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Old 07-12-2014, 06:55 AM   #9
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Location: China

Join Date: Feb 2009
Posts: 116

Originally Posted by kmcarr View Post
So if I am following your logic you:

1. Map your original reads to some reference with Tophat2.

2. Extract the unmapped reads from unmapped.bam to a new FastQ

3. Map the unmapped reads to some reference with Tophat2. I am going to assume that this mapping is to some different reference since trying to align unmapped reads back to the same reference wouldn't make sense.

I have highlighted the problem in red above. You correctly state "The base quality is phred+33", but then you incorrectly set "--solexa-quals" in your tophat command, which is telling tophat that your FastQ file is phred+64. Drop "--solexa-quals" from your second tophat alignment (step 3). Phred+33 is the default for tophat2 so there is no command line option used to specify it.
thank you kmcarr, as you found, I have wrongly set the quality parameter for tophat2.

Your suggestion is very useful and help me a lot.

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