Understanding library pooling for Illumina enrichment kits

Illumina Enrichment kits (DNA and RNA based) help to isolate and enrich specific regions of interest in a genome or transcriptome for sequencing. For example, Illumina DNA Prep with Enrichment (formerly Nextera Flex for Enrichment) with the TruSight Cancer Panel targets 94 genes and 284 SNPs that are associated with various cancers.

This article describes the principles of an enrichment reaction, and the commonly used terminology when describing Illumina enrichment kits. With enrichment kits, ‘Reaction’ (abbreviated “rxn”) refers to the number of enrichment reactions that are provided with the kit. ‘Plexity’ (abbreviated “plex”) refers to the number of pre-enriched libraries that are pooled together in an enrichment reaction. For example, an 8-rxn x 12-plex kit has enough reagents to perform eight enrichments. In each enrichment reaction, 12 libraries can be pooled. Therefore, the total number of samples that can be prepared with this kit is 96 samples. The following figure illustrates how three libraries are pooled in one enrichment reaction.

In this illustration, the three pre-enriched libraries are prepared then pooled together at equal amounts (by mass) for hybridization with target-specific enrichment probes (3-plex pooling). These probe-bound fragments are then captured with streptavidin beads. Any unbound fragments are washed away, and the bound fragments, enriched for the sequences of interest, will be eluted off the streptavidin beads and sequenced.

DNA Enrichment kits - enzymatic fragmentation of the DNA

Illumina DNA Prep with Enrichment (formerly known as Nextera Flex for Enrichment) is based on the enzymatic fragmentation of the DNA with a transposome enzyme. The following probe panels are used with Illumina DNA Prep with Enrichment:

• Illumina Exome Panel (for the entire coding transcriptome)

• TruSight Cancer

• TruSight One

• TruSight One Expanded

• TruSight Cardio

• TruSeq Neurodegeneration - for selecting more targeted regions of the genome.

• Illumina Custom Enrichment Panel, where the software DesignStudio is used to generate a custom panel of interest.

DNA Enrichment kits - mechanical fragmentation of the DNA

• TruSeq DNA exome captures the coding exome, after mechanical fragmentation of the DNA (requires a Covaris).

RNA enrichment-based kits

• Illumina RNA Prep with Enrichment uses On-Bead Tagmentation and captures coding RNA.

• TruSight RNA Pan-Cancer panel and TruSight RNA Fusion are used for analysis of the cancer transcriptome.

For any feedback or questions regarding this article (Illumina Knowledge Article #1246), contact Illumina Technical Support [e-mail].