CuffDiff on different data type

Pruexel · 07-08-2013, 06:07 AM

Hello,
I'm working on some RNA-seq data using TopHat/Cufflinks tools.
I need to compare a tissue (3 replicates for each of the 4 time points, paired end) with two other tissues (2 samples, single end, only one time point), is it possible to use CuffDiff?!
I try to run it, but the differential splicing between the first tissue and the second (for example, "day0 tissue1" vs "sample1 tissue2" or "day0 tissue1" vs "sample2 tissue2") doesn't work, but I see a different splicing in expression bar plot in some genes.

Thank you very much.

sqcrft · 07-08-2013, 07:21 AM

please share your code and what did you mean by "doesn't work"?

Pruexel · 07-08-2013, 07:45 AM

cuffdiff -o /CDout -b /Mus_musculus_UCSC_mm9/Mus_musculus/UCSC/mm9/Sequence/Bowtie2Index/genome.fa -p 30 -u /merged_asm/merged.gtf -L day0,day3,day6,day12,BH1,BH2 /dayo_R1/thout/accepted_hits.bam,/day0_R2/thout/accepted_hits.bam,/day0_R3/thout/accepted_hits.bam /day3_R1/thout/accepted_hits.bam,/day3_R2/thout/accepted_hits.bam,/day3_R3/thout/accepted_hits.bam /day6_R1/thout/accepted_hits.bam,/day6_R2/thout/accepted_hits.bam,/day6_R3/thout/accepted_hits.bam /day12_R1/thout/accepted_hits.bam,/day12_R2/thout/accepted_hits.bam,/day12_R3/thout/accepted_hits.bam /tissue2_Sample1/thout/accepted_hits.bam /tissue2_Sample2/thout/accepted_hits.bam
This is the code of CuffDiff, from day0 to day 12 are data from a tissue and at the end there are two sample from a different tissue.
My problem is that I need the different splicing between the tissues (but in tissue 1 must remain the time points) but the test fails (I check the splicing.diff file) when it calculates the JS distance between tissue 1 in a time points and each sample of tissue 2.
Another thing: I can see the different splicing in expression bar plot when I consider some genes with more than one isoforms.

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07-08-2013, 06:07 AM	#1
Pruexel Junior Member Location: Italy Join Date: May 2013 Posts: 4	CuffDiff on different data type Hello, I'm working on some RNA-seq data using TopHat/Cufflinks tools. I need to compare a tissue (3 replicates for each of the 4 time points, paired end) with two other tissues (2 samples, single end, only one time point), is it possible to use CuffDiff?! I try to run it, but the differential splicing between the first tissue and the second (for example, "day0 tissue1" vs "sample1 tissue2" or "day0 tissue1" vs "sample2 tissue2") doesn't work, but I see a different splicing in expression bar plot in some genes. Thank you very much.

07-08-2013, 07:21 AM	#2
sqcrft Member Location: boston Join Date: May 2012 Posts: 29	please share your code and what did you mean by "doesn't work"?

07-08-2013, 07:45 AM	#3
Pruexel Junior Member Location: Italy Join Date: May 2013 Posts: 4	cuffdiff -o /CDout -b /Mus_musculus_UCSC_mm9/Mus_musculus/UCSC/mm9/Sequence/Bowtie2Index/genome.fa -p 30 -u /merged_asm/merged.gtf -L day0,day3,day6,day12,BH1,BH2 /dayo_R1/thout/accepted_hits.bam,/day0_R2/thout/accepted_hits.bam,/day0_R3/thout/accepted_hits.bam /day3_R1/thout/accepted_hits.bam,/day3_R2/thout/accepted_hits.bam,/day3_R3/thout/accepted_hits.bam /day6_R1/thout/accepted_hits.bam,/day6_R2/thout/accepted_hits.bam,/day6_R3/thout/accepted_hits.bam /day12_R1/thout/accepted_hits.bam,/day12_R2/thout/accepted_hits.bam,/day12_R3/thout/accepted_hits.bam /tissue2_Sample1/thout/accepted_hits.bam /tissue2_Sample2/thout/accepted_hits.bam This is the code of CuffDiff, from day0 to day 12 are data from a tissue and at the end there are two sample from a different tissue. My problem is that I need the different splicing between the tissues (but in tissue 1 must remain the time points) but the test fails (I check the splicing.diff file) when it calculates the JS distance between tissue 1 in a time points and each sample of tissue 2. Another thing: I can see the different splicing in expression bar plot when I consider some genes with more than one isoforms.