Question: Differential expression Tophat/cufflinks
0
gravatar for bdeonovic
5.2 years ago by
bdeonovic180
United States
bdeonovic180 wrote:

I am a bit confused on how to run tophat/cufflinks properly. I have (paired) RNAseq data from pig. I have 8 samples (4 disease, 4 control). For each sample I also have data from 2 lanes (so was each sample run twice?) 

I want to ultimately do differential expression analysis using cuffdiff. 

When I run tophat do I input all of the reads I have into one tophat command?

 

e.g. tophat Sample_1/left_read, Sample_2/left_read, ... Sample_1/right_read, Sample_2/right_read

 

or do I run 8 separate tophat runs

e.g.

tophat Sample_1/left_read Sample_1/right_read

tophat Sample_2/left_read Sample_2/right_read 

...

rna-seq tophat • 2.7k views
ADD COMMENTlink modified 5.2 years ago by oigl60 • written 5.2 years ago by bdeonovic180
2
gravatar for Devon Ryan
5.2 years ago by
Devon Ryan92k
Freiburg, Germany
Devon Ryan92k wrote:

You would do

tophat some_options_here sample1_lane1_left.fastq,sample1_lane2_left.fastq sample1_lane1_right.fastq,sample1_lane2_right.fastq
tophat some_options_here sample2_lane1_left.fastq,sample2_lane2_left.fastq sample2_lane1_right.fastq,sample2_lane2_right.fastq

and so on. BTW, I don't think cuffdiff can handle paired-sample designs (e.g., experiments in which each patient has a matched control, which it sounds like you have), so you'll need to use something else (DESeq2, edgeR, limma, etc.).

ADD COMMENTlink modified 5.2 years ago • written 5.2 years ago by Devon Ryan92k

They are not case-control matched, just 4 of each. Thanks for the answer :) 

ADD REPLYlink written 5.2 years ago by bdeonovic180

Ah, then cuffdiff would work for you.

ADD REPLYlink written 5.2 years ago by Devon Ryan92k
1
gravatar for oigl
5.2 years ago by
oigl60
oigl60 wrote:

You can run the tools through the UGENE graphical interface. For details see this page. A brief video introduction is here.

To start you will need to download and install a full or NGS UGENE package for your operating system. All tools are available out of the box for Linux and Mac OS X. Then you open UGENE, select "Tools > Workflow Designer", open the samples and select the "RNA-seq analysis with Tuxedo tools". The wizard will appear in which you should input the required data and run the pipeline. Dashboard is also available to monitor the pipeline execution process and to manage the results. 

ADD COMMENTlink written 5.2 years ago by oigl60
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