Conversion read counts to TPM for microRNAs

Question

[lessismore]

Dear all,

im planning to make some visualization with microRNA data. I have a doubt. I received the read counts for a number of identified microRNAs and i want to convert these numbers to TPM. According to the formula here http://www.rna-seqblog.com/rpkm-fpkm-and-tpm-clearly-explained/ you need to :

1 - Divide the read counts by the length of each gene in kilobases. This gives you reads per kilobase (RPK).

this for miRNA doesnt make much sense so could be skipped, correct?

2 - Count up all the RPK values in a sample and divide this number by 1,000,000. This is your “per million” scaling factor.

doubt: all reads in a sample? or all reads mapped to identified miRNAs (so the sum of the read counts i received)?

3 - Divide the RPK values by the “per million” scaling factor. This gives you TPM.

thanks in advance for your help (or references if you have)

[Sam]

TPM converting formula:

 TPM (transcript per million) values TPM = (miRNA total reads/total clean reads) × 1000000

Answer 1

[Buffo]

TPM is not suitable for miRNAs, you can not normalize by transcript length for matures miRNAs. You can find some alternatives for miRNAs here