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Old 04-02-2014, 10:17 AM   #6
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Originally Posted by kajot View Post

Update: I just ran the counting script with -s reverse and it all seems to work. I have now almost 6 million reads that are empty, 1.5 milion ambigous and the rest (around 72.5 million) counted. Since I specified fr-firststrand in Tophat, isn't it somehow affecting my alignment ? Shouldn't it be fr-secondstrand then ? Or am I mixing something up here now ?
fr-firststrand is the correct setting to use in Tophat for TruSeq Stranded libraries so you are fine. What 'fr-firststrand' means is the the first read of the pair (or only read for single end reads) matches the 'first strand of cDNA synthesized', which is anti-sense to the RNA.
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