Question: confused about cuffdiff sample design
0
gravatar for F
3.2 years ago by
F3.4k
Iran
F3.4k wrote:

hi,

in my experiment, before high temperature treatment (48 c), Arabidopsis cotyledons were pre-heated for 2, 8 and 24 hours. in the other hand i have cotyledons keeping in room temperature for 2, 8 and 24 hours, i mean without pre-heating these cotyledons directly face the high temperature. i don have any replication

something like below

pre-heated-2h

pre-heated-8h

pre-heated-24h

unpre-heated-2h

unpre-heated-8h

unpre-heated-24h

i got totally confused if i should use this syntax in cuffdiff

cuffdiff -o diff_out -b genome.fa -p 8 -u merged_asm/merged.gtf  pre-heated-2h.bam, pre-heated-8h.bam, pre-heated-24h.bam, unpre-heated-2h.bam, unpre-heated-8h.bam, unpre-heated-24h.bam

or 

cuffdiff -o diff_out -b genome.fa -p 8 -L pre,unpre -u merged_asm/merged.gtf  pre-heated-2h.bam, pre-heated-8h.bam, pre-heated-24h.bam, unpre-heated-2h.bam, unpre-heated-8h.bam, unpre-heated-24h.bam

using the first syntax the cummerband result was nonsense..and the number of differentially expressed genes was 0, how possible

i am going to know the effect of pre-heating on heat stress tolerance

please help me to be clear a little bit

thank you 

rna-seq next-gen • 808 views
ADD COMMENTlink modified 3.2 years ago by Devon Ryan89k • written 3.2 years ago by F3.4k
3
gravatar for Devon Ryan
3.2 years ago by
Devon Ryan89k
Freiburg, Germany
Devon Ryan89k wrote:

Assuming there's no effect due to duration of treatment (or that it's minimal):

cuffdiff -o diff_out -b genome.fa -p 8 -L pre,unpre -u merged_asm/merged.gtf  pre-heated-2h.bam,pre-heated-8h.bam,pre-heated-24h.bam unpre-heated-2h.bam,unpre-heated-8h.bam,unpre-heated-24h.bam

 

ADD COMMENTlink written 3.2 years ago by Devon Ryan89k

thank you,

sorry, no way to consider the effect of time points too?

ADD REPLYlink written 3.2 years ago by F3.4k
1

Not with cuffdiff, no.

ADD REPLYlink written 3.2 years ago by Devon Ryan89k

thank you dear Devon

ADD REPLYlink written 3.2 years ago by F3.4k
4
gravatar for geek_y
3.2 years ago by
geek_y9.4k
Barcelona/CRG/London/Imperial
geek_y9.4k wrote:

You should at least put some effort and time ( may be 10 min ? ) to read the manual and copy paste ?

If you look at the manual, it clearly states:

cuffdiff [options]* <transcripts.gtf> \
<sample1_replicate1.sam[,…,sample1_replicateM.sam]> \
<sample2_replicate1.sam[,…,sample2_replicateM.sam]> … \
[sampleN.sam_replicate1.sam[,…,sample2_replicateM.sam]]

if you apply that exact command to your data:

cuffdiff -o diff_out -b genome.fa -p 8 -L pre,unpre -u merged_asm/merged.gtf \
pre-heated-2h.bam,pre-heated-8h.bam,pre-heated-24h.bam \
unpre-heated-2h.bam,unpre-heated-8h.bam,unpre-heated-24h.bam 

 

ADD COMMENTlink modified 3.2 years ago • written 3.2 years ago by geek_y9.4k
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