Targeting the genome

Genomic target selection using individually synthesised capture probes may translate cutting edge research in the laboratory into personalised medicine in the clinic

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 analysed 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-synthesised 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.

[caption id="attachment_35031" align="alignleft" width="200" caption="Figure 1: Individually synthesised oligonucleotide capture probes yield high coverage. The data show that 1,000 individually synthesised oligonucleotides targeting a 133kb region provided greater coverage than array/RNA baits."][/caption]

Foundation Medicine is a molecular information company that has developed FoundationOne a fully informative genomic profile for solid tumours to identify a patient’s individual molecular alterations and match them with relevant targeted therapies and clinical trials. This cancer genomic profile assay helps physicians make personalised therapy decisions for patients. The test, run in the company’s CLIA-certified laboratory, has been optimised for formalin fixed paraffin embedded (FPPE) samples and small specimens, such as core needle biopsies.

Using next generation sequencing (NGS) methods, tumour samples are sequenced for the entire coding sequence of hundreds of genes as well as a number of introns frequently rearranged in cancer. To ensure sensitivity and specificity >99% for alterations with a minor allele frequency of ?5%, sequencing of unique library molecules to 500–1000X coverage is required. This deep sequencing of clinical samples is achieved by enriching for the ~1Mb target of interest. Scientists at Foundation Medicine are continually researching methods to enhance their current commercial assay, which uses RNA baits.

As part of their assay improvement process, Foundation Medicine scientists analysed a target region of ~130kb within the human genome, representing 57 clinically-relevant, potentially actionable cancer-related genes. Solution hybrid selection of an adaptor ligated sequencing library was performed using xGen Lockdown Probes, individually synthesised 120 nucleotide (nt) 5?-biotinylated DNA oligonucleotides (Integrated DNA Technologies).

[caption id="attachment_35032" align="alignright" width="200" caption="Figure 2: Proprietary techniques enable short hybridization times without sacrificing performance."][/caption]

Hybridisation reactions were incubated for 24 hours and enriched libraries were sequenced on a HiSeq 2000 system (Illumina) using 49 x 49 paired-end reads. xGen Lockdown Probes facilitated an approximately 5000-fold enrichment of this ~130kb region. The researchers also observed high coverage over the entire targeted region (Figure 1), with minimal GC bias. By combining the probes with proprietary hybridisation techniques, the researchers were able to reduce hybridisation times to as little as 2.5 hours, instead of the standard 24-72 hours, without sacrificing the depth or uniformity of coverage (Figure 2).

Individually synthesised oligonucleotide probes were also spiked into array-synthesised RNA baits to improve coverage of areas with high GC content. Either 1,000 xGen Lockdown Probes targeting a ~133kb region, or three probes targeting a single exon, were mixed with ~1Mb RNA baits and hybridised 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 using 49 x 49 paired-end reads.

Supplementing the array-derived RNA baits with 1,000 xGen Lockdown Probes enhanced the coverage of many GC-rich targets, such as first exons (Figure 3A). Further, adding only three probes targeted at a single GC-rich exon improved coverage of that exon (Figure 3B).

[caption id="attachment_35033" align="alignleft" width="200" caption="Figure 3a"][/caption]

The researchers at Foundation Medicine successfully used individually synthesised 5?-biotinylated oligonucleotide capture probes to overcome some of the limitations of array-synthesised oligonucleotides. They also demonstrated that when used alone, xGen Lockdown Probes provide more uniform coverage and less GC bias than array-derived RNA baits. Additionally, the researchers find that individually synthesised probes are much more cost-effective on a per-reaction basis, and are particularly excited to have reduced hybridisation times while maintaining high-target coverage. The researchers continue to evolve their

[caption id="attachment_35034" align="alignleft" width="200" caption="Figure 3: Spiking in individually synthesized oligonucleotide baits improves sequencing coverage of array-derived RNA baits. (A) Addition of a set of 1,000 xGen Lockdown Probes to array-derived RNA baits enhanced the coverage of many GC-rich targets, such as first exons; (B) supplementing RNA baits with three xGen Lockdown Probes targeting a single GC-rich exon improved coverage of that exon."][/caption]

technologies to keep abreast with advances in NGS.

Author: Nicola Brookman-Amissah is a scientific writer at Integrated DNA Technologies. She obtained her PhD in Biochemistry at University College London, UK

Contact: xgen@idtdna.com. www.foundationone.com