Expression measures need to be normalized to remove biases that can be introduced during sequencing, such as sequencing depth and length of the RNA transcript. Geneious calculates three expression level measures on individual samples, which are normalized so that genes expressed in the same sample can be compared:
RPKM
Reads per kilobase per million normalizes the raw count by transcript length and sequencing depth.
RPKM = (CDS read count * 109) / (CDS length * total mapped read count)
FPKM
Same as RPKM except if the data is paired then only one of the mates is counted, ie. fragments are counted rather than reads.
TPM
Transcripts per million (as proposed by Wagner et al 2012) is a modification of RPKM designed to be consistent across samples. It is normalized by total transcript count instead of read count in addition to average read length.
TPM = (CDS read count * mean read length * 106) / (CDS length * total transcript count)
Counting
The above metrics are calculated by normalizing the count of reads that map to each CDS annotation. If a read at least partially intersects at least one interval from a CDS annotation, then it will be treated as though that read mapped to that CDS annotation. For reads that map to multiple locations, or reads that map to a location that intersect multiple CDS annotations, these may either be counted as partial matches, excluded from the calculations, or counted as full matches to each location they map to. We recommend counting reads as partial matches, for example if a read maps to two locations, then it will be counted as if 0.5 reads mapped to each of the two locations. When calculating statistics, reads that don’t map or map outside of a CDS annotation are ignored.