March 15, 2014 (Vol. 34, No. 6)

Use of Adapter Blocking Oligos for In-Solution Hybridization Improves Target Capture Performance

In-solution hybridization (target capture) has been used since the early days of next-generation sequencing (NGS) to enrich for genomic regions of interest. Hybridization protocols have routinely included Cot-1 DNA, which blocks repetitive sequences to prevent nonspecific binding of hybridization probes. However, researchers continued to experience poor signal-to-noise ratios, diminishing the cost and performance benefit of target capture.

Most of this background signal was caused by nonspecific binding of cross-reacting adapter sequences. These adapter sequences, which are present on every single library fragment, are required for sequence recognition by the different sequencing platforms. To overcome this interaction between adapter sequences, researchers began using synthetic oligonucleotides, called blocking oligos, to bind to the adapters and prevent cross-reactivity. Blocking oligos are now an essential and integral component of the NGS workflow.

Figure 1. Increased on-target read percentage with blocking oligos: Higher on-target percentage is achieved by adding oligonucleotide blockers before performing target capture. Increases in on-target reads from 35% to 59% [left] and 32% to 64% [right] were obtained by the addition of blocking oligos. This twofold increase in on-target reads allows for multiplexing of a greater number of samples and increases read depth for easier identification of rare alleles.

Maximizing On-Target Reads

Blocking oligos are essential for obtaining high-quality sequence data from focused NGS applications. By binding to platform-specific adapter sequences, blocking oligos prevent daisy chaining between target and off-target library fragments to improve the efficiency and performance of target capture. Failure to use blocking oligos during target enrichment can result in 50% reduction in on-target reads (Figure 1).

xGen® Blocking Oligos

Blocking oligos are synthetic oligonucleotide sequences that hybridize to specific sequences, including adapters, in a sequencing library to prevent cross-hybridization between library fragments. They are incubated with library fragments and Cot-1 DNA before the target enrichment probes are added (Figure 2). IDT offers two types of blocking oligos—xGen® Universal Blocking Oligos and xGen Standard Blocking Oligos.

xGen Universal Blocking Oligos are functionally validated oligonucleotides that incorporate proprietary modifications to enable a simpler workflow and higher throughput. Unlike regular adapter-specific blocking oligos, a single Universal Blocking Oligo can block multiple adapter sequences with no degradation in sequencing performance. Therefore, researchers can pool multiple barcoded samples before target capture, greatly reducing the cost of enrichment per sample.

xGen Standard Blocking Oligos are suitable for lower-throughput sequencing needs (up to 24 samples). Standard Blocking Oligos are made-to-order oligonucleotides that target specific adapter sequences.They can be designed against traditional (Illumina, Ion Torrent) or nontraditional (home-made, third party) adapter-index sequences. xGen Blocking Oligo sequences can be customized to suit researchers’ needs and are not required to be ordered as part of a kit. It is recommended that Standard Blocking Oligos be end terminated using a C3 spacer, and HPLC purified; however, other options are available to suit users’ needs.

Figure 2. Use of xGen Blocking Oligos: xGen Blocking Oligos bind to platform-specific next-generation sequencing adapters to prevent cross-hybridization between the adapters during target capture and, thereby, reduce the daisy-chain effect. Incorporating Blocking Oligos before hybridization with enrichment probes increases on-target capture and sequencing efficiency.

How Much Blocking Oligo do You Need?

Typically, at least one pair of xGen Blocking Oligos are required to bind to the complementary adapters located at the 3´ and 5´ ends of the genomic fragments in a prepared sequencing library. The protocol and number of Blocking Oligos used are dependent on the type of capture being performed.

Here, we provide the recommended amounts of xGen Blocking Oligos to be used with 500 ng fragmented, adapter-ligated sequencing library. We also show how to calculate the required amounts of Blocking Oligos when using multiple indexed adapters.

xGen Universal Blocking Oligos Increase Multiplexing Capabilities

When performing multiplexed target enrichment, researchers often use inosine-modified oligonucleotides to block multiple indexes in an attempt to reduce the number of blocking oligos in the reaction. We compared the efficacy of using xGen Universal Blocking Oligos to unmodified and inosine-modified Standard Blocking Oligos in 4-plex target enrichment.

Human genomic DNA (Coriell) was fragmented in four separate reactions and ligated to Illumina TruSeq LT adapters bearing a unique barcode for each sample. The adapter-ligated libraries were then combined for use in subsequent steps. Target enrichment was performed using 500 ng aliquots of the adapter-ligated library mix according to the xGen Lockdown® Probes Rapid Protocol using a 1.2 Mb xGen Lockdown Probe panel. Each aliquot of the library mix was combined with one type of blocking oligo and 5 µg Cot-1 DNA in three separate reactions. Sequencing was performed on an Illumina MiSeq® platform. Unique on-target reads were normalized to data obtained with unmodified Standard Blocking Oligos.

Incorporating unmodified Standard Blocking Oligos into target-enrichment produced 40% unique on-target reads (data not shown). Use of Universal Blocking Oligos increased unique on-target reads by approximately 30%, while a 15% drop in unique on-target reads was observed with inosine-modified Standard Blocking Oligos (Figure 3).

Figure 3. A Single xGen Universal Blocking Oligo enhances multiplex sequencing. xGen Universal Blocking Oligos were compared to Standard Blocking Oligos with or without inosine modifications. The data show fourplex sequencing of an enriched fraction of genomic DNA. Target capture was performed using a 1.2 Mb xGen Lockdown Probe panel and sequences were run on an Illumina MiSeq® platform. The average percentage on-target read obtained using unmodified Standard Blocking Oligos was set at 1 and compared to data from the other types of blocking oligos. Using xGen Universal Blocking Oligos increased on-target performance for multiplexed enrichment by 30%. Adding inosine residues to Standard Blocking Oligos reduced on-target performance by an average of 15%.


Blocking oligos are an essential component of targeted NGS through in-solution hybrid capture. However, having to use multiple blocking oligos for multiplex target capture can be cost prohibitive and complicated, and may eventually lead to degradation of sequencing performance. IDT provides two types of blocking oligos, xGen® Standard Blocking Oligos and xGen Universal Blocking Oligos. Standard Blocking Oligos are a low-cost option suitable for low-throughput applications. Universal Blocking Oligos are recommended for researchers who require low complexity and excellent performance for high-throughput sequencing.

Singleplex Target Capture
• 1 reaction Blocking Oligo for Adapter A
• 1 reaction Blocking Oligo for Adapter B

Multiplex Target Capture
xGen Standard Blocking Oligos
Standard Blocking Oligos are delivered dry with instructions for resuspension that yield a 1 µM solution.

• X nmol Blocking Oligon for each Adapter An
• Y nmol Blocking Oligon for each Adapter B
X=1/(number of unique side “A” adapters)
Y=1/(number of unique side “B” adapters)
n = the index number of the barcode

xGen Universal Blocking Oligos
Universal Blocking Oligos are delivered dry with instructions for resuspension to yield the appropriate concentration for 1 µL per reaction.
• 1 reaction of Blocking OligoA for Adapter A
• 1 reaction of Blocking OligoB for Adapter B

Note: Universal Blocking Oligos for the traditional Illumina Single-Indexed Adapter (TruSeq LT style) are optimized by index/barcode length. Therefore, for a 6 nt index, the corresponding 6 nt Universal Blocking Oligo must be used. If using both 6 nt and 8 nt length index regions (balancing bases included), then the 6 nt and the 8 nt Universal Blocking Oligos must be mixed in the same ratio as the respective indexes.

Nicola Brookman-Amissah, Ph.D.([email protected]), is senior scientific writer and Ibrahim Jivanjee is NGS product manager at Integrated DNA Technologies.

Previous articleNovo Nordisk Building $100M Purification Pilot Plant
Next articleTop R&D Spenders of 2013