RNA Seq Pipeline Question (CuffMerge)

Question

[akash27519]

I am trying to implement a pipeline for RNA-seq Analysis using the Tuxedo suite of tools. I am planning on doing the following Cufflinks ---> CuffMerge ---> CuffDiff. The analysis I am trying to conduct contains 2 different conditions with two samples each for a total of four files.

Condition 1: Sample 1 & 2 Condition 2: Sample 3 & 4

My question is for the CuffMerge step should I be merging all four samples or just do merges within the conditions?

All 4 of these samples are from the same cell line so I assume that the best practice would be to merge all 4 samples and then use CuffDiff to calculate the FPKM values at the condition and sample level. Is this a correct assumption?

[WouterDeCoster]

You should know that the old 'Tuxedo' pipeline of Tophat and Cufflinks is no longer the "advisable" tool for RNA-seq analysis. The software is deprecated/ in low maintenance and should be replaced by HISAT2, StringTie and ballgown. See this paper: Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown. (If you can't get access to that publication, let me know and I'll -cough- help you.) There are also other alternatives, including alignment with STAR and bbmap, or pseudo-alignment using kallisto or salmon.

Answer 1

[lakhujanivijay]

Whether it is cuffmerge from old tuxedo or stringtie-merge from new tuxedo protocol as mentioned by WouterDeCoster both of them merge together all the gene structures found in any of the samples. This step is required because transcripts in some of the samples might be only partially covered by reads. The merge step creates a set of transcripts that is consistent across all samples, so that the transcripts can be compared in subsequent steps.

Hence, you can take all samples together.