FPKM & RPKM in RNA-Seq: What They Are & How They Differ

Raw read counts from RNA-Seq analysis cannot be directly compared across genes or samples.

This is because longer genes naturally accumulate more mapped reads, and samples with a higher total number of sequenced reads will have more reads mapped to every gene.

To address these biases, various normalization methods have been proposed. This page explains FPKM/RPKM. Note that FPKM/RPKM has been criticized for not accurately representing gene expression levels, and TPM has become the more commonly used alternative.

FPKM stands for 'Fragments Per Kilobase of exon per Million mapped reads', and RPKM stands for 'Reads Per Kilobase of exon per Million mapped reads'. As the names imply, both metrics normalize the total mapped read count to one million and the transcript length to 1,000 bases. The only difference between FPKM and RPKM is whether fragments or reads are counted; the underlying formula is the same.

The formula is as follows, where \(q_i\) is the read count and \(l_i\) is the transcript length:

The exact FPKM/RPKM calculation can vary slightly between software tools. For instance, some tools use the effective length rather than the actual transcript length for \(l_i\).

The effective length is calculated as follows:

\(μ_{FLD}\) represents the average fragment length.

Using the effective length in FPKM/RPKM calculations is considered to provide a more accurate correction for length bias.