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xGen™ Hybridization Capture Core Reagents

Research reagents for the xGen NGS Hybridization Capture workflow

The xGen Hybridization Capture Core Reagents provide reliable targeted sequencing functionality across a range of panel sizes and multiplexing levels. They work with xGen Hyb Panels for a complete, high quality target enrichment research solution.

xGen NGS—made to maximize.

Ordering

  • Achieve uniform coverage and reliable capture functionality across a broad range of xGen Hyb Panels
  • Generate targeted NGS data with quick and easy workflow results
  • Updated, automation-friendly protocol enables various levels of throughput
  • xGen Universal Blockers effectively block a variety of index adapter designs with proprietary sequence modifications, improving on-target results

For customers using the legacy xGen™ Lockdown reagents, these products can be ordered here.

Product details

The xGen™ Hybridization and Wash Kit has been designed for use with xGen Hyb Panels and xGen Universal Blockers. The kit is compatible with the protocol, xGen hybridization capture of DNA libraries for NGS target enrichment, to provide a complete, high-quality hybridization capture solution for targeted NGS research.

Prevent adapter cross-hybridization by using xGen Universal Blockers in your hybridization capture reactions. Adapter sequences are ligated to all library fragments, both on-target and off-target, before enrichment. These adapter sequences can hybridize with each other during enrichment, creating a "daisy-chain" effect, with off-target fragments being captured alongside on-target fragments, which can impact sequencing efficiency.

xGen Universal Blockers bind to platform adapter sequences on a designated strand (usually the inverse of the synthetic adapter) to help prevent non-specific binding. Blocking the adapter sequences significantly improves capture results [1,2].

Automation

We have partnered with several instrument vendors to develop automation scripts for research using the xGen hybridization capture protocol on liquid handling robots. Scripts are currently available for the following:

  • Beckman Coulter Biomek™ i5 and i7 Automated Workstations
  • Hamilton NGS STAR™ for Library Prep Workstation
  • Perkin Elmer Sciclone™ Workstation

Please contact the instrument vendors for more information regarding script availability and installation or contact IDT Customer Care for an updated list of qualified automation scripts for liquid handling robots.

Product data

Achieve uniform sequence coverage

Figure 1.  Example of uniform sequence coverage with xGen Hybridization Capture Core Reagents in research studies. DNA libraries were created from 100 ng of human genomic DNA (Coriell) using xGen Stubby Adapter and Unique Dual Index Primer Pairs. These libraries were enriched either as 1-plex captures (n=3) or in a single 12-plex capture using the xGen Hybridization and Wash Kit, xGen Universal Blockers, and xGen Exome Hyb Panel v2. The enriched libraries were sequenced (2x100) on a NextSeq™ instrument (Illumina) and subsampled to 5 Gb. The data show deep, uniform coverage with a flanked on-target rate of 94.7%, mean target coverage of 64.5X, and a duplication rate of 3.3% (calculated with Picard).

Consistent on-target rates manually and with automation

Figure 2. Consistent on-target rates independent of workflow format. Sequencing libraries with an average insert size of ~300 bp were prepared from replicate captures from a single human genomic DNA (Coriell NA12878) input. DNA libraries were enriched using the xGen AML Cancer Hyb Panel and the indicated combinations of methods (manual or automated) and formats (plate or tubes) were performed on three (3) days (n = 16 singleplex captures for automation and manual plate, n=8 singleplex captures for tube). The xGen Hybridization and Wash Kit was used according to the protocol in xGen hybridization capture of DNA libraries for NGS target enrichment. Automated runs were performed using the Perkin Elmer Sciclone™  Workstation with automation scripts developed jointly by IDT and Perkin Elmer. On-target rates were calculated using 150-base flanks added to the ends of target regions.

Increased on-target rates

Figure 3. An example of improved on-target rate delivered by xGen Universal Blockers across TruSeq (A) and Nextera (B) adapter styles. (A) 1ug input DNA libraries were prepared from human genomic cell line NA12878 (Coriell) using either 8nt or 10nt adapters , and enriched using the xGen AML Cancer Hyb Panel (singleplex, n=3 for each condition) with xGen Universal Blockers—TS Mix, xGen Universal Blockers 10bp – TS Mix or without blockers. Sequencing was performed on a NextSeq500™ System (Illumina) to generate 2 x 150 bp, paired-end reads. (B) 50 ng cell line NA12878 (Coriell) gDNA was used for Nextera™ library preparation. Amplified libraries were enriched in 4-plex reactions (n=3) using the xGen AML Cancer Hyb Panel, or in 12-plex (n=1) using the xGen Human ID Hyb Panel, with xGen Universal Blockers—NXT Mix or without blockers (n=1). Sequencing was performed on a NextSeq™ system (Illumina) to generate 2 x 75 bp paired-end reads.

High on-target and uniform coverage with xGen Universal Blockers—10 bp TS Mix

Figure 4. (A) Example of consistent results with multiplexed samples using the xGen Universal Blockers—10 bp TS Mix.DNA libraries (500 ng input per sample) from cell line NA12878 (Coriell) were enriched in single (n=8) or multiplex reactions (n=3) using the xGen AML Cancer Hyb Panel. The libraries were created using 10 bp TruSeq™ adapters with unique dual indexes. Sequencing was performed on a NextSeq™ 500 System (Illumina) with 2 x 150 bp paired-end reads. Coverage was consistent across all multiplexing levels tested. (B) Example of improved on-target rates delivered by xGen Universal Blockers—10 bp TS Mix. DNA libraries (500 ng input per sample) from cell line NA12878 were enriched in singleplex reactions (n=3) using the xGen AML Cancer Hyb Panel with and without xGen Universal Blockers—10 bp TS Mix. Sequencing was performed on a NextSeq™ 500 System to generate 2 x 150 bp, paired-end reads. On-target values (with 150 bp flank) were averaged across experiments.

References

  1. Blumenstiel B, Cibulskis K, et al. (2010) Targeted exon sequencing by in-solution hybrid selection. Curr Protoc Hum Genet, Chapter 18:Unit 18.4.
  2. Hodges E, Rooks M, et al. (2009) Hybrid selection of discrete genomic intervals on custom-designed microarrays for massively parallel sequencing. Nat Protoc 4(6):960–974.

*RUO—For research use only. Not for use in diagnostic procedures. Unless otherwise agreed to in writing, IDT does not intend for these products to be used in clinical applications and does not warrant their fitness or suitability for any clinical diagnostic use. Purchaser is solely responsible for all decisions regarding the use of these products and any associated regulatory or legal obligations.

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