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.
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 an example where three libraries are pooled in one enrichment reaction, which we would refer to as a 3-plex enrichment.
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 Coding Exome Panel (for the entire coding transcriptome)
Illumina Custom Enrichment Panel V2, where the software DesignStudio is used to generate a custom panel of interest.
Illumina DNA Prep with Exome 2.5 Enrichment V2
Uses the Twist BioScience for Illumina Exome 2.5 Panel with optional Twist Bioscience for Illumina Mitochondrial Panel spike in.
Illumina 5-Base DNA Prep with Enrichment
Uses Illumina Custom Enrichment Panel V2 panels, which can be designed via Design Studio.
This prep accepts either sonication sheared gDNA or cfDNA input. I offers 5-base information, i.e. additional methylation (5-MeC) data in conjunction with standard sequencing information.
Illumina Cell-Free and FFPE DNA Prep with Enrichment
Uses Illumina Custom Enrichment Panel V2 panels, which can be designed via Design Studio.
Note that Illumina Cell-Free DNA Prep with Enrichment and Illumina FFPE DNA Prep with Exome 2.5 Enrichment follow different protocols, but use the same library prep kits.
RNA enrichment-based kits
Illumina RNA Prep with Enrichment uses On-Bead Tagmentation and captures coding RNA. Same protocol is used for Illumina Respiratory Pathogen ID/AMR Enrichment Panel Kit and Illumina Respiratory Virus Enrichment Kit.
Illumina Viral Surveillance Panel v2 uses On-Bead Tagmentation and enrichment to perform whole-genome sequencing and characterization of >200 viruses and subtypes.
Illumina Rapid RNA Prep with Enrichment uses rapid, single tube reaction to combine cDNA synthesis and adapter tagging into a single step.
Illumina Rapid Respiratory Pathogen ID/AMR Enrichment Panel Kit uses a rapid, single tube reaction to detect respiratory pathogens (180+ bacteria, 50+ fungi, and 40+ viruses) and antimicrobial resistance (1200+).
Illumina Rapid Respiratory Virus Enrichment Kit uses a rapid, single tube reaction, offering highly sensitive detection and characterization of common respiratory viruses, including SARS-CoV-2 strains.
Illumina Rapid Viral Surveillance Panel v2 uses a rapid, single tube reaction, to perform whole-genome sequencing and characterization of >200 viruses and subtypes
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 techsupport@illumina.com.
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