Prepare for Pooling

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The index adapter plates contain a unique combination of dual i5 and i7 indexes per well. There are a total of 96 unique dual (UD) index combinations per plate. Index adapter plates from sets A and B offer different UD index combinations per set. For index adapter sequences and how to record them, refer to Illumina Adapter Sequences.

For additional recommendations to plan indexing and pooling strategies for library prep, refer to Index Adapters Pooling.

Illumina FFPE DNA Prep with Exome 2.5 Enrichment reagents are configured and tested at 4-plex enrichment plexity.

• Enrichment plexity—The number of pre-enriched libraries pooled together in one enrichment reaction for hybridization with the enrichment probe panel. For example, combining four pre-enriched libraries together creates a 4-plex enrichment pool.
• Enrichment reaction—The number of unique enrichment reaction preparations, regardless of the number of pre-enriched libraries pooled per reaction. For example, a single enrichment reaction prepares a 4-plex enrichment pool.

Pre-enriched libraries are pooled during the hybridized probes procedure. 4-plex library pools are processed in the same manner through the enrichment steps and normalization workflows.

To calculate the total number of enriched libraries, multiply the enrichment plexity per reaction by the number of enrichment reactions. For example, a single enrichment reaction of a 4-plex enrichment pool produces a pool of four enriched libraries.

Pooling libraries from samples of different DNA quality in the same 4-plex library pool increases the chance of unequal index representation during sequencing.

Do not mix tumor and matched normal libraries in the same tube. Libraries prepared from tumor samples must be mixed in one 4-plex library pool. Libraries prepared from matched normal samples must be mixed in a separate 4-plex library pool.

Enriched libraries are pooled for sequencing after the library normalization step. Libraries must be pooled with nonoverlapping indexes. Each sequenced tumor and matched normal library must achieve at least 500x and 150x on-target coverage respectively for optimal variant detection. Refer to the denature and dilute instructions for your sequencing system for the recommended library plexity for sequencing, and to confirm that each sample achieves sufficient on-target coverage.