Question: Significant Expression Difference
0
gravatar for Adrian Pelin
5.7 years ago by
Adrian Pelin2.3k
Canada
Adrian Pelin2.3k wrote:

Hello,

I am doing some preliminary RNA-Seq and I have 2 libraries, 1 for condition A and one for condition B. I know how to use bowtie and cufflinks to estimate FPKM values for genes of interest, but how can I get a list of genes which have a significant expression difference so that I may test them with qRT-PCR?

Thank you.

cufflinks rna-seq bowtie • 2.0k views
ADD COMMENTlink modified 5.7 years ago by Devon Ryan91k • written 5.7 years ago by Adrian Pelin2.3k
2
gravatar for Devon Ryan
5.7 years ago by
Devon Ryan91k
Freiburg, Germany
Devon Ryan91k wrote:
  1. Without replicates you will never get meaningful p-values. You might consider just sorting things by fold-change.
  2. If you've already run cufflinks, just use cuffdiff (it'll give you p-values, but they're not very meaningful without replicates).
ADD COMMENTlink written 5.7 years ago by Devon Ryan91k

My thinking is that I would use q-RTPCR to validate the data after finding potential candidates. Do you think the approach is reasonable?

ADD REPLYlink written 5.7 years ago by Adrian Pelin2.3k

Yeah, though keep in mind that your validation rate might be really low. At least if you're using Taqman assays, it usually ends up being cheaper to just sequence more samples (then you end up wasting fewer assays).

ADD REPLYlink written 5.7 years ago by Devon Ryan91k
1

and wasting time. just sequence one or two additional samples for each group. If you only look for expression, you don't have to go deep, so you can multiplex all your samples in one lane.

ADD REPLYlink modified 5.7 years ago • written 5.7 years ago by Nicolas Rosewick8.1k

Very true, though the trick is to have someone else do the qPCRs for you. Wasting time only sucks when it's your time being wasted :p

ADD REPLYlink written 5.7 years ago by Devon Ryan91k

It's not my project, I am just in charge of the bioinfo part:) They will be using SYBR Green super mix, and they are only interested in a few genes, so hopefully we don't waste too much money.

We did 50bp single end sequencing, I think that's enough since we aren't trying to reconstruct transcriptomes here...

ADD REPLYlink written 5.7 years ago by Adrian Pelin2.3k

You can try DESeq, there is a "without replicate" mode

ADD REPLYlink written 5.6 years ago by Nicolas Rosewick8.1k
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