In tophat-cufflink-cuffmerge pipeline, order of gtf file in assembles.txt file

Question

[illinois.ks]

Hello

I have somehow naive questions.

I have two conditions which I would like to compare. In each condition, we have three replicates.

When, I try to do the cuffmerge command (based on nature protocol paper, 2012,

assemblies.txt file says

./C1_R1_clout/transcripts.gtf
./C2_R2_clout/transcripts.gtf
./C1_R2_clout/transcripts.gtf
./C2_R1_clout/transcripts.gtf
./C1_R3_clout/transcripts.gtf
./C2_R3_clout/transcripts.gtf

but I think that the second and fourth lines should be changed as follows.. Is it right? (I am not sure)

(since current Corrigendum doesn't mention about it, I am wondering whether I am right or not..)

./C1_R1_clout/transcripts.gtf
./C2_R1_clout/transcripts.gtf
./C1_R2_clout/transcripts.gtf
./C2_R2_clout/transcripts.gtf
./C1_R3_clout/transcripts.gtf
./C2_R3_clout/transcripts.gtf

Also, cuffdiff command,

cuffdiff \
  -o diff_out \
  -b genome.fa \
  -p 8 \
  -L C1,C2 \
  -u merged_asm/merged.gtf \
  ./C1_R1_thout/accepted_hits.bam,./C1_R2_thout/accepted_hits.bam,./C1_R3_thout/ accepted_hits.bam \
  ./C2_R1_thout/accepted_hits.bam,./C2_R3_thout/accepted_hits.bam,./C2_R2_thout/ accepted_hits.bam

changed to:

cuffdiff \
  -o diff_out \
  -b genome.fa \
  -p 8 \
  -L C1,C2 \
  -u merged_asm/merged.gtf \
  ./C1_R1_thout/accepted_hits.bam,./C1_R2_thout/accepted_hits.bam,./C1_R3_thout/ accepted_hits.bam \
  ./C2_R1_thout/accepted_hits.bam,./C2_R2_thout/accepted_hits.bam,./C2_R3_thout/ accepted_hits.bam

Is it right? OR I am missing something?

I realized that the order of gtf file in assemblies.txt makes difference in cuffmerge result.

So, I want to make sure I am right..

Thanks in advance

Answer 1

[Devon Ryan]

For cuffmerge the order shouldn't matter, since you're constructing a merged annotation file and not trying to do any quantification.

For cuffdiff, the order within groups shouldn't matter (in essence, you're comparing group means and that's order independent), just don't mix up samples between groups. Having said that, I think most of us would use the order you prefer just for the sake of sanity.

[illinois.ks]

Thank you so much!! :) Got it!

K.s