Denature and dilution FAQ for Illumina DNA PCR Free

Which Denature & Dilute protocol to follow for NovaSeq 6000 and for other supported instruments? If protocol A/standard protocol, is NaOH added twice? (Once mid-protocol (HP3 reagent) and then again as per Denature & Dilute guide protocol)

• Follow Protocol A for standard workflow for the NovaSeq 6000 with standard loading and other instruments, and yes, add the NaOH during denaturation step. It will prevent formation of any secondary structures.

• For the NovaSeq 6000 Xp workflow, follow Protocol B.

Do libraries need to be heat shocked/heat treated to make sure it is single stranded DNA like with Nextera XT, which also uses bead-based normalization? If so, where are the instructions? No. NaOH denaturation will take care of secondary structures. Additional heat denaturation is not required.

If denaturation prior to clustering was omitted—will the data be okay? What is the performance impact? This method of clustering is not supported. However, because Illumina DNA PCR-free generates single stranded DNA (ssDNA) libraries, the libraries may still cluster and generate high quality data, though this cannot be guaranteed. It is possible to see lower quality, lower data output (lower cluster density or percent occupancy), or run failure.

For any feedback or questions regarding this article (Illumina Knowledge Article #3444), contact Illumina Technical Support [e-mail].