Index color balancing for XLEAP SBS reagents on the NextSeq 1000/2000 and NovaSeq X/X Plus

Selecting Index Adapters with diverse index sequences that optimize color balance within pooled libraries is crucial for successful demultiplexing and data analysis. As a general guideline, Illumina recommends selecting index sequences such that signal will be present in both channels for each index cycle when sequencing libraries on a 2-channel system.

Like Illumina's other sequencers utilizing 2-color chemistry, the XLEAP-SBS reagents for the NextSeq 1000/2000 and NovaSeq X/X Plus systems uses two fluorescent dyes and two images to encode data for the four base calls. However, the XLEAP-SBS chemistry has updated C to the dual-color base and A to the single, blue-channel only base. This differs from other two-color SBS chemistry where A is the dual-color base.

For more information on this, see the Knowledge article Chemistry and Imaging on the NovaSeq X/X Plus Instruments.

T = Green C = Blue + Green A = Blue G = Dark (no label)

Due to this change, indexing combinations need to be reviewed and potentially revised to make sure that signal is present in both channels for each index cycle during runs on XLEAP chemistry.

When sequencing using XLEAP chemistry, combine index sequences so that signal is present in both channels for every cycle whenever possible. It is acceptable to have signal only in the green channel from the T or C bases, if needed. Illumina recommends avoiding index combinations which only have signal in the blue channel from A or A+G in any given cycle, or no signal (only G) present in any given cycle.

For larger pools, it is recommended to maintain base and color balance for all pooled indexes in each lane. Testing is required to confirm index combinations not listed in Index Adapters Pooling Guide, and for third-party or custom index combinations to make sure the index selections work reliably.

When sequencing on a two-channel system, either of the first two cycles of the Index Read must start with at least one base other than G. If an Index Read starts with two G bases, signal intensity is not generated and this can cause signal registration issues. The NextSeq 1000/2000 sequences index 2 in the reverse-complement orientation, but DRAGEN requires index 2 to be entered in forward orientation, thus index 2 cannot end with CC (while index 1 cannot start with GG) in the sample sheet. This criteria is more relevant for lower plexity pools. Higher plexity pools are able to pass registration due to larger percentage of signal from neighboring clusters.

Please note, even optimally color-balanced pools can display base calling errors if the run is severely overloaded or underloaded. Libraries must be sequenced at their optimal loading concentration to ensure the best demultiplexing results.

Example Low-Plexity Index Adapter Combinations*

• For 2-plex it may not be possible to have both green and blue representation in every cycle, but all recommended pairs have signal in the green channel (highlighted green) for every cycle. 2-plex examples below:

  
• For 3-plex and above: recommended pooling combinations have representation in both green (C or T) and blue (C or A) channels. 3-plex examples below:

  

*For more information on index selection for low-plexity pools, and additional low-plexity combinations of Illumina indexes, see the Illumina Index Adapters Pooling Guide and the Illumina Adapter Sequences Guide.

For any feedback or questions regarding this article (Illumina Knowledge Article #8422), contact Illumina Technical Support [e-mail].