Introducing the NovaSeq 6000 v1.5 reagents

This article outlines the changes and improvements the new NovaSeq 6000 v1.5 reagents bring to sequencing on the NovaSeq 6000 system. What’s new:

• Usage of NovaSeq 6000 v1.5 reagents is enabled by an upgrade to NovaSeq 6000 Control Software (NVCS) v1.7. The upgrade is required to run the new reagents and can be self-installed when upgrading from NVCS v1.5 or higher.

• Updates to Quality Score Specifications (Table 1).

• Extended minimum guaranteed shelf life from three months to six months.

• Change in Default Indexing Workflow from the Forward Strand Workflow (see Fig. 1) to the Reverse Complement Workflow with v1.5 reagents, which uses the Solution-Based Primer (see Fig. 2).

• Support for Unique Molecular Identifiers (UMIs) increased the number of available cycles. NovaSeq 6000 v1 kits had 23 additional cycles for dual indexing and 30 additional cycles for single indexing, with the NovaSeq 6000 v1.5 reagents there are now an additional 38 cycles for both single and dual indexing runs.

• New NovaSeq 6000 reagent kit configuration, introducing the S4 v1.5 35 cycle kit.

• Supports the new Illumina DNA PCR-Free Prep with new primers included in the v1.5 cluster cartridges.

  

Table 1: Updated Q30 specs between v1.5 and v1.0 reagents.

Figure 1: NovaSeq 6000 v1.0 reagents use the Grafted Oligo Lawn workflow, or the Forward Strand workflow.

Figure 2: The NovaSeq 6000 v1.5 reagents with the NVCS v1.7 use the Solution-Based Primer, or Reverse Complement Workflow.

Table 2: Comparison of indexing strategy, consumable version, and the NVCS version.

Frequently asked questions:

• What changes were made to the NovaSeq 6000 v1.5 Reagent Kits? The SBS and PE cluster cartridges were updated to v1.5. The flow cell, buffer cartridge, and library tube components were not changed.

• What is the change in loading concentration between v1.0 and v1.5? The loading concentration does not change between v1.0 and v1.5 reagents. We recommend continuing to run your libraries at the validated concentration.

• Can v1.0 reagents be run with NovaSeq Control Software v1.7? Yes, the NovaSeq 6000 Control Software v1.7 can run both v1.0 reagents and v1.5 reagents. The instrument will still use the Forward Strand indexing workflow for v1.0 reagents, however.

• Will sample sheets need to be configured differently between v1.0 and v1.5 reagents? Overall, the sample sheets have the same format, but when using v1.5 reagents the i5 index needs to be in the reverse orientation rather than the forward orientation. Illumina Experiment Manager v1.19.1 provides an option to reverse-complement the index sequence when selecting NovaSeq v1.5 reagents.

• Is an engineer required for the upgrade to NovaSeq 6000 Control Software 1.7? If the current version of NovaSeq 6000 Control Software is v1.5 or v1.6, the software upgrade is customer installable. The release notes, updated instructions, and installer can be found here.

• Can v1.0 XP kit be used for v1.5? Yes, v1 XP kit can be used for both v1.0 and v1.5 reagents and v1.5 XP kit can also be used for both v1.0 and v1.5 reagents.

• Can v1.0 and v1.5 reagents be mixed? The SBS and PE cluster cartridges cannot be mixed, both must be v1.0 or v1.5. The flow cells and buffer cartridges are both still v1.0 and can be used interchangeably.

• When running UMIs, can the run be set up with a 159+10+10+159 configuration? The new reagents will allow for enough cycles to run 159 cycles (or 259 cycles with the SP kits) in a single direction. There will be a software warning that the run is set up for over 151 cycles, but the run can proceed.

• With the new solution-based primer indexing strategy, can custom Index 2 (i5) primers be used? After installing NovaSeq 6000 Control Software v1.7, customers can select the use of custom indexes during run setup. If Custom Index is selected, primers from the custom primer well position will be used for both the Index 1 (i7) and Index 2 (i5) reads when using NovaSeq 6000 v1.5 reagents. If Custom Index is not selected, the Illumina primers will be used.

For any feedback or questions regarding this article (Illumina Knowledge Article #3717), contact Illumina Technical Support [e-mail].