The required input for Nextera Enrichment kits is 50 ng gDNA. Quantify the input using a fluorometric-based method specific for duplex DNA, such as the Qubit dsDNA BR Assay system.
When the prep is complete, 500 ng is used as input for the enrichment portion of the protocol. Use PicoGreen or the Qubit Fluorometric Quantitation system to quantify the input. Adding > 500 ng library DNA into the enrichment assay (up to 1 ug per sample) might produce greater mean coverage per sample.
The Illumina Experiment Manager (IEM) will notify you if improper combinations are used when creating a sample sheet for use with CASAVA, so it is highly recommended to create your sample sheet prior to performing sample prep/pooling. There are also low plexity pooling guidelines in the Nextera, TruSeq DNA, and TruSeq RNA sample preparation guides. Always pool samples with valid index combinations to avoid image registration failures.
The Nextera Enrichment DNA Sample Preparation kit supports pre-enrichment pooling of up to 12 different indexed samples. For pooling libraries prior to enrichment, Illumina recommends pooling libraries so all Index 1 (i7) indexes are unique. With this pooling approach, samples can be sequenced using a single index read workflow, as described in the relevant instrument User Guide. If Index 1 (i7) indexes are not unique, ensure that libraries with different Index 2 (i5) indexes are included (eg, N703/E501 and N703/E502). With this approach, samples can be sequenced using a dual index read workflow, as described in the relevant instrument User Guide. For additional details regarding sample pooling, please refer to the Nextera Enrichment Sample Prep Guide.
Nextera Enrichment Kits are provided as a bundle with reagents for sample prep, enrichment and the indexes included together under a single catalog part number. To order a Nextera Custom Enrichment kit, DesignStudio is an online design tool that supports custom design and ordering for Nextera Custom Enrichment projects. Please go to http://designstudio.illumina.com/ for more information or to design a project. Note that a MyIllumina account is required to access DesignStudio.
Two different Nextera enrichment kits are available: an exome enrichment kit and a custom enrichment kit. Both kits use the fast, simple, high-throughput protocol of Nextera technology with a comprehensive enrichment solution.
This is difficult to predict, but the complexity of the custom oligo library may yield lower enrichment rates and lower coverage uniformity. Additionally, low complexity libraries (eg, less than 2 Mb capture) or high GC content areas may have lower enrichment or coverage uniformity as well.
No. The Nextera Rapid Capture Custom Enrichment kits contain unique reagents that are not provided in other Nextera kits, including Index 2 primers with an "E" prefix. Index 2 primers from other Nextera library prep kits should not be used.
Samples enriched with Nextera Enrichment kits can be sequenced on the MiSeq System. Full bioinformatics analyses may have to be performed offline, and support for enrichment workflows is expected in a future MiSeq Reporter software update.
Illumina does not support, and strongly advises against, running libraries prepared with different library prep kits in the same lane of a flow cell. Running libraries prepared by different library prep kits in different lanes of the same flow cell or spiking in Illumina PhiX control library in the same lane as any user-prepared libraries is supported.
If different library types are run in different lanes, dual index recipes and the Dual Index Primer Box are required. The indexes for TruSeq HT and Nextera are unique and not shared.
Based on performance data and range of library sizes, Illumina recommends 2 × 36 bp to 2 × 50 bp read lengths for runs on a HiSeq system. For MiSeq runs, 2 x 150 bp runs are recommended.