Correcting PCR amplification errors in unique molecular identifiers to generate absolute numbers of sequencing molecules.

Unique Molecular Identifiers (UMIs) are random oligonucleotide sequences that remove PCR amplification biases. However, the impact that PCR associated sequencing errors have on the accuracy of generating absolute counts of RNA molecules is underappreciated. We show that PCR errors are the main source of inaccuracy in both bulk and single-cell sequencing data, and synthesizing UMIs using homotrimeric nucleotide blocks provides an error correcting solution, that allows absolute counting of sequenced molecules. ### Competing Interest Statement A.P.C, U.O and M.P are co-founders of Caeruleus Genomics Ltd and are inventors on several patents related to sequencing technologies filed by Oxford University Innovations. T.B. Jr. is a director and shareholder of ATDBio. T.B. Sr. is a director of ATDBio. The other authors declare no competing interests.