Transcript Quantification

Note that we use absolute read counts because we are going to compare counts across samples.

Other metrics for comparison of genes within the same sample are:

CPM (Counts Per Million)

Each gene count is divided by the corresponding library size expressed in millions of reads.

RPKM (reads per kilobase of exons per million mapped reads)

Each gene count is divided by the sum of the lengths (in kilobases) of exons of the gene and then divided by the library size expressed in millions of reads.

TPM (Transcript per Million)

1. Divide the read counts by the length of each gene in kilobases. This gives you reads per kilobase (RPK).
2. Sum up all the RPK values in a sample and divide this number by 1,000,000. This is your “per million” scaling factor.
3. Divide the RPK values by the “per million” scaling factor. This gives you TPM

Note that the advantage of the TPM metrics is that it is intra and inter normalized, allowing to compare the level of expression of genes inside a sample or the level of expression of one gene between samples.

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