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Old 07-16-2013, 09:09 AM   #1
JJP
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Default Tophat2 normal alignment rate

Hi there, I am a complete RNA seq newbie, so let me excuse myself preventively for any stupid questions.

Recently I started working with mouse RNA seq data, after running the data though Tophat2 I got a 91.0% overall read alignment rate and a 83.1% concordant pair alignment rate.

Could somebody with more experience in the field tell me if this are good alignment values, and if not which are the normal values I should expect ?

Thanks in advanced.
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Old 07-17-2013, 08:37 PM   #2
Jon_Keats
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what read length, not bad if 100mers
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Old 07-18-2013, 01:19 AM   #3
JJP
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Sorry I knew I had forgotten to mention something, the reads have a length of 101.

Last edited by JJP; 07-18-2013 at 04:56 AM.
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Old 07-30-2013, 06:07 PM   #4
jp.
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Question

Dear Jon
I have posted the same problem here;
http://seqanswers.com/forums/showthr...098#post112098
any solution ?

Quote:
Originally Posted by Jon_Keats View Post
what read length, not bad if 100mers
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Old 08-08-2013, 01:55 PM   #5
rnastar
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I just finished running tophat on about 20 cancer samples and my concordant pair alignment rate ranged between 50-60% for 50 bp reads. Should I be worried? Will this have an impact on the downstream cufflinks/cuffdiff analysis?
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Old 08-09-2013, 06:07 AM   #6
westerman
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I presume you are talking about *human* samples. The 50-60% rate is low. I suspect that since the samples are more-or-less uniform in their mapping rate then you won't have too many problems with cufflinks/cuffdiff aside from not having as much data to work with.

Did you run non-cancer controls? Or are those part of the "20" you mention above. How about replicates -- technical and biological?
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Old 08-12-2013, 06:08 AM   #7
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Yes, human samples. We did run non-cancer controls but they did not have this problem. What do you think this means? Is this more a reflection of biology or an indication of a technical issue? We do not have any technical replicates, only biological.
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Old 08-12-2013, 09:16 AM   #8
westerman
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The lack of technical replicates is not a big concern -- although they can help troubleshooting usually the technical process is flawless. Although when problems do occur you really wish that you had technical reps. :-)

As for the question "is a 50-60% concordant rate normal for cancer samples when the non-cancer samples have a higher concordant rate?" ... I am going to have to skip on answering this. I simply do not have enough experience with human cancer samples to say one way or another. Cancer genome rearrangements can be widespread so may 50-60% if expected. Can anyone with more human experience chime in?

BTW: You may wish to make a new thread on this topic with the keywords "human"
and "cancer" in it. That might attract more knowledgeable people.
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