Best Way To Measure Mirna-Seq Abundance

Question

[gundalav]

I know RPKM is most commonly used for RNA-Seq. But I'm not sure how reliable it is for single end reads mIRNA-Seq.

Are any of these more suitable: FPKM, TPM? Or others?

Answer 1

[Istvan Albert]

The reason for introducing the RPKM and other measures was to account for the fact that long transcripts produce more sequencing reads at the same abundance levels.

For miRNA a single read will cover an entire miRNA so there is no need for length based normalization methods.

Of course whether or not simple counting and normalizing by coverage (and how that should be done) is source of a lot of disagreement.

[Thind amarinder]

I would like to use miRNA abundance for co-expression analyses, I am thinking of using count per million (CPM)? Do you have any feedback or suggestions for this? thanks

[Istvan Albert]

you need to ask new questions separately, this here is 7 year old thread.

[Thind amarinder]

I don't think it is a unique question and so new one usually ends up with penalties or with a comment of someone that "it is asked somewhere before". However, here is it interaction prediction of circRNA and miRNA and co -expression network

Answer 2

[support]

As you normalize, be aware of high duplication rates and serious microRNA:adapter preferences that really introduce significant bias:

Jayaprakash, A. D., Jabado, O., Brown, B. D., & Sachidanandam, R. (2011). Identification and remediation of biases in the activity of RNA ligases in small-RNA deep sequencing.

Sorefan, K., Pais, H., Hall, A. E., Kozomara, A., Griffiths-Jones, S., Moulton, V., & Dalmay, T. (2012). Reducing ligation bias of small RNAs in libraries for next generation sequencing.

Zhang, Z., Lee, J. E., Riemondy, K., Anderson, E. M., & Yi, R. (2013). High-efficiency RNA cloning enables accurate quantification of miRNA expression by deep sequencing.

-- Genohub

Answer 3

[Nicolas Rosewick]

You should normalize. Each sample has a different sequencing depth. The workflow I suggest :

1. htseq-count using miRNA gtf on each bam file. It gives you an expression matrix

2. DESeq or edgeR for normalization and DE analysis