Library denaturation and compatibility for the iSeq 100 system

Libraries loaded onto the iSeq 100 system must be double-stranded DNA, as libraries are automatically denatured into single strands onboard the instrument. Bead-normalized libraries are single-stranded and are not compatible with the iSeq 100.

The following Illumina library prep protocols include an optional bead normalization procedure:

• Nextera XT

• TruSeq Bovine Parentage Sequencing

• TruSeq Genotype NeKit

• TruSight Myeloid

If using any of the workflows above with iSeq 100, omit the bead normalization steps and proceed with manual normalization of the libraries.

For more information on manual normalization, see Best practices for manually normalizing library concentrations.

Other library prep workflows listed below also yield single-stranded libraries and are additionally incompatible with iSeq 100 due to requirements for output or for custom primers.

• TruSight Oncology 500

• TruSight Oncology 500 ctDNA

• TruSight Oncology 500 High Throughput

• TruSight Tumor 170

Important Note: The lone exception to the requirement of double-stranded DNA is the Illumina DNA PCR-Free library preparation workflow. Although this workflow yield single-stranded DNA, it has been validated for use on the iSeq 100 when utilizing iSeq Control Software v3.0. This is due to the requirement of VP10 and VP14 as primers for Read 1 and Index Read 2, respectively. These are sold separately (catalog # 20041797) and must be loaded as custom primers on board the instrument, hence the requirement for updating to iSeq Control Software v3.0.

For any feedback or questions regarding this article (Illumina Knowledge Article #1705), contact Illumina Technical Support [e-mail].