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 <jia...@iupui.edu
<mailto:jia...@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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