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Genomic Target Selection Using Individually Synthesized Capture Probes

Next Generation Sequencing with Biotinylated Ultramer® Oligonucleotides

Genomic target selection enables faster development of clinical sequencing tests for disease diagnosis, stratification, and informed therapy selection by allowing smaller, specific regions of the genome to be sequenced and analyzed at high depth in a cost-effective manner. Faster, targeted sequencing could also accelerate development of effective therapies for various diseases. Currently available commercial target capture reagents use biotinylated, array-synthesized oligonucleotides to generate DNA or RNA probes, often referred to as “baits”. However, baits that are generated by these methods often present performance challenges, such as failure to capture regions of high GC content.

High Target Coverage from Individually Synthesized, Biotinylated Oligonucleotides

Foundation Medicine, Inc. has developed a cancer diagnostic genomic test for solid tumors that provides a fully informative profile to help physicians make personalized therapy decisions for patients with cancer. This assay, run in the company’s CLIA-certified laboratory, has been optimized for FFPE samples and small specimens such as core needle biopsies. Using next generation sequencing (NGS) methods, they sequence tumor samples for the entire coding sequence of 182 cancer-related genes plus 37 introns from 14 genes frequently rearranged in cancer. To ensure sensitivity and specificity >99% for mutations with a minor allele frequency of ≥5%, they require sequencing at 500–1000X coverage, which is achieved by enriching for the ~1 Mb target of interest. Foundation Medicine’s current commercially available assay uses RNA baits for enrichment; however, the company plans to use DNA baits and an expanded assay for a forthcoming product enhancement. We spoke to Mirna Jarosz and Phil Stephens about the pioneering work being performed at Foundation Medicine, which is translating cutting edge research to the clinic.

“We are in a sweet spot between large-scale whole genome sequencing and extremely targeted ‘hotspot’ sequencing, and this is where the IDT Ultramer® Oligonucleotides are particularly useful to us.”—Dr Mirna Jarosz

In a poster presented at the Advances in Genome Biology and Technology (AGBT) 2012 annual meeting, Foundation Medicine researchers analyzed a target region within the human genome of ~130 kb, representing 57 clinically-relevant, potentially actionable cancer-related genes. Using 2 μg from an amplified sequencing library, in-solution hybrid selection was performed with 1000 individually synthesized 120 nt 5’-biotinylated IDT Ultramer® Oligonucleotides  spanning the targeted region.

Hybridizations were performed for 24 hr and enriched libraries were sequenced on a HiSeq 2000 system (Illumina) using 49 x 49 paired-end reads. Approximately 5000-fold enrichment was achieved for this ~130 kb region using individually synthesized Ultramer Oligonucleotide capture probes. The researchers also observed high coverage over the entire targeted region (Figure 1), with minimal GC bias. Using proprietary hybridization techniques and Ultramer Oligonucleotides, the researchers can reduce hybridization time to as little as 2.5 hr, instead of the standard 24–72 hr, without sacrificing depth or uniformity of coverage (Figure 2).

Figure 1. Individually Synthesized Oligonucleotide Capture Probes Yield High Coverage. The data show that 1000 oligonucleotides targeting a 133 kb region provided greater coverage than array/RNA baits.

Figure 2. Proprietary Techniques Enable Short Hybridization Times Without Sacrificing Performance.

Improved Coverage of Array Synthesized “Baits”

Individually synthesized 5’-biotinylated Ultramer Oligonucleotides were also spiked into commercial, array-synthesized RNA baits to improve coverage of areas with high GC content. Either 1000 Ultramer Oligonucleotide baits targeting a ~133 kb region, or 3 Ultramer Oligonucleotide baits targeting a single exon, were mixed with 1.1 MB RNA baits and hybridized using protocols developed by Foundation Medicine, followed by target capture using streptavidin. Enriched targets were sequenced to high coverage on a HiSeq® 2000 sequencing system (Illumina) using 49 x 49 paired-end reads.

Adding the 1000 Ultramer Oligonucleotide set to array-derived RNA baits enhanced the coverage of many GC-rich targets, such as first exons (Figure 3A), and supplementing RNA baits with 3 Ultramer Oligonucleotides targeting a single GC-rich exon improved coverage of that exon (Figure 3B).

Figure 3. Spiking In Individually Synthesized Oligonucleotide Baits Improves Sequencing Coverage of Array-Derived RNA Baits. (A) Addition of a set of 1000 individually synthesized Ultramer® Oligonucleotides to array-derived RNA baits enhanced the coverage of many GC-rich targets, such as first exons; (B) supplementing RNA baits with 3 Ultramer Oligonucleotides targeting a single GC-rich exon improved coverage of that exon.

Individually Synthesized Oligonucleotides are a Superior Alternative to Array-Derived “Baits” for Genome Target Capture

The scientists at Foundation Medicine have successfully used individually synthesized 5’-biotinylated Ultramer Oligonucleotide capture probes to overcome some limitations of array-synthesized oligonucleotides. They have also demonstrated that when used alone Ultramer oligonucleotides provide a more uniform coverage than the more widely used, arrayderived RNA baits, with reduced GC bias. Additionally, they find individually synthesized baits are much more cost-effective on a per-reaction basis. The researchers are particularly excited to have been able to reduce hybridization times while maintaining high-target coverage, and intend to continue evolving their technologies with advances in NGS.

Ultramer® Oligonucleotides and xGen® Lockdown® Probes

IDT proprietary synthesis systems and chemistries allow high fidelity synthesis of Ultramer® Oligonucleotides, long oligonucleotides up to 200 nt. These oligos are purified by desalting to remove small molecular impurities and are delivered deprotected. Standard desalted Ultramer Oligonucleotides ship in 2–4 business days.

xGen® Lockdown® Probes are 60–120 nt 5’-biotinylated Ultramer Oligonucleotides that have been designed specifically for NGS target capture. xGen Lockdown Probes are suitable for developing custom capture panels or supplementing existing panels. The probes are available at different scales (mini, standard, and XL), supplied in 96- or 384-well plates or as pools of up to 2000 oligos, and ship in 7–10 business days.


Personalized Medicine in the Clinic

Foundation Medicine’s first clinical product—a fully informative genomic profile for solid tumors—is run from the company’s CLIA-certified lab in Cambridge, MA. This assay complements traditional cancer treatment decision tools and can expand options by matching each patient with targeted therapies that are relevant to the molecular changes in their tumor. This genomic assay identifies all classes of genomic alterations, including substitutions, copy number alterations, insertions, deletions, and select rearrangements, in hundreds of cancer-related genes. Foundation Medicine’s test is the first commercially available targeted sequencing assay that uses NGS in routine cancer specimens such as FFPE and core needle biopsies. The company’s goal is to make this type of analysis available for all patients, helping to achieve the realization of personalized therapy for cancer.

About Foundation Medicine

Foundation Medicine is a molecular information company dedicated to delivering a transformation in cancer care in which treatment is informed by an understanding of the genomic changes that contribute to each patient’s unique cancer. The company has developed a fully informative genomic profile to identify a patient’s individual molecular alterations and match them with relevant targeted therapies and clinical trials. Foundation Medicine’s molecular information platform aims to improve day-to-day care for patients by serving the needs of clinicians, academic researchers, and drug developers to help advance the science of molecular medicine in cancer. For more information, visit the company’s website at www.foundationmedicine.com.

Mirna Jarosz, PhD, Director, Molecular Biology and Sequencing leads a team in the R&D group responsible for continuous improvement of Foundation Medicine’s NGS assay for comprehensive sequencing of clinical tumor specimens.

Before joining Foundation Medicine, Dr Jarosz was manager of sequencing development at Helicos Biosciences, where she helped develop the world’s first commercial single molecule DNA sequencer. Dr Jarosz has a PhD in chemistry from MIT.







Phil Stephens, PhD, Vice President, Cancer Genomics drives R&D at Foundation Medicine, including internal research efforts and external collaborations.

Dr Stephens is a world-renowned expert in NGS and cancer genome analysis and has authored numerous publications in Nature, Cell, and other high-profile journals. Prior to joining Foundation Medicine, Dr Stephens held various senior research positions at the Wellcome Trust Sanger Institute under the direction of Professor Michael Stratton. During this time, Dr Stephens was a member of the team that sequenced the first two comprehensive melanoma and lung cancer genomes, and was co-lead author in the discovery of BRAF in melanoma and ERBB2 in lung cancer. Dr Stephens received a PhD from Oxford University.


Author: Nicola Brookman-Amissah is a Scientific Writer at IDT.

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.

Product details »



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.

Product details »



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.

Product details »


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.