De novo RNAseq analyses

Question

[agata88]

Hi all!

I have 3 samples of RNAseq in 3 different conditions (no replicates). This is Illumina MiSeq 2x150bp reads. No reference genome available.

First, I've performed quality filtering with trimmomatic, de novo assembly with Trans-Abyss (k-mer size = 96), then I've mapped reads onto contigs with BWA. I have also performed clustering transcripts by Corset. Now I would like to perform differentiation analysis and GO analysis.

So my questions are:

1. How to perform differentiation analysis for data with no replicates? Which tool would you recommend?
2. What tool for GO analysis?

Thank you in advance,

Agata

Answer 1

[h.mon]

This question has been asked plenty of times here:

Advice on RNASeq analysis without biological replicates for differential gene expression.

Analyzing RNA-seq without replicates

best workflow for RNA data analysis without replications

I do not recommend differential expression with no replicates - you will have very low power and a high rate of false positives. There are some software designed to analyse no-replicates differential expression, such as GFOLD or NoiSeq, and even edgeR has pointers in its manual on how to proceed.

What tool for GO analysis?

For GO annotation, you may try dammit or Trinotate. The perform and enrichment test with the list of "differentially expressed" genes with GOseq.