Question: How To Calculate The Rpkm For Isoforms
2
gravatar for camelbbs
5.2 years ago by
camelbbs610
China
camelbbs610 wrote:

Hi I want to ask a question about calculate the RPKM.

I see the previous people calculate the RPKM for a gene, by using all the reads included in intron and exons belong to a gene.

If I just want to get the RPKM for different isoforms. Can I just pick up the reads mapped to exons.

For example, Gene A has 4 exons:

Chr2 MSU_osa1r6 gene 4542759 4544980 . + . ID=13102.t00754;Name=unknown gene
Chr2 MSU_osa1r6 CDS 4543031 4543177 . + 0 Parent=13102.m00974
Chr2 MSU_osa1r6 CDS 4543287 4543709 . + 0 Parent=13102.m00974
Chr2 MSU_osa1r6 CDS 4543836 4543952 . + 0 Parent=13102.m00974
Chr2 MSU_osa1r6 CDS 4544064 4544423 . + 0 Parent=13102.m00974

There are 4,011 reads that map to this gene (between positions 4542759 and 4544980).

There are 4 exons for this particular gene which contain a total of 1,043 base pairs.

So the RPKM for this particular gene is ((4,011 reads/1.043kb of exon)/31.8mill mapped reads) = 120.9RPKM

If I just calculate the RPKM for different isoforms, I just want to use the reads in exons, so it is about 3000 reads.

So my RPKM is ((3000/1043)/31.8mill mapped reads)=90RPKM.

So if I just want to compare the expression level of this isofoms across samples, is it ok to do that?

Thanks in advance.

rpkm • 2.1k views
ADD COMMENTlink modified 5.2 years ago by JC6.4k • written 5.2 years ago by camelbbs610
0
gravatar for JC
5.2 years ago by
JC6.4k
Mexico
JC6.4k wrote:

I think is better to use Cufflinks to do that. It reports nicely the gene and transcript RPKM expressions in the output.

ADD COMMENTlink written 5.2 years ago by JC6.4k

Cufflinks assemble the isoforms first, right? I just want to use the known isoforms in refseq.

ADD REPLYlink written 5.2 years ago by camelbbs610

No, you can run it with a GTF file, then it doesn't assemble.

ADD REPLYlink written 5.2 years ago by Mikael Huss4.5k

As I think, the isoforms expression level should not include the intron reads, since we only need mRNA expression.

ADD REPLYlink written 5.2 years ago by camelbbs610
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