Question: Cuffmerge GTF versus known annotation GTF used with Cuffdiff
0
gravatar for adoorprabha
5 months ago by
adoorprabha0 wrote:

I am performing an RNA-Seq analysis on two different plants with two different conditions.

  1. Susceptible
  2. Resistant

I have done the steps upto cufflinks. Now my doubt is should i perform cuffmerge or should i directly go for cuffdiff for differential expression because the two datas i have selected for a plant are not biological replicates they are of two different conditions since i am taking two different conditions of the same plant. Please reply as soon as possible.

(admin edit: reformat text)

ADD COMMENTlink modified 5 months ago by Jennifer Hillman Jackson25k • written 5 months ago by adoorprabha0
0
gravatar for Jennifer Hillman Jackson
5 months ago by
United States
Jennifer Hillman Jackson25k wrote:

Hello,

If you are interested in discovery, use the Cuffmerge step. If not, you can skip it.

Why?

  • If the annotation GTF given to Cuffdiff is from Cuffmerge, the results will include novel splice-variant transcripts and/or novel genes (if any) present in your data.

  • If the annotation GTF is instead directly from a public source, the results will be summarized based on only those known transcripts/genes.

Should you be not sure of which to pick, perhaps run both then review the differences to make a decision. Full details are in the Cufflinks' manual, FAQs, and publications: http://cole-trapnell-lab.github.io/cufflinks/

Note: These tools are considered a bit older and newer RNA-seq DE methods are available. If interested, please see the Galaxy tutorials here: https://galaxyproject.org/learn/

Thanks! Jen, Galaxy team

ADD COMMENTlink modified 5 months ago • written 5 months ago by Jennifer Hillman Jackson25k

My question was should i perform cuffmerge for non-biological replicates?As my study is based on two different conditions of the same plant i.e susceptible and resistant and i am having only one sequence data for each condition.Should i perform cuffmerge on datas of two different conditions.

ADD REPLYlink written 5 months ago by adoorprabha0

Having replicates or not does not impact whether Cuffmerge should be used or not. The decision is based on whether you want to include novel isoforms or not (eg: any not included in public annotation GTF datasets).

Whenever you want to include all Cufflinks discovered isoforms in the final Cuffdiff result, use CuffMerge. This is the first option above and merges data from different conditions and/or replicates and optionally a reference annotation GTF - all will be combined together to create a "master" non-redundant set of transcripts/genes for Cuffdiff.

If you are not interested in discovery (novel isoforms), a public annotation GTF can be used. This is the second option above.

ADD REPLYlink written 5 months ago by Jennifer Hillman Jackson25k
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