Question: RNA-seq triple replicates
0
gravatar for biolab
4.9 years ago by
biolab1.2k
biolab1.2k wrote:

Dear all,

I have triple replicates of RNA-seqs  for both control and mutant samples.  All of these six seqs are single-end un-stranded RNA-seqs.   I ran tophat2 for all six RNA-seqs, but then do not know how to run cufflinks, cuffmerge and cuffdiff commands.   Can I merge three control and three mutant seqs?  Thank you very much for any of your suggestions.

cufflinks tophat2 rnaseq • 2.0k views
ADD COMMENTlink modified 4.9 years ago by Evgeniia Golovina1.0k • written 4.9 years ago by biolab1.2k
2
gravatar for Devon Ryan
4.9 years ago by
Devon Ryan95k
Freiburg, Germany
Devon Ryan95k wrote:

You run cufflinks on each of the 6 samples separately, run cuffmerge once with all 6 GTFs, and then run cuffdiff once with the results.
 

ADD COMMENTlink written 4.9 years ago by Devon Ryan95k

Hi, Devon Ryan, your answer is really helpful. I just have one more question: previously I ran cuffdiff using the following command, however this time I have six bam files rather than two bam files, could you please provide me a cuffdiff command? Thank you very much!

cuffdiff -o wt_mut_cuffdif -b ~/at/TAIR10.fas -p 4 -L wt,mut -u merged_asm/merged.gtf wt_tophat/accepted_hits.bam mut_tophat/accepted_hits.bam
ADD REPLYlink modified 6 months ago by RamRS27k • written 4.9 years ago by biolab1.2k
1
cuffdiff -o something -b foo.fa -p 4 -L WT,MUT -u some.gtf WT1/accepted_hits.bam,WT2/accepted_hits.bam,WT3/accepted_hits.bam MUT1/accepted_hits.bam,MUT2/accepted_hits.bam,MUT3/accepted_hits.bam

Note that there are no spaces between the sample names and commas within a group, but the groups themselves are separated by a space. Adding aberrant spaces is a very common mistake to make at this point.

ADD REPLYlink written 4.9 years ago by Devon Ryan95k

Hi, Devon,

This command and your description are really really helpful!  Thanks a lot.

ADD REPLYlink written 4.9 years ago by biolab1.2k
1
gravatar for Evgeniia Golovina
4.9 years ago by
New Zealand
Evgeniia Golovina1.0k wrote:

Hi,

  1. yes, you can merge three control and three mutant samples - in order to get 2 bam files. But it's optional step:

    samtools merge <out.bam> <in1.bam> <in2.bam> [...]
    
  2. to calculate isoform abundance, you need to quantify FPKM (Fragments Per Kilobase of exon per Million fragments mapped) values:

    cuffquant -u <annotation.(gtf/gff)> <aligned_reads.(sam/bam)>
    
  3. And you can perform analysis between two groups of samples. Actually, you can specify all your 6 samples in cuffdiff command. Look more about cuffdiff command here:

    cuffdiff <other options> --labels conditionA,conditionB <annotations file> sample1.bam,sample2.bam sample3.bam,sample4.bam
    
ADD COMMENTlink modified 6 months ago by RamRS27k • written 4.9 years ago by Evgeniia Golovina1.0k

It should be noted that (2) should not be done on the output of (1), but rather on the unmerged files.

ADD REPLYlink written 4.9 years ago by Devon Ryan95k

Yes. You're right. Thank you!

ADD REPLYlink modified 4.9 years ago • written 4.9 years ago by Evgeniia Golovina1.0k
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