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xGen® Blocking Oligos

Enhance the Performance of Target Capture for Next Generation Sequencing

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, improve the number of reads on-target, and increase the depth of enrichment.

Benefits

  • Increase sample throughput by effectively blocking multiple pooled library adapters with a single blocking oligo
  • Easy and flexible ordering of predesigned sequences that correspond to commonly available adapter library preparation kits

Features

  • A single xGen® Universal Blocking Oligo effectively blocks multiple barcoded adapters
  • Easily order from predesigned xGen Standard Blocking Oligos for low throughput target capture


xGen® Universal Blocking Oligos

Illumina TruSeq® and Other  Single-Index Adapters

These xGen Universal Blocking Oligos are designed for single-indexed adapters. There is 1 Universal Blocking Oligo that binds to the p5 (universal) adapter and 2 Universal Blocking Oligos are designed for 6 nt and 8 nt index versions of the p7 adapter. 

For more information on compatibility and instructions for use, please review the "How to Use xGen Universal Blocking Oligos" section under the Support Tab.

For Illumina TruSeq® HT and Other  Dual-Index Adapters

These xGen Universal Blocking Oligos are designed for dual-indexed adapters. They are compatible with any dual indexing scheme that uses adapters with an 8-base index region, such as Illumina TruSeq HT adapters.

For Ion Torrent Barcoded Adapters

These consist of 2 xGen Universal Blocking Oligos predesigned to bind to the P1 and barcoded A adapters used in the Ion Torrent platform.

xGen® Standard Blocking Oligos

For Illumina TruSeq® and Other Single-Index Adapters

These xGen Standard Blocking Oligos are predesigned to bind to the most common single-index library adapters that comprise 1 universal adapter and 24 barcoded adapters. The 24 Standard Blocking Oligos designed against the barcoded adapters are labeled 1–27, corresponding to the numbering scheme used in Illumina, NEB, and Bioo Scientific single-index adapter kits.

For Illumina TruSeq® HT and Other Dual-Index Adapters

These xGen Standard Blocking Oligos are predesigned against 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

These xGen Standard Blocking Oligos are predesigned against 2 non-barcoded adapters (A and P1) and 32 barcoded A adapters used in the Ion Torrent platform.

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 Roche Nimblegen in the SeqCap LR V3.0 protocol.

xGen® Custom Blocking Oligos

Custom xGen® Blocking Oligos

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

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 submit this form to receive a custom quote.

Prevent Adapter Cross-Hybridization

Adapter sequences specific to the platform being used for 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.

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 (Figure 1). HPLC purification of your blocking oligos can improve binding to adapter sequences. Introducing end-terminating modifications, such as a 3' C3 spacer, helps to inhibit spurious amplification of blocking oligos.

Enhance Capture Performance by Blocking Platform Adapters

Figure 1. Blocking Oligos Increase On-Target Read Percentage. Several published articles have shown markedly higher on-target percentage by adding oligo blockers. For example, Blumenstiel et al. [1] increased on-target reads from 35% to 59% by including blocking oligos before target capture; Hodges et al. [2] achieved even better performance with blocking oligos, improving their on target reads from 32% before blocking oligos to 64% after the addition of blocking oligos. This approximately two-fold increase in on-target reads allows for multiplexing of a greater number of samples and increases read depth for easier identification of rare alleles.

High and Low Throughput Options with Excellent Peformance

Figure2. A Single xGen® Universal Blocking Oligo Effectively Enhances Multiplex Sequencing. Blocking oligos improve on-target capture percentage by binding to adapter sequences to prevent cross-hybridization from occurring between library fragments. xGen Universal Blocking Oligos enhance performance and enable higher multiplexing by blocking multiple barcoded adapters using a single oligonucleotide sequence. The data show 4-plex sequencing of genomic DNA enriched using a 1.2 Mb xGen Lockdown® panel and run on an Illumina MiSeq® platform. The percent on-target includes a 500bp buffer region at each end of the target. Error bars indicate standard deviation.

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.

Resuspension Instructions

Final concentration should be 1 nmole/µL or 1 reaction/µL.

Product Scale Resuspension Volume (1X)
10 reaction (or 10 nmole) 10 µL TE Buffer pH 7.5
25 reaction (or 25 nmole) 25 µL TE Buffer pH 7.5

How to Use xGen Universal Blocking Oligos

For Illumina TruSeq® LT Libraries 

Important: If you are using a combination of 6 nt (adapters 1–12) and 8 nt (adapters 13–27) barcoded TruSeq® LT adapters, use the formulas below to determine the fractions of 6 nt (X) and 8 nt (Y) blocking oligos you will need. X + Y must equal 1.

    X = (number of libraries with adapters 1–12 or 6nt barcodes) / (total number of barcoded libraries)
    Y = (number of libraries with adapters 13–27 or 8nt barcodes) / (total number of barcoded libraries)
500 ng Illumina TruSeq® LT Library
5 µg Cot-1 DNA
1 μL xGen® Universal Blocking Oligo – TS-p5
X μL xGen® Universal Blocking Oligo – TS-p7 (6 nt)
Y μL xGen® Universal Blocking Oligo – TS-p7 (8 nt)

Compatibility Guide:

TS-p5 and TS-p7 (6nt Index)

  • TruSeq LT Adapters 1–12
  • NEB Adapters 1–12
  • Bioo Adapters 1–6, 7–12, 13–24
  • Any single-index adapter with a 6-nucleotide index

TS-p5 and TS-p7 (8nt Index)

  • TruSeq LT Adapters 13–27
  • NEB Adapters 13–27
  • Bioo Adapters 96-index
  • Any single-index adapter with an 8-nucleotide index

For Illumina TruSeq® HT Libraries*

500 ng Illumina TruSeq® HT Library
5 µg Cot-1 DNA
1 μL xGen® Universal Blocking Oligo TS HT-i5
1 μL xGen® Universal Blocking Oligo TS HT-i7

* No calculations are necessary for determining the required amounts of TruSeq HT blocking oligos because the lengths of the barcoded regions are fixed.

For Ion Torrent® Libraries

500 ng Ion Torrent® library
5 µg Cot-1 DNA
1 μL xGen® Universal Blocking Oligo – IT-P1
1 μL xGen® Universal Blocking Oligo – IT-A*

* Assumes that the A adapter contains a barcode sequence. If the A adapter does not contain a barcode sequence, use xGen® Standard Blocking Oligos.


How to Use xGen Standard Blocking Oligos

Total blocking oligo material should be equivalent to 1 nmole each for the 3' and 5' ends of the library fragments. For multiplexed library preparation, the sum of all blocking oligos used to block barcoded adapters should equal 1 nmole.

Example 1: Dr Jane is performing target capture for a singleplex (1 sample) sequencing experiment on the Illumina MiSeq® sequencing platform. During enrichment, she will need 1 nmole (10 μL of 1 mM) blocking oligo against the universal sequencing adapter and 1 nmole indexed adapter complementary to the one used in sample preparation.

Example 2: Dr Jane is performing a capture on 24 pre-barcoded and pooled samples prepared using the Illumina TruSeq LT sample preparation kit. She will need 1 nmole of the universal sequencing adapter, and 42 pmole (1/24) of the blocking oligo corresponding to each indexed adapter (a total of 25 blocking oligos).

Available Modifications and Services

Description10 nmole xGen™ Blocking Oligo25 nmole xGen™ Blocking Oligo
HPLC Purification$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 oligos for your specific application. In the event that the standard modifications or yields do not meet your needs, please submit this form to receive a custom quote.

Note: If you have previously ordered your blocking oligos as custom oligos, you can go to your order history in your online account and add the same products to your cart without having to get a new quote.