Question: Combining all biological replicates
1
gravatar for melati
3.6 years ago by
melati10
Malaysia
melati10 wrote:

Hi,

I have RNA-seq data with 3 isolates (Isolate 1, isolate 2, isolate 3), for each isolate there are 2 samples(Media A, Media B) and each sample have 3 biological replicates (R1, R2, R3). So, is there any way to combine these biological replicates to be representative as one using Cuffdiff ? Please let me know your opinions. Thank you.

 

 

rna-seq cuffdiff • 2.1k views
ADD COMMENTlink modified 3.6 years ago • written 3.6 years ago by melati10
1

Why do u want to use cuffdiff ?  You can create a design matrix and use edgeR or DESeq for more robust analysis ?

ADD REPLYlink modified 3.6 years ago • written 3.6 years ago by geek_y9.3k

What if they want to use Cuffdiff instead? I'm thinking the same way as them, is there any possibility to combine the biological replicate? Otherwise, is it practical enough if simply pick any biological replicate when making the differential expression analysis?

ADD REPLYlink written 3.6 years ago by CikLa90

I second Goutham Atla suggestion to use a method that explicitly takes into account replicates within design variables (i.e. linear models as implemented in edgeR/DEseq). Picking one representative replicate or combining replicates will result in less data being used and/or ignoring the variation between replicates. (I haven't used cuffdiff for a long time, so I don't know if a better handling of replicates is possible)

ADD REPLYlink written 3.6 years ago by dariober9.9k

Isolates as in 3 strains of different bacteria/virus ?

ADD REPLYlink written 3.6 years ago by wanziyi8950

The isolates came from same pathogen with different level of pathogenecity which is isolate 1 with most virulent, isolate 2 is medium and isolate 3 is less virulent.

ADD REPLYlink written 3.6 years ago by melati10

Now, what you want to compare ? The effect of media with in each isolate ? or the DE genes between isolates ?

ADD REPLYlink written 3.6 years ago by geek_y9.3k

I want to campare DE genes between isolates. Somehow it makes me confuse. Let say.. can I compare isolate1 (replicate1) with isolate2 (replicate1) or.. maybe isolate1 (replicate2) with isolate2 (replicate3) etc?

ADD REPLYlink written 3.6 years ago by melati10
2

If you want to compare DE genes between isolates, better to create a design matrix and use edgeR/DESeq. I don't think so Cuffdiff can handle this experimental design. I don't think you are "interested" to use cuffdiff, may be you feel its easy to use or you don't have proper annotations for organism of your interest.

ADD REPLYlink modified 3.6 years ago • written 3.6 years ago by geek_y9.3k
1

The best method is to compare all of the replicates from isolate1 with all of them from isolate2. Do not merge the samples or otherwise just use only 1 of them! If you want to include the samples for both media types as well then you can't use cuffdiff, it's simply not flexible enough. Use edgeR, DESeq2, or limma/voom instead.

ADD REPLYlink written 3.6 years ago by Devon Ryan88k
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