Cuffdiff with more than two samples

zeam · 10-17-2011, 01:54 AM

Recently,I'm using Cufflinks to process my RNA-seq data,and I have 4 tissues(more than two).In the mannual of cuffdiff,you said,if more than two are provided, Cuffdiff tests for differential expression and regulation between all pairs of samples. But when I used the cuffdiff,it only report one pairs of samples.There was only one gene_exp.diff in the output directory.Command was just as below,
======================================
cuffdiff -o DEG_cuffdiff -p 20 cuffcmp.combined.gtf ./tophat_sd/accepted_hits.bam ./tophat_em/accepted_hits.bam tophat_en1/accepted_hits.bam ./tophat_en2/accepted_hits.bam
======================================
What's my problem in using cuffdiff?
Also I want to compare gene expression of duplicate gene pairs, and is it neccesery to remove the reads that mapped to multiple location in the BAM file generated by Tophat as duplicate gene pairs have high sequence similarity ?
Thanks!

taozuo · 10-17-2011, 02:34 PM

For Question 1:

Yes, Cuffdiff would only generate one output even though you have more than one comparisons. However, all other comparisons are also included in that file. Check the output file more carefully.

zeam · 10-17-2011, 05:49 PM

In fact, I have checked the outpu directory, and there are only 11 files as follows:cds.diff , cds_exp.diff,genes.fpkm_tracking, isoforms.fpkm_tracking ,splicing.diff, tss_groups.fpkm_tracking , cds.fpkm_tracking , gene_exp.diff isoform_exp.diff , promoters.diff , tss_group_exp.diff. And I did not find any other comparison files between genes.
I have posted this problem on the other thread ,and his met the similar issue.
http://seqanswers.com/forums/showthr...4104#post54104

taozuo · 10-17-2011, 08:28 PM

I am not quite sure right now for your case. But I did this before and I can get all the comparisons. For example, if I open the gene_exp.diff file, I will have the q1 vs q2 first. And when I continue check the result by clicking pagedown, I will find q1 vs q3 and etc.

zeam · 10-17-2011, 11:36 PM

Now I know all of results of the comparisons betweem pairs are saved in one file.
Thanks!

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10-17-2011, 01:54 AM	#1
zeam Member Location: USA Join Date: Oct 2010 Posts: 40	Cuffdiff with more than two samples Recently,I'm using Cufflinks to process my RNA-seq data,and I have 4 tissues(more than two).In the mannual of cuffdiff,you said,if more than two are provided, Cuffdiff tests for differential expression and regulation between all pairs of samples. But when I used the cuffdiff,it only report one pairs of samples.There was only one gene_exp.diff in the output directory.Command was just as below, ====================================== cuffdiff -o DEG_cuffdiff -p 20 cuffcmp.combined.gtf ./tophat_sd/accepted_hits.bam ./tophat_em/accepted_hits.bam tophat_en1/accepted_hits.bam ./tophat_en2/accepted_hits.bam ====================================== What's my problem in using cuffdiff? Also I want to compare gene expression of duplicate gene pairs, and is it neccesery to remove the reads that mapped to multiple location in the BAM file generated by Tophat as duplicate gene pairs have high sequence similarity ? Thanks!

10-17-2011, 05:49 PM	#3
zeam Member Location: USA Join Date: Oct 2010 Posts: 40	I have checked the outpu directory. In fact, I have checked the outpu directory, and there are only 11 files as follows:cds.diff , cds_exp.diff,genes.fpkm_tracking, isoforms.fpkm_tracking ,splicing.diff, tss_groups.fpkm_tracking , cds.fpkm_tracking , gene_exp.diff isoform_exp.diff , promoters.diff , tss_group_exp.diff. And I did not find any other comparison files between genes. I have posted this problem on the other thread ,and his met the similar issue. http://seqanswers.com/forums/showthr...4104#post54104

10-17-2011, 11:36 PM	#5
zeam Member Location: USA Join Date: Oct 2010 Posts: 40	Thanks for your help Now I know all of results of the comparisons betweem pairs are saved in one file. Thanks!

10-17-2011, 02:34 PM	#2
taozuo Junior Member Location: ames, IA Join Date: Jul 2011 Posts: 8	For Question 1: Yes, Cuffdiff would only generate one output even though you have more than one comparisons. However, all other comparisons are also included in that file. Check the output file more carefully.

10-17-2011, 08:28 PM	#4
taozuo Junior Member Location: ames, IA Join Date: Jul 2011 Posts: 8	I am not quite sure right now for your case. But I did this before and I can get all the comparisons. For example, if I open the gene_exp.diff file, I will have the q1 vs q2 first. And when I continue check the result by clicking pagedown, I will find q1 vs q3 and etc.