Overview
The Illumina DNA Prep with Exome 2.5 Enrichment kit provides library prep and enrichment reagents, clean up/size selection beads, human exome panel, and indexes needed to make high quality, human, whole exome libraries. These libraries include hybridization-based enrichment of the exome portion of the library.
Illumina DNA Prep with Exome 2.5 Enrichment:
| • | Enables the construction of 96 libraries followed by enrichment of those libraries in eight hybridization reactions. Each reaction contains 12 libraries (12-plex enrichment) for a total output of 96 exome-enriched, human libraries. |
| • | The number of libraries in each enrichment reaction may be reduced but requires optimization and reduces the total number of samples enriched with each kit. |
* Comparative bioinformatic analysis of target region coverage between this exome panel and the targets in the various public databases. Visit the product support page to download the complete BED file for this design.
The Twist Bioscience for Illumina Exome 2.5 Panel is a 120 bp, double-stranded oligo panel. The Exome 2.5 Panel may be combined with the Twist Bioscience for Illumina Mitochondrial Panel, and/or an Illumina Custom Enrichment Panel v2 (120 bp) to add or boost target coverage. Both the Mitochondrial Panel and the recommended Illumina Custom Enrichment Panel v2 are 120 bp, double-stranded oligo panels. Spike-in panels in formats other than these 120 bp, double-stranded oligo products have not been tested.
The Twist Bioscience for Illumina Mitochondrial Panel is designed to cover the 37 genes (16,659 base pairs) of the human mitochondrial genome. The panel is purchased separately and can be used as a spike-in with the Illumina DNA Prep with Exome 2.5 Enrichment kits. Refer to Product Contents for descriptions and part numbers.
The Illumina Custom Enrichment Panel v2 is a user-defined custom enrichment panel designed on the DesignStudio platform. Refer to Product Contents for descriptions and part numbers.
This documentation explains how to prepare up to 96 uniquely dual-indexed, paired-end, whole exome, and mitochondrial genome libraries from DNA using the Illumina DNA Prep with Exome 2.5 Enrichment workflow.
The Illumina DNA Prep with Exome 2.5 Enrichment workflow:
| • | Uses on-bead tagmentation, an enzymatic reaction, to both fragment the input DNA and add adapter sequences in only 15 minutes. |
| • | Uses innovative sample normalization at inputs ≥ 50 ng so that library quantification is not required before hybridization. |
| • | Provides sufficient volume of each reagent to enable automation on most liquid handler platforms. |
| • | Enables preparation of libraries directly from whole blood or saliva samples when using our recommended protocol. Refer to Supplemental Procedures for more information. |
How the Illumina DNA Prep with Exome 2.5 Enrichment Assay Works
The Illumina DNA Prep with Exome 2.5 Enrichment workflow uses a bead-based transposome complex to tagment genomic DNA. Tagmentation is a process that fragments DNA and then tags the DNA with adapter sequences in one step. After it is saturated with input DNA, the bead-based transposome complex fragments a set number of DNA molecules. This fragmentation provides flexibility to use a wide DNA input range to generate normalized pre-enrichment libraries of consistent tight fragment size distribution. Following tagmentation, a limited-cycle PCR adds adapter sequences to the ends of a DNA fragment. This step enables compatibility across all Illumina sequencing systems. A subsequent target enrichment workflow is then applied. Following pooling, the double-stranded DNA libraries are denatured and biotinylated oligonucleotide probes are hybridized to the denatured library fragments. After hybridization, Streptavidin Magnetic Beads (SMB3) then capture the complexes containing the biotinylated exome panel probes hybridized to the complementary exome. The exome-enriched and indexed libraries are eluted from beads and further amplified before sequencing.
A subsequent Illumina Purification Beads (IPB) cleanup step then purifies libraries for use on an Illumina sequencing system.
