Values used for calculating FPKM Value
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6.2 years ago
bassanio ▴ 40

Dear All,

I had got a Transcriptome Library with 10M PE reads( R1->10M & R2 ->10M ).  When I mapped the same to the genes from a particular genome using Bowtie2  80% of them mapped (8M).

Now I need to calculate the Expression rate for these genes. For calculating the FPKM Value I use the formula below

FPKM= MG*(10^9))/N*Length of Gene

Where;

MG= Count of PE mapped to the genes (20,000)

N= Library size or Mapped count ?(10M/8M?)

My question is What is the value of N should be is it 10M or 8M

FPKM Bowtie RPKM • 3.4k views
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6.2 years ago

It should be 8M. For example from edgeR rpkm() function, its

lib.size: library size, defaults to ‘colSums(x)’
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If I use the same transcript data and alligned with another genome(B) where aligned reads is 9M and then if I do a DeSeq between Sample A(8M) mapped reads and Sample B(9M) mapped reads then the data is Skewed more towards Sample A as the normallization  value is not same between SampleA and Sample