Cuffdiff Error: Cuffdiff Requires At Least 2 Sam Files

Question

[Lisanne]

Dear All,

I am really confused about the cuffdiff function in cufflinks. I thought that cuffdiff needs at least to BAM or SAM files. I have 2 accepted_hits.sam files and one combined_transcripts.gtf. I try this:

cuffdiff -o A1_A3 -q A1_A2.combined.gtf A1/A1_accepted_hits.sam A3/A3_acctepted_hits.sam ERROR: cuffdiff requires at least 2 SAM files

And for the BAM files: cuffdiff -o A1_A3 -q A1_A2.combined.gtf A1/A1_accepted_hits.bam A3/A3_acctepted_hits.bam ERROR: cuffdiff requires at least 2 SAM files

Can anyone please tell me what I am doing wrong? Thanks!

Answer 1

[Dinesh Cyanam]

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.

Answer 2

[Sta]

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]

i use version 1.3.0

Answer 3

[Bontus]

@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

[aditisk]

I am also facing a similar issue. My command is as follows:

cuffdiff -p 6 -o ./UM108_cuffdiff -u --compatible-hits-norm -M Homo_sapiens.GRCh37.75.gtf \ 61327_tophat.bam \ 61328_tophat.bam

I also tried the same command as a single line and I still get this error - "Error: cuffdiff requires at least 2 SAM files"

Could someone please help me out with this ?

Thanks, Aditi