Hi JIwen,
As the seqanswers thread shows, there is some debate about this. One of the last posts there makes the most sense - where "fragment length" is defined as the total genome bases covered by the aligned paired sequences: tip of 5' start, through the gap, to the very 3' tail end and where "mean inner distance" is the gap in the middle (between the paired seqs) where there is no alignment. This could be tested with smaller samples of data and compared to your fragment selection, to see how it matches up. But, for a definitive answer, asking the tool authors is the best bet. The contact information is: tophat.cufflinks@gmail.com
If you are given a reply that explains the calculation, it would be great if the TopHat documentation itself were updated. http://tophat.cbcb.umd.edu/manual.html
And we would be very glad to hear of the details, so that here at Galaxy we could add the information to our RNA-seq help and the searchable mailing list archives.
Great question! If we find out ourselves meanwhile, an update will be posted back here to the mailing list,
Best,
Jen Galaxy team
On 3/6/12 12:23 PM, 杨继文 wrote:
Hi all, When mapping pair end RNA-seq reads using tophat, we need to type in "Mean Inner Distance between Mate Pairs". In galaxy, we can read the following information:
This is the expected (mean) inner distance between mate pairs. For, example, for paired end runs with fragments selected at 300bp, where each end is 50bp, you should set -r to be 200. There is no default, and this parameter is required for paired end runs.
I think the size of fragment (here 300bp) includes not only the length of pair end reads, but also the length of adaptors. so, maybe the Mean Inner Distance between Mate Pairs should be : fragment length - pair end read length - adaptor length. Am I right? or did I miss something?
Is it a must to type in the accurate value?
Looking forward to your reply
JIwen
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