error in cuffdiff ---(mutiple sample comparision)

Question

[megha.24may]

Hello Everyone,

I am using cuffdiff to compare my 2samples against the control here I am using following command for analysis.

cuffdiff \
  -p 10 \
  -o /home/pinky/Desktop/NGS/diff_out/son/ \
  --labels conditionA1, conditionB1, conditionC1 \
  -b /home/pinky/Desktop/NGS/conindex/rice_exc.fasta \
  -u /home/pinky/Desktop/NGS/cuffmerge/sonexp/transcripts.gtf \
  /home/pinky/Desktop/NGS/tophat_fasta/sample19/accepted_hits.bam, /home/pinky/Desktop/NGS/tophat_fasta/sample20/accepted_hits.bam /home/pinky/Desktop/NGS/tophat_fasta/sample21/accepted_hits.bam /home/pinky/Desktop/NGS/tophat_fasta/sample25/accepted_hits.bam, /home/pinky/Desktop/NGS/tophat_fasta/sample26/accepted_hits.bam, /home/pinky/Desktop/NGS/tophat_fasta/sample27/accepted_hits.bam /home/pinky/Desktop/NGS/tophat_fasta/sample31_1/accepted_hits.bam, /home/pinky/Desktop/NGS/tophat_fasta/sample32/accepted_hits.bam, /home/pinky/Desktop/NGS/tophat_fasta/sample33/accepted_hits.bam

Warning: Your version of Cufflinks is not up-to-date. It is recommended that you upgrade to Cufflinks v2.2.1 to benefit from the most recent features and bug fixes (http://cufflinks.cbcb.umd.edu).
Error: number of labels must match number of conditions

but instead of proper run I am getting this error. So I manipulated it in this way to resolve this.

cuffdiff \
  -p 10 \
  -o /home/pinky/Desktop/NGS/diff_out/son/ \
  --labels conditionA1, conditionA2, conditionA3, conditionB1, conditionB2, conditionB3, conditionC1, conditionC2, conditionC3 \
  -b /home/pinky/Desktop/NGS/conindex/rice_exc.fasta \
  -u /home/pinky/Desktop/NGS/cuffmerge/sonexp/transcripts.gtf /home/pinky/Desktop/NGS/tophat_fasta/sample19/accepted_hits.bam, /home/pinky/Desktop/NGS/tophat_fasta/sample20/accepted_hits.bam /home/pinky/Desktop/NGS/tophat_fasta/sample21/accepted_hits.bam /home/pinky/Desktop/NGS/tophat_fasta/sample25/accepted_hits.bam, /home/pinky/Desktop/NGS/tophat_fasta/sample26/accepted_hits.bam, /home/pinky/Desktop/NGS/tophat_fasta/sample27/accepted_hits.bam /home/pinky/Desktop/NGS/tophat_fasta/sample31_1/accepted_hits.bam, /home/pinky/Desktop/NGS/tophat_fasta/sample32/accepted_hits.bam, /home/pinky/Desktop/NGS/tophat_fasta/sample33/accepted_hits.bam

but still the problem remains same. so will you please guide or suggest something.

Thanks
Megha Varshney

[megha.24may]

Thanks @Goutham Atla and @ Daniel, I got solved my problem as previously I had made some mistakes.

Answer 1

[Daniel]

Remove the spaces between your commas, as cuffdiff is interpreting them as separate strings (it is expecting one comma separated list).

What you want to provide is the same number of strings as your conditions, so it looks like this but with full paths:

cuffdiff -p 10 -o diff_out/son/ --labels conditionA1,conditionB1,conditionC1 -b rice_exc.fasta -u transcripts.gtf sample1.bam,sample2.bam,sample3.bam sample4.bam,sample5.bam,sample6.bam sample7.bam,sample8.bam,sample9.bam

If it would make it easier to read, you can separate your string lines with a \ (press: space-backslash-newline) which only counts as a space, but separates your text, like so:

cuffdiff -p 10 -o diff_out/son/ --labels conditionA1,conditionB1,conditionC1 -b rice_exc.fasta -u transcripts.gtf \
sample1.bam,sample2.bam,sample3.bam \
sample4.bam,sample5.bam,sample6.bam \
sample7.bam,sample8.bam,sample9.bam

Answer 2

[GouthamAtla]

From your command, it looks like that you have four groups of bam files (Each group is separated by space, within each groups replicates are separated by comma) and 8 conditions. Each condition separated by comma.

Basic format is

-L  A,B,C   SampleA_rep1.bam,SampleA_rep2.bam,SampleA_rep3.bam      SampleB_rep1.bam,SampleB_rep2.bam,SampleB_rep3.bam    SampleC_rep1.bam,SampleC_rep2.bam,SampleC_rep3.bam

The documentation may be misleading as it says "Specify a label for each sample, which will be included in various output files produced by Cuffdiff" but label should be given for each group.