This product was formerly named Nextera Flex for Enrichment.
The sequencing data specifications of Illumina DNA Prep with Enrichment libraries are dependent on the enrichment probe panel.
See the Illumina DNA Prep with Enrichment Data Sheet for example data sets.
The chemistry used in Illumina DNA Prep with Enrichment with Illumina Custom Enrichment Panel is based on bead-linked transposome technology that is different from the Nextera Rapid Capture Custom workflow.
Illumina DNA Prep with Enrichment works with the Illumina Custom Enrichment Panel with comparable, and possibly improved, library quality and sequencing metrics.
Illumina DNA Prep with Enrichment offers significantly improved chemistry and shorter assay time (6.5 hours for 12 samples).
Illumina DNA Prep with Enrichment, (S) Tagmentation (16 Samples, catalog # 20025523 and 96 Samples, catalog # 20025524) contain pre-enrichment library preparation reagents and enrichment reagents (enrichment probe panel and index adapters are not included and must be ordered separately) for 16 or 96 samples, using the Illumina DNA Prep for Enrichment workflow.
Illumina DNA Prep, (S) Tagmentation (16 Samples, catalog # 20025519 and 96 Samples, catalog # 20025520) only contain pre-enrichment library preparation reagents (index adapters are not included and must be ordered separately) without enrichment reagents for 16 or 96 samples, using the Illumina DNA Prep with Enrichment workflow.
To complete the Illumina DNA Prep with Enrichment workflow and generate libraries for sequencing, the following components are needed:
See the Product Contents section in the Illumina DNA Prep with Enrichment Reference Guide for a list of supported Enrichment Probe Panels and user-supplied consumables and equipment that are needed to complete the library prep.
Agencourt AMPure XP beads (Beckman Coulter, catalog # A63880) are not included and must be purchased separately.
To be environmentally friendly, minimize the use of dry ice, and improve the unpackaging experience, Illumina formulated Illumina DNA Prep with Enrichment reagents to be compatible with 4oC shipping conditions. This shipping condition is not expected to impact reagent or workflow performance.
Promptly store the reagents at the temperature stated on the reagent package.
Specifically, store the Illumina DNA/RNA Prep Tagmentation PCR Reagents and Illumina DNA Fast Hyb Enrichment PCR + Buffers boxes at -25°C to -15°C.
Illumina DNA Prep with Enrichment, (S) Tagmentation (16 Samples, catalog # 20025523) is configured to support 16 pre-enrichment library preparation and sixteen 1-plex enrichments.
Illumina DNA Prep with Enrichment, (S) Tagmentation (96 Samples, catalog # 20025524) is configured to support 96 pre-enrichment library preparation and eight 12-plex enrichments.
Illumina DNA Prep with Enrichment is an enrichment-based library prep workflow for generating targeted next generation sequencing libraries compatible with all Illumina sequencing platforms.
Illumina DNA Prep with Enrichment uses an innovative, bead-based transposome complex to fragment and tag (tagment) genomic DNA with adaptors. Following tagmentation, a limited-cycle PCR step adds indexed adapter sequences to the ends of a DNA fragment. A subsequent target enrichment workflow is then applied by hybridizing biotinylated oligonucleotide probes. After hybridization, Streptavidin Magnetic Beads (SMB) are used to capture the targeted library fragments within the regions of interest. The captured, indexed libraries are eluted from beads and further amplified before sequencing.
Illumina DNA Prep with Enrichment works with a wide variety of input DNA such as:
Refer to the Input Recommendations section in the Illumina DNA Prep with Enrichment Reference Guide. The blood lysis and saliva lysis protocols are also provided in Appendix A.
Input DNA should have an 260/280 ratio of 2.0-2.2 and 260/230 ratio of 1.8-2.0. Additionally, input DNA should not contain EDTA (> 1 mM), SDS, phenol, ethanol or other inhibiting agents such as:
The Bio-Rad CFX96 Touch Real-Time PCR Detection System is recommended. Other systems have not been tested and may require validation.
No, using more blood will inhibit the reaction and is not recommended. Use 10 μl of blood as indicated in the Blood Lysis protocol.
Quantification of the DNA is not necessary but can be performed with a Qubit, using 1 μl sample diluted with 4 μl purified water. Quantification using a BioAnalyzer or Fragment Analyzer is not recommended.
Use of frozen or clotted blood with the Flex Lysis Reagent Kit and Blood Lysis protocol with Illumina DNA Prep with Enrichment is not supported or tested by Illumina.
Use of blood spots on cards with the Flex Lysis Reagent Kit and Blood Lysis protocol for Illumina DNA Prep with Enrichment is not supported or tested by Illumina.
This protocol has been developed to work with the Oragene saliva collection tubes. Saliva samples collected into other tube types may not be stable under the same conditions and therefore the use of other collection tubes is not recommended.
The recommended saliva volume is 30 μl. Although the DNA yield from saliva sample can vary due to biological differences, the library yield by Illumina DNA Prep with Enrichment with these samples is normalized. Quantification of saliva DNA before tagmentation is not necessary but can be done with 1 μl sample diluted with purified water at 1:5 ratio, then measured with Qubit. Quantification using a BioAnalyzer or Fragment Analyzer is not recommended.
Illumina recommends storing at 2ºC to 8ºC for up to 30 days.
Illumina DNA Prep with Enrichment has not been tested with non-human species but DNA from other species may be feasible. Customer validation is necessary for those applications.
No, eBLT and BLT beads are not interchangeable. BLT cannot be used in Illumina DNA Prep with Enrichment due to different reagent formulations developed for different workflows.
Yes, you can use strip tubes. Pay close attention when aligning strip tubes on thermal cycler to ensure proper sitting and optimal heat transmission.
Over-drying eBLT beads will significantly reduce the amounts of samples that can be eluted or increase the chances of complete sample loss.
To prevent over drying of eBLT, process samples in 16 sample (2 column) increments with multichannel pipettes when preparing > 48 samples.
Enrichment Plexity is the number of pre-enriched libraries (1–12) pooled together in one enrichment reaction for hybridization with the enrichment probe panels. For example, combining 12 pre-enriched libraries together creates a 12-plex enrichment pool.
Enrichment Reaction is the number of unique enrichment reaction preparations, regardless of the number of pre-enriched libraries pooled per reaction. For example, a single enrichment reaction can prepare a 1-plex or 12-plex enrichment pool.
To calculate the total number of post-enriched libraries, multiply the enrichment plexity per reaction by the number of enrichment reactions. For example, a single enrichment reaction of a 12-plex enrichment pool produces a pool of 12 post-enriched libraries.
Uneven library input amount into enrichment is not expected to change enrichment efficiency. However, uneven library input amount will impact data representation in final sequencing data. Acceptable range of input depends on acceptable variation of data representation among samples for your experiments.
Illumina DNA Prep with Enrichment libraries constructed with FFPE samples have been tested at 1-plex enrichment with 500 ng per pre-enriched library when pooling by mass or 14 μl per pre-enriched library when pooling by volume. Other enrichment plexities might be possible but optimal results are not guaranteed.
For samples derived from high-quality DNA, as little as 100 ng per pre-enriched library can be used for enrichment.
Greater than 12-plex pooling for enrichment is not supported.
Yes. When pooling by mass, 500 ng per pre-enriched at 12-plex enrichment from high quality gDNA sample is supported (total of up to 6 μg DNA). As little as 100 ng per pre-enriched library can be used for enrichment. Additional optimization may be required to obtain suitable enrichment yield when pooling pre-enriched libraries < 250 ng. Optimal results are not guaranteed.
If proceeding with < 500 ng per pre-enriched library for enrichment, decrease the mass of each pre-enriched library to equal concentration based on your sample quantification results. Make sure the total DNA library mass (ng) remains 500–6000 ng. For example, if you have 250 ng per pre-enriched library, you can proceed with 12-plex enrichment with 3000 ng of total DNA library mass.
Illumina recommends 62ºC for all third-party panels. You can choose to test other hybridization temperatures recommended by other vendors at your own risk.
No, both strip-tubes and 96-well PCR plates have been tested. Be cautious that strip-tubes can easily be crushed in the thermal cycler.
Yes, you use the same volume of enrichment reagents for 12-plex enrichment as 1-plex enrichment.
Use the following steps to manually program the NF-HYB program:
If optimizing percent duplicates for third party panels is needed, increasing the 90 minute hold time to between 2.5 to 16 hours might improve performance. See example data sets with increased hybridization times in BaseSpace Public Data.
The enrichment reaction can be held at the NF-HYB final temperature for up to 24 hours with Microseal ‘B’ to prevent evaporation. Holding for more than 24 hours can lead to sample evaporation resulting in low yield, low percent enrichment efficiency, and/or low coverage uniformity.
No, the capture and wash reaction volumes are too high for 96-well PCR plates on a thermal cycler.
Use of a heat block is not recommended because temperature control on a heat block is less accurate than the Microheating System-Hybex System for Illumina.
Illumina recommends a paired-end run of 101 cycles per read (2x101) with 10 cycles for each index read (Read 1: 101, Index 1: 10, Index 2: 10, Read 2: 101) regardless of the enrichment probe panel or sample input type. You can sequence longer to 2x126 or 2x151 if you would like additional overlapped reads or additional raw coverage, but it is not required. The recommended minimum read length is 2x101 and the optional maximum read length is up to 2x151.
For more information on sequencing run set up adjustments to accommodate 10 cycle index reads, see IDT for Illumina - DNA/RNA UD Indexes.
If performing the sequencing run on HiSeq 2500 with a 200-cycle reagent kit, click Continue when prompted to allow sequencing without topping off sequencing reagents.
See the setup page for your instrument on IDT for Illumina - DNA/RNA UD Indexes for information on setting up your sequencing run and to download sample sheet templates or library kit definition files.
If you are using the standalone mode on iSeq, MiniSeq, NextSeq, HiSeq series, or NovaSeq, enter 10 bp as the index read.
Yes. Select IDT-ILMN DNA/RNA UD Indexes Set A for Illumina DNA Prep with Enrichment from the Library Prep Kit dropdown menu. This will default your sequencing run to 101 for Read 1 & 2 Cycles and Dual Index with 10 for Index 1 & 2 Cycles. If you only select one sample for the pool, Index 1 & 2 will default to 0 as indexing reads are not required, but you may change this to 10 if you would like to proceed with dual indexing reads.
The recommended sequencing depth or number of reads per sample depends on the enrichment probe panel used.
The following are the recommended coverage for germline variant calling with some of the supported panels:
Illumina Exome (CEX) – 50M PE reads
TruSight One – 22M PE reads
TruSight One Expanded – 33M PE reads
TruSight Cancer – 2M PE reads
TruSight Cardio – 2M PE reads
Illumina recommends the Enrichment App in BaseSpace Sequence Hub. For more information on how to use the Enrichment App, see the Enrichment App Online Help.
Human genome assembly hg19 is being used for analysis in Enrichment App v3.1.
See the Set Analysis Parameters section in the Enrichment v3.1 Online Help for more information.
Enrichment App v3.1 supports up to 96 samples per analysis.
Yes, the Enrichment App v3.1 supports CNV analysis. However, CNV analysis was not tested with Illumina DNA Prep with Enrichment.