Normalising miRNA for spike in control

Question

[PJC]

Hi

I am analysing differentially expressed miRNA in human plasma (4 groups). I have used the miARma-Seq pipeline (http://miarmaseq.idoproteins.com/) and DESEQ2 for the differential expression analysis.

We used a spike in control (c. elegans miR 39).

Can anyone advise the best method for standardising/ normalising the data to the spiked c.elegans miRNA.

Not sure if I should be using a different pipeline or if there was an established/validated means of doing this?

PC

Answer 1

[ATpoint]

The DESeq2 normalization function estimateSizeFactors has an option to provide "control genes" which you can use here, type ?DESeq2::estimateSizeFactors to see the help:

controlGenes:
optional, numeric or logical index vector specifying those genes to use for size factor estimation (e.g. housekeeping or spike-in genes)