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