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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