xGen Blocking Oligos
Improve on-target performance during targeted sequencing by reducing adapter-to-adapter hybridization
xGen Blocking Oligos bind to library adapter sequences to reduce off-target capture during library enrichment. Choose from convenient, optimized, predesigned mixes or build your own custom blocking oligo. xGen Universal Blockers are designed for use with Illumina indexed adapters. These ready-to-use mixes effectively block dual- or single-index adapters to significantly improve on-target performance.
- Reduce sequencing cost per sample by improving on-target performance
- Effectively block a variety of index adapter designs with proprietary sequence modifications
- Eliminate complicated blocker selection by using worry-free formulations compatible with library prep kits, such as Illumina’s ligation-based TruSeq® kit or Illumina’s Nextera® kit
- Reduce workflow steps with a convenient premixed liquid formulation
Custom xGen Blocking Oligos are also available for other platforms, including Ion Torrent, and specialized applications.
Product details
All xGen Blocking Oligos are compatible with any xGen Lockdown Panel or custom xGen Lockdown Probe set. Each oligo is individually synthesized and assessed by ESI mass spectrometry for quality control*.
* With the exception of mixed base oligos, which could potentially represent multiple sequences and therefore cannot be accurately evaluated by ESI mass spectrometry.
xGen Universal Blockers
IDT currently offers three different ready-to-use, universal blocker reagents. xGen Universal Blockers—TS Mix blocks Illumina LT (p5, p7 – 6 nt and 8 nt) and HT (i5, i7) adapters and is compatible with ligation-based library prep kits, such as Illumina TruSeq kits. xGen Universal Blockers—10 bp TS Mix is compatible with ligation-based library prep kits that use 10 bp index barcodes. xGen Universal Blockers—NXT Mix contains adapter blockers that are optimized for use with Illumina Nextera library prep kits.
xGen Universal Blockers are offered in 16-, 96-, and 4 x 96-reaction formats for convenient experiment design and for use with xGen Lockdown Panels or xGen Lockdown Probe sets.
Custom xGen Blocking Oligos
Custom blocking oligos can be ordered for specialized applications or other sequencing platforms, and are offered with specified modifications. While we have identified the most commonly used modifications and services required for effective blocking in next generation sequencing, we understand that certain modifications or services may improve the performance of blocking oligo performance for your specific application.
For more information about available blockers, please contact our customer care team.
Technical overview
Prevent adapter cross-hybridization by using xGen blocking oligos in your target 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 Blocking Oligos 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 performance [1,2].
References
- Blumenstiel B, Cibulskis K, et al. (2010) Targeted exon sequencing by in-solution hybrid selection. Curr Protoc Hum Genet, Chapter 18:Unit 18.4.
- 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.
Performance
Increased on-target performance with adapter blockers
Figure 1. Improved on-target performance delivered by xGen Universal Blockers. (A) DNA libraries were prepared from cell line NA12878 (Coriell) using a TruSeq Exome Library Prep Kit (Illumina), and enriched in single or multiplex reactions using the xGen AML Cancer Panel 1.0 with xGen Universal Blockers—TS Mix. Sequencing was performed on a MiSeq® System (Illumina) to generate 2 x 150 bp, paired-end reads. On-target values (with 150 bp flank) were averaged across experiments. (B) Cell line NA12878 (Coriell) was used for library preparation using the Nextera DNA Library Preparation Kit for Enrichment (Illumina). Amplified libraries were enriched in single or multiplex reactions using the xGen Exome Panel, xGen AML Cancer Panel, or the xGen Human ID Research Panel, with xGen Universal Blockers—NXT Mix. Sequencing was performed on a NextSeq® system (Illumina) to generate 2 x 75 bp paired-end reads.
Consistent on-target performance with multiplex capture
Figure 2. Consistent performance during multiplex enrichment using xGen Universal Blockers. DNA libraries (500 ng/sample) from cell line NA12878 (Coriell) were enriched in single or multiplex reactions using the xGen Exome Panel v1.0. The enriched libraries were created using (A) the TruSeq Exome Library Prep Kit (Illumina), adding xGen Universal Blockers—TS Mix during hybridization capture, or (B) the Nextera DNA Library Preparation Kit for Enrichment (Illumina), adding xGen Universal Blockers—NXT Mix during hybridization capture. Hybridization washes were done on the Sciclone® G3 liquid handling workstation (Perkin Elmer). Sequencing was performed on a NextSeq system (Illumina) to generate 2 x 75 bp paired-end reads. Minimal differences in performance were observed across multiplexing levels.
Equivalent, high target coverage of LT and HT adapter libraries
Figure 3. Effective blocking of LT and HT adapters using xGen Universal Blocker—TS Mix. DNA libraries prepared from cell line NA12878 (Coriell), and incorporating LT (n = 12) or HT (n = 12) adapters, were enriched using the xGen AML Cancer Panel v1. 0 with xGen Universal Blockers—TS Mix (IDT). Target enrichment was performed according to the protocol. Hybridization capture of DNA libraries using xGen Lockdown Reagents and Probes, v3. Enriched libraries were sequenced on a MiSeq system (Illumina) to generate 150 bp paired-end reads. Comparable target coverage was observed for the LT and HT adapter libraries.
High on-target and uniform coverage with xGen Universal Blockers—10 bp TS Mix
Figure 4. (A) Consistent performance with multiplexed samples using the xGen Universal Blockers—10 bp TS Mix. DNA libraries (500 ng/sample) from cell line NA12878 (Coriell) were enriched in single or multiplex reactions using the xGen Acute Myeloid Leukemia Cancer Panel v1.0. 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) Improved on-target performance delivered by xGen Universal Blockers—10 bp TS Mix. DNA libraries (500 ng/sample) from cell line NA12878 were enriched in singleplex reactions using the xGen Acute Myeloid Leukemia Cancer Panel v1.0 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.
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