Hi,

I think you need to first remove the adaptors and then trim the reads.
 That is probably the correct way.  As for the second part of the question,
you could try a rudimentary way to actually search for a sequence header.
 I have seen this different sizes in the r1 and r2 read files, but taken
together almost 90% turn out to be true the paired reads.

Hope this helps,
Sameet

On Wed, Feb 22, 2012 at 12:29 PM, Ravi Karra <ravi.ka...@gmail.com> wrote:

> Hello,
>
> I have Illumina 76bp paired end data for a zebrafish RNA-seq experiment
> and am basically stuck while trying to pre-process my data prior to using
> Tophat/CuffDiff.
>
> For each sample, I have a read1 fastq file and a paired read2 fastq file.
>  After using FASTQ Groomer, I trimmed the ends using FASTQ quality trimmer
> with a threshold quality score of 20 ans a window size of 1 (I think that
> will essentially lop off the end of the read until the quality score is >=
> 20).  Next, I trimmed the adapters using Clip.
>
> What I am left with is a modified read1 fastq file and a modified read2
> file, where the pairs are not in the same order and some reads are left
> without pairs. From what I have read, I don't think TopHat can incorporate
> paired end data that is out of order.. I tried to get around the ordering
> issue using FASTQ joiner, but this tool is not able to join the reads
> (return is 0 joined reads).  I am not really sure why FASTQ joiner didn't
> work for me and am looking for suggestions of what to try next.
>
> Thanks!
> ravi
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-- 
Sameet Mehta, Ph.D.,
Phone:  (301) 842-4791
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