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Old 03-19-2014, 07:45 PM   #1
edge
Senior Member
 
Location: China

Join Date: Sep 2009
Posts: 199
Default Problem faced to deal with multisample by using Cuffdiff

Hi,

I have 10 RNA-seq samples now.
Samples Times (hours after inoculation)
Susceptible NIL 0
Susceptible NIL 3
Susceptible NIL 6
Susceptible NIL 9
Susceptible NIL 12
Resistant NIL 0
Resistant NIL 3
Resistant NIL 6
Resistant NIL 9
Resistant NIL 12

My main objective is to identify and characterize resistant genes associated to disease/bacteria-infection in my plant samples.
I have a reference genome and reference annotation of my plant samples.
From my RNA-seq data, I don't have any biological replicate or technical replicate.

As shown in cufflink link, http://cufflinks.cbcb.umd.edu/tutorial.html
I have run the cuffdiff as shown in the "Differential analysis with gene and transcript discovery" section.
cuffdiff merged_asm/merged.gtf S_NIL_0.bam S_NIL_3.bam S_NIL_6.bam S_NIL_9.bam R_NIL_0.bam R_NIL_3.bam R_NIL_6.bam R_NIL_9.bam

From the above command, it generates a list of output file :
var_model.info
tss_groups.read_group_tracking
tss_groups.fpkm_tracking
tss_groups.count_tracking
tss_group_exp.diff
splicing.diff
run.info
read_groups.info
promoters.diff
isoforms.read_group_tracking
isoforms.fpkm_tracking
isoforms.count_tracking
isoform_exp.diff
genes.read_group_tracking
genes.fpkm_tracking
genes.count_tracking
gene_exp.diff
cds.read_group_tracking
cds.fpkm_tracking
cds_exp.diff
cds.diff
cds.count_tracking
bias_params.info

Do anybody has similar experience to use Cuffdiff for multisample analysis ?
I hope can plot some PCA plot, heat-map, MA-plot, etc in order to identify and characterize resistant genes associated to disease/bacteria-infection in my plant samples.

Below is one of the journal I refer so far, http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3223079/
I have a similar experiment design as the journal above.
But I have no much idea how to use Cuffdiff output for further analysis.

Thanks for any advice.
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Old 03-20-2014, 05:39 AM   #2
TiborNagy
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Location: Budapest

Join Date: Mar 2010
Posts: 329
Default

Try cummeRbund.
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Old 03-20-2014, 05:23 PM   #3
edge
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Location: China

Join Date: Sep 2009
Posts: 199
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Hi TiborNagy,

Thanks for advice.
I will have a try on it now.

Hope it works.
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