xGen® Blocking Oligos

xGen® Blocking Oligos prevent adapter cross hybridization and minimize off-target capture, increasing specificity during targeted sequencing.

xGen Universal Blockers—TS Mix is designed for use with Illumina® barcoded adapters. This ready-to-use mix effectively blocks dual- or single-index adapters to significantly improve on-target performance.

  • Improve on-target performance by reducing adapter-to-adapter hybridization
  • Effectively block multiple barcoded adapters using a single set of blocking oligos
  • Reduce workflow steps with a convenient premixed liquid formulation

Also available

  • xGen Standard Blocking Oligos
  • xGen Custom Blocking Oligos
  • xGen Blocking Oligos for diagnostics—contact GMPinfo@idtdna.com for information
  • xGen Blocking Oligos for Ion Torrent adapters

For previously offered xGen Universal Blocking Oligos delivered separately for dual- and single-index adapters, click here to order or download the resuspension protocol. For information about future availability of these legacy products, please contact applicationsupport@idtdna.com.

Heading

For single-index adapters (e.g., Illumina® TruSeq®)

Predesigned to bind to the most common single-index library adapters that comprise 1 universal adapter and 24 barcoded adapters. The 24 blocking oligos for the barcoded adapters are labeled 1–27, corresponding to the numbering scheme used in Illumina, NEB, and Bioo Scientific single-index adapter kits.

For dual-index adapters (e.g., Illumina TruSeq HT)

Designed to block Illumina TruSeq HT dual-index adapters, i5 and i7. The i5 oligos consist of numbers D501–D508 and the i7 oligos consist of D701–D712.

For Ion Torrent™ Barcoded Adapters

Predesigned against 2 non-barcoded adapters (A and P1) and 32 barcoded A adapters.

For Roche®/454 Adapters

Protocols for blocking Roche 454 GS and GS Jr adapters generally require only 2 blocking oligos. The adapters in this set are recommended by the Roche Nimblegen® SeqCap® LR V3.0 protocol.

xGen® Standard Blocking Oligos

Heading

Custom designs

ProductPricingLength
10 nmole xGen® Blocking Oligo$0.99 USD / Base20 - 75 Bases
25 nmole xGen® Blocking Oligo$1.99 USD / Base20 - 75 Bases

Available modifications and services

Description10 nmole xGen® Blocking Oligo25 nmole xGen® Blocking Oligo
HPLC$70.00 USD$104.00 USD
3' C3 Spacer$55.00 USD$85.00 USD
3' Dideoxy-C$125.00 USD$194.00 USD
Int deoxyInosine$12.00 USD$18.00 USD

Custom yield or modification quote

While we have identified the most commonly used modifications and services required for effective blocking in next generation sequencing, IDT understands that certain modifications or services may improve the performance of our oligonucleotides for your specific application. In the event that the standard modifications or yields do not meet your needs, please request a custom quote.

Note: If you have previously ordered your blocking oligos as custom oligos, you do not need to request a new quote. Simply add the custom blocking oligos to your cart from the order history in your online account.


xGen® Custom Blocking Oligos

Heading

xGen® Blocking Oligos for Ion Torrent platforms

xGen® Blocking Oligos

Prevent adapter cross-hybridization by using xGen Blocking Oligos in your target capture reactions. Adapter sequences specific to a given next generation sequencing platform 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 in which off-target fragments are captured along with 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].

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 mass spectrometry for quality control. Predesigned xGen Universal Blockers receive additional functional validation to improve performance and provide consistent results.

xGen Universal Blockers—TS Mix

xGen Universal Blockers—TS Mix is a specially formulated blend of IDT’s industry leading universal blocking oligos for Illumina® adapters. Based on proprietary technology, this ready-to-use, single tube mix blocks Illumina LT (p5, p7 – 6 nt & 8 nt) and HT (i5, i7) adapters to deliver optimal on-target sequencing performance and overall cost-effectiveness. Both 16- and 96-reaction formats are available for convenient experimental design and use with xGen Lockdown Panels or xGen Lockdown Probe sets.


xGen Standard Blocking Oligos

Standard blocking oligos are made-to-order oligonucleotides that target specified adapter sequences.


xGen Custom Blocking Oligos

Custom blocking oligos are available in either Universal or Standard formats.


xGen Blocking Oligos for Ion Torrent™ Adapters

These universal blockers are specific to the P1 and barcoded A adapters.

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.

Increased on-target performance with adapter blockers

Figure 1

Figure 1. xGen® Universal Blockers increase on-target read percentage. DNA libraries were prepared from cell line NA12878 (Coriell) using the KAPA Hyper DNA Library Prep Kit for Illumina (Kapa Biosystems). Libraries were enriched with the xGen AML Cancer Panel 1.0 (IDT), according 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. On-target values (with 150 bp flank) were averaged across experiments. The blockers performed equally well whether used as a preformulated mix (xGen Universal Blockers) or as individual blocking oligos.

Consistent on-target performance with multiplex capture

Figure 2

Figure 2. Consistent performance of multiplex enrichment using xGen® Universal Blockers. DNA libraries prepared from cell line NA12878 (Coriell) were enriched in singleplex HT (n = 12) or multiplex HT (n = 4) reactions using the xGen AML Cancer Panel v1. 0 with xGen Universal Blockers—TS Mix (IDT). For multiplex captures, 500 ng of each library was used, for a total of 2 µg per multiplex capture of 4 libraries. 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. No difference was observed in performance between singleplex and multiplex captures.

Equivalent, high target coverage of LT and HT adapter libraries

Figure 3

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.

Performance guarantee

Products have been functionally tested using the protocol, Hybridization capture of DNA libraries using xGen Lockdown Reagents and Probes, v3 or v2 (for blocker formulations other than xGen Universal Blockers), which are available online. Please contact IDT at xgen@idtdna.com for more information on tested protocols. Products are expected to be functional for up to 6 months after the "sell by" date listed on the tube.