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.

Plexity is the number of libraries combined in a reaction or pool. For example, if 12 libraries are combined in one pool, the plexity is 12.

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 instruments using XLEAP-SBS chemistry, refer to pooling strategies indicated for XLEAP-SBS.

Combinatorial dual (CD) indexes have unique dual pairs, but the index adapters share some sequences. In contrast, unique dual (UD) indexes have distinct sequences so each index adapter is unique.

• Single-indexed libraries add six base Index 1 (i7) sequences to generate uniquely tagged libraries.
• Dual-indexed libraries add Index 1 (i7) and Index 2 (i5) sequences to generate uniquely tagged libraries.
• UD indexes have distinct, unrelated index sequences for the i5 and i7 Index Read. Indexes are 8 or 10 bases long.
• CD indexes have a limit of eight unique pairs, resulting in most libraries sharing common indexes on the i5 or i7 end. Indexes are eight bases long.

Plate layouts for Illumina UD Indexes are designed with the proper color balance. Achieving this balance with CD indexes requires additional planning.