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.
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.