Differential expression Tophat/cufflinks

Question

[bdeonovic]

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
...

Answer 1

[Devon Ryan]

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.).

[bdeonovic]

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

[Devon Ryan]

Ah, then cuffdiff would work for you.

Answer 2

[oigl]

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.