which is obviously DNA) and my RNA-seq data is cDNA (so introns left out), then there should be a bias regarding the inner distance. The question came up when I tested the best value for the -r option by setting different (meaningful) values and checking them with samtools flagstat. On one value the statistics told me that there were around 30% properly mapped (seems bad) and the other value had 70% (rather good). When I assume that there are also introns in the DNA that are left out, which value fits better and is it even useful to give this parameter? Ok, TopHat forces me to do so ;-) and actually it cares about splice junctions. But the procedure is not clear to me and if this properly mapped statistic is a useful one in this context.
I'm happy about any advice!
Best,
Oliver
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