Can someone explain to me what the orientation in paired-end RNA-seq data means? Is forward/forward the standard orientation? How does this affect tophat options, just specify unstranded Rna-seq?
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What protocol did you use for library preparation? Normally for ScriptSeq Illumina libraries, the forward reads would correspond to the DNA coding strand while the reverse reads would be reverse complements, -
If you are only looking for a primer on the "paired end" part have a look at this blog entry: http://www.homolog.us/blogs/blog/201...looking-reads/ You are less likely to have the second kind of libraries mentioned in the post.Comment
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I'm not sure what the 'standard orientation' is.
But if you want to know more about read-orientation then take a look at these threads:
http://www.biostars.org/p/49152/
And while 'unstranded' is the default mode for tophat/bowtie, you need to change that if your library is something else.Comment
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Well, I think the quickest test to assess if they are directional and how forward to reverse are handled in the librarires, you could map to a couple of genes and have a look in e.g. IGV to see if it makes sense. Or easier extract some read pairs from the raw sequencing files, and have a look how they align with BLAT.Originally posted by Nick View PostComment
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