Hello,

I posted to the seqanswers forum, but have not received any feedback.  I am 
working with RNA-seq Illumina data files in Galaxy 
(http://main.g2.bx.psu.edu/). The two files are 100bp paired-end reads, 
multiplexed with barcoding to distinguish samples. The barcodes are the first 
four bases of the sequences in the s_7_1_sequence.txt file.

Would the following Galaxy workflow be correct?

1. Upload both s_7_1_sequence.txt and s_7_2_sequence.txt to Galaxy with the 
reference genome selected
2. Run NGS: QC and manipulation --> FASTQ Groomer on each file to convert to 
Sanger FASTQ
3. Run NGS: QC and manipulation --> FASTQ joiner to combine the data from the 
two files
4. Run FASTX-TOOLKIT FOR FASTQ DATA --> Barcode Splitter to generate separate 
FASTQ files for each barcode group
5. Run NGS: RNA Analysis --> Tophat to map the reads from each group to the 
reference genome

The problem I am having is that if I select paired-end for the library in 
Tophat, it requests two FASTQ files. Would I have to use FASTQ Splitter to 
separate the joined FASTQ files? If there is a more standard way to handle 
these types of barcoded files, I would appreciate hearing about this workflow.

Thanks very much in advance,
jjw

P.S. Galaxy is an incredibly useful resource.  Thanks!



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