Misuse of RPKM or TPM normalization when comparing across samples and sequencing protocols

In recent years, RNA-sequencing (RNA-seq) has emerged as a powerful technology for transcriptome profiling. For a given gene, the number of mapped reads is not only dependent on its expression level and gene length, but also the sequencing depth. To normalize these dependencies, RPKM (reads per kilobase of transcript per million reads mapped) and TPM (transcripts per million) are used to measure gene or transcript expression levels. A common misconception is that RPKM and TPM values are already normalized, and thus should be comparable across samples or RNA-seq projects. However, RPKM and TPM represent the relative abundance of a transcript among a population of sequenced transcripts, and therefore depend on…