Allele Enrichment

Boreal’s OnTarget™ allele enrichment system allows for enrichment of rare mutant alleles from tumor samples and blood plasma, enabling reliable clinical detection of rare variants below 0.01% abundance. By simultaneously enriching multiple specific mutations while depleting the sample of wild type sequence, we are able to increase the sensitivity and specificity of current mutation detection assays.

Wild-type alleles tend to overwhelm low-abundance mutations, generating false positives and limiting mutant detection sensitivity. By enriching mutant sequences over the unwanted wild-type allele, OnTarget™ improves the sensitivity of downstream detection regardless of the specific assay.

Mutation detection assays are limited by two common problems. Enzymatic errors in PCR amplification actually generate mutant sequences from wild-type templates, which are then further amplified and detected. Mutation detection methods also have limited specificity distinguishing between closely related sequences such as single base substitutions. Both of these errors can result in false-positives, limiting mutant detection sensitivity.

In 2nd (Next) Generation Sequencers (NGS), these errors arise from PCR steps in flow cell and during locus enrichment, library preparation, cluster generation, and in the sequencing itself. When these error rates exceed the mutation abundance, e.g. for clinical mutation detection from plasma, the mutant cannot be reliably detected. This problem cannot be overcome simply with increased depth of coverage.

Similar problems also plague PCR-based mutation assays making mutant detection in the presence of a large amount of wild-type sequence difficult. Erroneous amplification generates mutant molecules. Detection strategies employing reporter molecules or probes can interact with wild type molecules at low but significant efficiency. All of these errors generate mutant signal, even if no mutant DNA is present, limiting positive calls to samples where the mutant signal is well above background. The result is that most assays are limited in sensitivity of the mutation to ~ 1-20% mutation abundance compared to wild-type. While some methods exist to block the amplification of the wild-type allele and other methods employing digital PCR attempt to segregate the mutant into individual reaction drops to avoid wild-type contamination, all these methods are limited in either sensitivity or ability to be multiplexed over multiple mutations.

What is needed is a method for enriching a sample for mutation alleles while rejecting the wild-type sequence, over at least 100 different mutations, thus enabling multiplexed mutation detection from plasma as can be done by Boreal’s OnTarget™ allele enrichment system.

Non-linear electrophoresis
OnTarget™ allele enrichment is based on a sequence-specific version of our core SCODA electrophoresis technology. SCODA is a non-linear electrophoresis method described here, but in the OnTarget™ system, non-linearity is created by incorporating sequence-specific probes within the concentration matrix. We use the same electric fields as in regular SCODA, but engineer a stronger non-linearity in the nucleic acid velocity by using an acrylamide gel that is covalently decorated with short oligonucleotide probes. The probes, typically 20 bases long, are single stranded and designed to match a sequence that is uniquely present in a target DNA and not present in background DNA. The gel is operated at an elevated temperature near the melting temperature of the DNA duplex that would form between the probe and the single stranded target. The sample is denatured and injected into the gel, where the target sequences transiently interact with the probes. The interactions are not static hybridizations. Instead, they are short, sequence-specific interactions that are denatured during peaks in the electric field cycle. These interactions result in a high non-linearity in target velocity with respect to electric field, which is dependent on a perfect match to the probe sequences. Consequently, only target sequences that match the probes are focused, while non-complementary DNA in the sample can be removed by means of a weak DC field. The large electrophoretic non-linearity induced on target molecules as a result of the interactions with the gel oligos also dramatically increases the speed of focusing, allowing injection and focusing to occur in less than 1 hour.