Hello!

                I have 10 human RNA-Seq samples consisting of 3 groups (2 
replicates per group).  I have already run each of them through TopHat and 
Cufflinks on the Penn State Galaxy instance.  I am now at a head-scratching 
moment.  I want to use CuffCompare next (in the end I will want to run CuffDiff 
so that I can determine the gene/isoform expression between these 3 groups) but 
am unsure of the best way to do this.  After reading several Galaxy posts, I've 
come across a couple of ideas:

1.       Run CuffCompare on two Cufflinks output files.  When that is finished 
take the CuffCompare output file and run it again in CuffCompare with the third 
Cufflinks output file sample.  When this is finished, take that CuffCompare 
output file and run it again in CuffCompare with the fourth Cufllinks output 
file sample, etc... (I hope you catch my drift as to where this is going).  In 
a nutshell I will be repeatedly merging Cufflinks outputs in CuffCompare.  Then 
when all 10 have been put through CuffCompare, then I can run CuffDiff and set 
up 3 groups in CuffDiff with their appropriate BAM files from TopHat.

2.       Add all 10 Cufflinks output files in CuffCompare using the "add new 
GTF input file" option.

                I chose step two because it looked the simplest and from the 
posts I read, it sounded like this was a fully functional option.  I was also 
using a reference annotation file as well (that file has worked before in the 
past on non-replicate analyses).  However, I came across an error:
Error running cuffcompare. You are using Cufflinks v1.0.3, which is the most 
recent release.
No fasta index found for ./input1. Rebuilding, please wait..
Error: sequence lines in a FASTA record must have the same length!


cuffcompare v1.0.3 (2403)

cuffcompare -o cc_output  -r 
/galaxy/main_database/files/002/678/dataset_2678888.dat  -R  -s  ./input1 
./input2 ./input3 ./input4 ./input5 ./input6

                Any suggestions as to why this is happening?  Am I trying 
something that shouldn't be attempted yet?  Is there a better alternative to 
analyzing replicates?  Any suggestions/ideas/workflows/you name it would be 
greatly appreciated!!

Thanks,
David


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