How can I add UMIs to my QuantSeq libraries?

Unique Molecular Identifiers (UMIs) can be incorporated into QuantSeq FWD libraries during second strand synthesis. The UMIs are 6 nt long and are positioned 5' of the random priming sequence in the primers used for second strand synthesis. In addition to the standard QuantSeq FWD Library Prep Kit, the UMI Second Strand Synthesis Module for QuantSeq FWD (Illumina, Read 1, Cat. No. 081) is required. This module provides the UMI Second Strand Synthesis Mix (USS) that contains the UMI-tagged random primers for second strand synthesis.

The UMI Second Strand Synthesis Module is used at step 7 of the QuantSeq protocol instead of the standard Second Strand Synthesis Mix 1 (SS1) from the standard QuantSeq FWD Kit. With this simple exchange solution there are no additional steps added to the QuantSeq library prep protocol. The figure below shows how UMIs are added to QuantSeq libraries during second strand synthesis.