Hi, I believe you are receiving the error because you misspelled accepted in your A3 file. The file name should be A3_accepted_hits.sam

Also, make sure that the GTFs and the SAM and BAM files are in the given path and I would use the combined GTF of sample A1 and A3 and not A1 and A2 as you mentioned above.

i have the same prblem with my data.

I use the command:

cuffdiff - diffout -b /home/thx/genome.fa -p 8 -L /home/sta/Gen/accepted_hits.bam /home/sta/Gen2/accepted_hits.bam -u /home/sta/gtf/mm9.gtf

ERROR: cuffdiff requires at least 2 SAM files

but... if i try 2 convert bam into sam it is also not working!

Command here: samtools view -bt /home/sta/index/mm9.fa accepted_hits.sam

Can anyone help here?

@STA: Given the information in the manual http://cufflinks.cbcb.umd.edu/manual.html#cuffdiff

you use the flag for labelling groups -L without specifying any group labels in this command: cuffdiff - diffout -b /home/thx/genome.fa -p 8 -L /home/sta/Gen/accepted_hits.bam /home/sta/Gen2/accepted_hits.bam -u /home/sta/gtf/mm9.gtf

for using labels, try: cuffdiff -o diffout -b /home/thx/genome.fa -p 8 -L C1,C2 -u /home/sta/gtf/mm9.gtf /home/sta/Gen/accepted_hits.bam /home/sta/Gen2/accepted_hits.bam

or without: cuffdiff -o diffout -b /home/thx/genome.fa -p 8 -u /home/sta/gtf/mm9.gtf /home/sta/Gen/accepted_hits.bam /home/sta/Gen2/accepted_hits.bam