Overview

This resource provides recommendations for optimizing color balance across all Illumina systems when pooling indexed libraries. Pooling combines at least two libraries to sequence in one run.

The recommendations are designed to form low-plex, color-balanced pools. Higher plexity pools are inherently color-balanced, so any index adapter combinations are acceptable. To develop alternative pooling strategies, review index adapter sequences in Illumina Adapter Sequences to ensure color balance for your system.

Use this resource as a reference to plan indexing and pooling for library prep. Refer to the library prep kit support pages on the Illumina support site for additional resources. These resources include software, training, and compatible products. Always check the support pages for the latest versions.

Pooling strategies differ based on the type of chemistry used. For NovaSeq X Series or NextSeq 1000/2000 instruments using XLEAP-SBS chemistry, refer to pooling strategies indicated for XLEAP-SBS. For MiSeq i100 Series instruments, refer to standard pooling strategies. While the MiSeq i100 Series utilizes XLEAP-SBS reagents similar to those on the NovaSeq X Series and NextSeq 1000/2000, the following minor changes make it unique:

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The MiSeq i100 Series XLEAP-SBS reagents use three fluorescent dyes and two images to encode data for the four base calls.

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The MiSeq i100 Series uses A as the dual-color base and C as the single blue channel only base, similar to the MiniSeq, NextSeq 500/550, NextSeq 1000/2000 standard reagents, and NovaSeq 6000.