Question: How does miRdeep2 normalise sequences
0
gravatar for K.patel5
10 weeks ago by
K.patel530
K.patel530 wrote:

Hi, I have mirdeep2 output that looks like this.

#miRNA        read_count precursor     total    seq   seq(norm)
mmu-let-7a-5p   43271   mmu-let-7a-1    43271   43271   7658.26
mmu-let-7a-1-3p 784     mmu-let-7a-1    784     784     138.76
mmu-let-7a-5p   43224   mmu-let-7a-2    43224   43224   7649.94

I think using the seq(norm) would be appropriate, but I cannot find how the sequences have been normalised. I think it is something like TPM but am not sure. Does anyone know?

Best, Krutik

sequencing mirdeep2 mirna • 139 views
ADD COMMENTlink modified 10 weeks ago by ATpoint46k • written 10 weeks ago by K.patel530
1
gravatar for ATpoint
10 weeks ago by
ATpoint46k
ATpoint46k wrote:

https://github.com/rajewsky-lab/mirdeep2/blob/master/FAQ.md

How are miRNA expression values reported by the qunatification module normalized?

Expression values are normalized by library size and multiplied by a factor of 1E6 which corresponds technically to counts per million mapped miRNA reads (RPM). The library size is the total number of reads mapping to miRNA precursors.

So a pretty simple and suboptimal normalization. Better use something like edgeR or DESeq2 for a proper normalization, depending on your downstream analysis. What is the analysis goal?

ADD COMMENTlink written 10 weeks ago by ATpoint46k

I would like to do a standard differential expression analysis.

ADD REPLYlink written 10 weeks ago by K.patel530
1

Then I would simple make a count matrix, genes being rows, samples being columns, with the raw counts (read_count) the values, and feed it into e.g. DESeq2. This tool needs raw counts.

ADD REPLYlink written 10 weeks ago by ATpoint46k
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