Question: In tophat-cufflink-cuffmerge pipeline, order of gtf file in assembles.txt file
1
gravatar for illinois.ks
3.9 years ago by
illinois.ks140
Korea, Republic Of
illinois.ks140 wrote:

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 say that 

 

./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 bold line 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 

also changed as 

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

 

 

ADD COMMENTlink modified 3.9 years ago by Devon Ryan89k • written 3.9 years ago by illinois.ks140
2
gravatar for Devon Ryan
3.9 years ago by
Devon Ryan89k
Freiburg, Germany
Devon Ryan89k wrote:

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.

ADD COMMENTlink written 3.9 years ago by Devon Ryan89k

Thank you so much!! :) Got it!

 

K.s 

ADD REPLYlink written 3.9 years ago by illinois.ks140
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