SAN FRANCISCO (GenomeWeb) – Boreal Genomics has developed a sequencing technique that it said can improve the detection of low-frequency mutations in cell-free DNA assays, while keeping sequencing costs down.
The firm, which described the technique recently in a paper posted to the BioRxiv preprint server, said that it could be complementary to previous technology it has developed, OnTarget, which targets known activating mutations in cancer genes.
The method, called Proximity Sequencing (Pro-Seq), involves linking both strands of a double-stranded DNA fragment together so that they populate one cluster on an Illumina flow cell. That increases the chances that sequencing errors are compensated for by comparing the two strands of the DNA molecule.
In the study, the Boreal team described both a targeted and a whole-genome approach, using DNA from a cell line and cell-free DNA. They found that on average, they could detect mutations down to a .003 percent frequency and that per-base specificity was 99.9997 percent.
Andre Marziali, Boreal's chief scientific officer, said that the goal was to develop a technique that would be as good as its OnTarget assay at identifying low-frequency mutations, but would also be able to be broader. The premise behind the OnTarget technology is that it selectively enriches for known mutations, which is useful for analyzing oncogenes like KRAS and BRAF, which have known hotspots, but less useful for tumor suppressor genes like TP53, where pathogenic mutations can occur nearly anywhere, Marziali said. In addition, the method had to be relevant for clinical use, so costs couldn't be too high.
"We wanted a technology that was broader in order to look at tumor suppressor genes, but that still had the same sensitivity of OnTarget," he said. "And, for liquid biopsies and other assays that are meant to be used broadly, the cost has to be low."
In addition, Joel Pel, head of technology development at Boreal, said that the method takes around eight hours, from extracted DNA to libraries loaded onto the sequencer.Â
For the whole-genome method, a double-stranded DNA molecule is ligated, followed by a strand displacement extension step, where the molecule is extended using strand displacing polymerase, which creates linked templates. Those can then be loaded onto the sequencer.
For targeted, cell-free DNA applications, the first step relies on droplet PCR, such that one template of double-stranded cfDNA, along with universal linked primers and multiplexed gene-specific primers, is contained in a drop. Those droplets are loaded onto the PCR machine, which creates linked molecules from each drop, with the regions of interest amplified. Then, the emulsion is broken, unlinked strands are digested, and the linked DNA strands are cleaned up and loaded onto the sequencer..
To test the method, the Boreal team first ran a 7-amplicon panel on wild-type plasma-derived cfDNA and found that the false positive rate was 2.6 errors per million bases. In a 19-amplicon panel, the false positive rate was 1.1 in one million for an analytical specificity of 99.9997 percent.
To measure sensitivity, the researchers used cell lines with known mutations and spiked them into wild-type plasma-derived DNA at varying concentrations. The team found that in samples containing 1.5 copies of each mutant, more than 70 percent were detected. When the number of mutants per sample was increased to 4.5 copies and higher, the researchers achieved 100 percent detection. Thus, at .003 percent frequency, four out of five mutants were detected above the background, while at .01 percent mutant fraction, all of the mutants were detected.
Marziali said the company is in the midst of discussions with undisclosed partners to commercialize the technology. "We don't expect to offer assays ourselves, but instead to license it to groups to use the technology."
He said that one option being explored is to include the Pro-Seq technology alongside the OnTarget technology in a clinical study of early cancer detection. Already, the company is running a pilot study using its OnTarget assay that targets 96 hotspot mutations to analyze more than 1,000 individuals between the ages of 55 and 75 who have never had a cancer diagnosis. The researchers aim to determine whether the OnTarget assay can detect cancerous mutations in patients' cell-free DNA before the patients are diagnosed. The study will determine both a positive predictive value and a negative predictive value of the OnTarget assay.
That study launched last October, and Marziali said that the team would consider including Pro-Seq in a second study. "A nice combination could be to use OnTarget for oncogenes and Pro-Seq for tumor suppressors," he said.
The Pro-Seq technology is one of several that have been developed in the last couple of years to enable detection of mutations present at low frequencies, an important ability for liquid biopsy applications.
Roche is commercializing cfDNA assays based on a method originally developed by Stanford University researchers known as CAPP-seq, which includes error correction to enable detection of mutations present at .004 percent frequency.
A spinout from the University of Washington, TwinStrand Biosciences, plans to commercialize technology known as duplex sequencing. This involves tagging both strands of DNA with different, but complementary primers. It is similar to the Pro-Seq method in that it enables errors to be filtered by comparing the complementary DNA strands, but it does not physically link the strands.
Companies such as Inivata and AccuraGen are developing their own methods for better detection of rare variants for liquid biopsy applications.
Compared to duplex sequencing, Marziali said that the Pro-Seq technology was similar in its ability to detect low-frequency variants, but that it should be less expensive since it requires around 10-fold fewer sequencing reads. Pro-Seq needs fewer reads to achieve the same sensitivity because the DNA molecules are physically linked, whereas with duplex sequencing, extra templates end up being sequenced to ensure that there is enough redundancy to create consensus.
Similarly, compared to Roche's Avenio assay, which is based on the CAPP-seq technology, Pel said that the Pro-Seq technology has a lower reported error rate and so would be able to detect low frequency variants with less sequencing.
Marziali said that the Pro-Seq technology would also have other applications. For instance, Boreal has been working on incorporating it into a target enrichment protocol.