Boreal has developed a unique molecular enrichment technology that enables highly sensitive analysis of circulating tumor DNA (ctDNA) in plasma with applications that include therapy selection, monitoring, and early detection of disease.

Even in late stage cancer, plasma may contain very low tumor content that can confound standard assays, particularly when the few tumor molecules that may be present are massively outnumbered by normal DNA. Boreal’s OnTarget™ technology selectively enriches the sample for targeted DNA sequences to reveal tumor mutations which may help help guide clinical decision making and improve clinical trials or drug development programs.

The fundamental challenge of ctDNA analysis is one of finding needles in a haystack. 

We have a simple, yet innovative solution – we take the haystack, keep the needles, and throw out the hay.

Example of how OnTarget™ enrichment improves detection sensitivity for rare tumor mutations over conventional assays

 


 

Challenges in ctDNA analysis

Sensitivity

Blood plasma has been shown to carry cell-free DNA containing tumor mutations, indicating that plasma tests can be a non-invasive alternative to tissue biopsies.  However, detection of cell-free DNA in plasma poses a significant challenge as tumor DNA is heavily diluted by DNA from normal cells, to abundances that can be as low as 0.01% and present in as low as single molecule abundance.

Accuracy

The challenge facing many current technologies lies in the fact that each mutant allele may be outnumbered by the nearly identical wild-type allele by more than 10,000 to 1. Errors made on the abundant wild-type allele lead to false positives from the wild-type alleles and make the low abundance mutant signal indistinguishable from noise.

Multiplexing

There is increasing evidence that many mutations play a role in drug response or resistance, suggesting that in order to be informative, assays will need to test for multiple mutations without sacrificing sensitivity. Background mutation levels in healthy individuals (~1 mutation in 1,000,000) as well as evolving mutation profiles, can confound assays that are limited to analyzing only a few mutations. Correct interpretation of assay results will require a comparison of relative abundance over a large number of tumor-related mutations in order to identify significant mutational events that signal emerging disease or drug resistance.

 

 

 

 


 

How OnTarget works

The OnTarget™ technology is based on targeted enrichment of mutant alleles over wild-type DNA and can be incorporated into a fully integrated sample-to-answer workflow.

 

Quantify

DNA extracted and quantified with qPCR

Barcode

Limited-cycle PCR with sample-ID barcodes

Enrich Mutations

Mutant alleles selected over the wild-type

Construct Library

Samples prepared for sequencing and pooled

Sequence

Mutations sequenced on a MiSeq

Analyze

Mutations quantified and reported

OnTarget™ enrichment is based on a synchronous thermal-electrophoretic separation to remove virtually all wild-type alleles from a sample prior to analysis.  Without amplification and generation of new nucleic acid strands, this greatly improves the ratio of target allele to wild-type DNA, making subsequent detection of mutations simple and accurate.

This unique approach to enrichment also means that OnTarget™ can analyze short, degraded, or damaged DNA fragments that would be missed by other methods – accessing more signal from precious samples.

The OnTarget™ assay provides quantitate results through the use of reference nucleic acid sequences and internal controls – to learn more about the OnTarget™ technology, please contact us to schedule a technical discussion.

Learn more about how OnTarget™ works in this clip from our webinar.