Great advice Sean! Jianguang, this is the correct analysis - mapping the data to test the actual insert size of the library as sequenced. The experimental notes at SRA are just a starting place, the data is truth. A sample through TopHat itself might produce more precise results. I suspect the coverage on your top Blastn HSP is not complete, breaking off where it hits a splice. And that you have some bias for sequences/hits that cross junctions near ends. But overall, none of this would likely make that much of a difference in the analysis as a whole. Good luck! Jen Galaxy team On 8/15/12 8:39 AM, Sean Davis wrote:
On Wed, Aug 15, 2012 at 11:13 AM, Du, Jianguang <jiandu@iupui.edu <mailto:jiandu@iupui.edu>> wrote:
Dear All,
I am analyzing the downloaded RNA-seq datasets. However I am not sure how much is Mean Inner Distance between Mate Pairs for these paired-end datasets.
Take a paired-end RNA-seq dataset as an example, there is a description for this dataset in SRA database of NCBI: "Layout: PAIRED/, Orientation: /5'-3'-3'-5'/, Nominal length: /400/, Nominal Std Dev: /20"
At first I thought the Mean Inner Distance between Mate Pairs should be 325bps because the length of reads on both ends is 36bps. However when I aligned the sequence of the paired reads on to transcripts and genome using BLASTn, the distance between the paired reads is about 200bps. How should I decide the Mean Inner Distance between Mate Pairs in my case?
The information from SRA is likely only an approximation. SRA does not validate these details, I do not think.
You can probably use the distribution from your data as the best estimate.
Sean
Thanks.
Jianguang Du
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