How can I add UMIs to my QuantSeq FWD libraries?

Unique Molecular Identifiers (UMIs) can be incorporated into QuantSeq FWD libraries during second strand synthesis. The UMI tags (6 nt long + 4nt spacer) are located at the 5' end of the random primer used for the second strand synthesis.

To do so, both the standard QuantSeq FWD Library Prep Kit (Cat. No. 191-196) and the UMI Second Strand Synthesis Module for QuantSeq FWD (Illumina, Read 1, Cat. No. 081) are required. The UMI module provides the UMI Second Strand Synthesis Mix (USS) that contains the UMI-tagged random primers for second strand synthesis. This mix is to be used at step 7 of the QuantSeq protocol instead of the standard Second Strand Synthesis Mix 1 (SS1) provided in 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.