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.

On Wed, Feb 22, 2012 at 12:29 PM, Ravi Karra <ravi.karra@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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