DGE using trinity

Question

[loly.pearl86]

Hi everyone,

I am doing gene expression study for 2 different treatments in order to comparing them and I follow this methods:

RNA extraction,sequencing by illumina ,QC, trimmomated, assembly using de novo, CEGMA, then I start Differential Expression Analysis Using a Trinity Assembly in command line following this steps:

• Alignment-based abundance estimation methods
• Build Transcript and Gene Expression Matrices
• Counting Numbers of Expressed Transcripts or Genes(no biological replications)

Now I want run Extracting and clustering differential expressed transcripts but not sure should I do TMM normalisation before or no?! if yes How can I do TMM normalisation using trinity in command line?!

Thanks

Answer 1

[Antonio R. Franco]

I believe you use Trinity only to assemble a transcriptome to use as reference for mapping your reads with programs such as tophat, BBmap, star, kallisto an others After mapping you count reads with RTSeq-Count or alike, and then process an normalize the data with packages such as DESeq2, NOISeq, edgeR, etc

[loly.pearl86]

Thanks for u reply

Yes this what I did but I stick now in this step How can I do normalization?

thanks