Next Gen Sequencing

Protocols and Support

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Blocking Oligos for NGS Target Enrichment Applications

xGen™ Blocking Oligos

xGen™ Blocking Oligos are essential compo­nents of the target enrichment process for next generation sequencing (NGS). During sample preparation, platform-specific adapters are added to all library fragments. These adapter sequences can interact with each other during hybridization-based en­richment, causing a daisy-chain effect, and thereby increasing undesired enrichment of off-target sequences. Blocking oligos, added during hybridization of the capture reagents, bind to the adapter sequences to prevent them from hybridizing to each other. Using high-quality blocking oligonu­cleotides enhances this effect and increases the number of “reads on-target”, a measure of the amount of sequence information that corresponds to the target region(s) of interest, observed after sequencing.

xGen Blocking Oligos are available at a defined yield of 10 or 25 nmoles and can be ordered using our intuitive online interface. Now you can easily:

  • Obtain a complete set of barcode- specific blocking oligonucleotides.
  • Replace individual oligos in an existing set.
  • Configure “universal” blocking oligos by incorporating deoxyinosine modifica­tions or degenerate bases.
  • Enhance binding efficiency by request­ing HPLC purification.
  • Add terminal modifications, such as a 3’ C3 Spacer, to prevent nonspecific ampli­fication of the blocking oligos.

Common xGen Blocking Oligo Sets

Illumina® TruSeq®

Illumina® TruSeq® HT

Roche 454 Adapters

Ion Torrent™ Adapters

For more information, or to order xGen Blocking Oligos, go to www.idtdna.com/xgen and select xGen Blocking Oligos, or email us at xgen@idtdna.com.

 

Additional Resources

xGen® Lockdown® Panels

Stocked Enrichment Panels for Targeted Next Generation Sequencing

xGen® Lockdown® Panels are stocked enrichment panels for targeted next generation sequencing. Lockdown Panels consist of individually synthesized and quality controlled Lockdown Probes that have been internally validated to provide the highest level of performance.



The xGen® AML Cancer Panel v1.0 comprises 11,743 individually synthesized and quality controlled xGen Lockdown® Probes that deliver deep enrichment of targets from more than 260 genes associated with the AML disease pathway.

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The xGen® Pan-Cancer Panel v1.5 consists of 7816 individually synthesized and quality controlled xGen Lockdown® Probes that provide deep enrichment of 127 significantly mutated genes implicated across 12 tumor types.

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The xGen® Inherited Diseases Panel v1.0 comprises 116,355 xGen Lockdown® Probes targeting 4503 genes and 180 SNPs associated with inherited diseases. The panel is available in 2 sizes—16 reactions and 96 reactions.

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Design Method

The design of xGen® Lockdown® Panels is typically based on 1X tiling of xGen Lockdown Probes (Figure 1).

Figure 1. Schematic of 1X Tiling Density. Probes are tiled end-to-end to allow each base within the region of interest to be covered at least once.

Synthesis

xGen® Lockdown® Probes are synthesized individually using the IDT Ultramer® synthesis platform, which incorporates the highest coupling rate (~99.6%) in the oligonucleotide manufacturing industry to provide high quality, long oligonucleotides.

The quality of each probe is then assessed by electrospray ionization mass spectrometry (ESI-MS; Figure 2 shows a typical trace).   Even probes with high GC content can be synthesized. Probes that do no pass QC are documented, unlike probes from an array pool, where there is no way of knowing beforehand of their presence or absence.

 

Figure 2. ESI-MS of a Typical xGen® Lockdown® Probe. The trace of an xGen Lockdown Probe with 78.3% GC content is shown. Sequence: 5′ biotin–GCG GCG AGC GGA GAT CCG GGG CCT GCG CTG CGC ACT CGA GCC TGG CGG GCC GGC ACG GTG CGG GCC ATG AGC GGG GCG GTG CCC CAG GAC CTA GCG GTG AGT GGC GGC CGA GTC GGG CAC


Supplementing xGen® Lockdown® Panels

xGen® Lockdown Panels can be customized by supplementing with additional probes. When supplementing a Lockdown Panel, it is important to add the new probes at the same molar concentration as the probes in the original pool. The following table lists the molar concentration of each probe for a single hybrid capture reaction.

Panel
Number of Probes
fmol per Reaction
xGen® Acute Myeloid Leukemia Cancer Panel v1.0 11,743 0.255
xGen® Pan-Cancer Panel v1.0 7,816 0.384
xGen® Inherited Diseases Panel v1.0 116,355 0.026

For step-by-step instructions on how to supplement Lockdown Panels with additional probes, consult the addendum to the xGen Rapid Capture Protocol, Probe Supplementation (Spike-In) Protocol v1.0.

Performance Guarantee

All products have been functionally tested using the xGen® Rapid Capture Protocol, which is 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.