With the help of new funding from the National Human Genome Research Institute, Tucson, Ariz.-based HTG Molecular Diagnostics is looking to extend its proprietary nuclease protection probe technology into a multiplexed gene expression and variant quantifying tool that can be used in conjunction with high-throughput sequencing instruments.
"We've modified the chemistry so that instead of taking the prepared sample and reagents and reading them on our reader in an array format, we can now quantitate those in the next-gen sequencers," Tim (T.J.) Johnson, president and CEO of HTG Molecular, told In Sequence.
Earlier this month, the company announced that is has secured a $1 million phase II Small Business Innovation Research, or SBIR, grant from NHGRI to develop the sequencing-compatible scheme, known as EdgeSeq.
So far, much of HTG Molecular's focus has been on using its nuclease probe protection approach for RNA-related applications — from measuring levels of messenger RNA transcripts to quantifying small RNAs or long, non-coding RNAs.
As it moves through phase II of the EdgeSeq project, though, the company plans to develop protocols for quantifying specific DNA sequences, too, noted Vijay Modur, HTG Molecular's vice president of translational science and chief medical officer.
"The way we envision that working is to target specific sequences in the DNA which have high frequencies of mutations or [SNPs]," Modur told IS.
For the two-year first phase of the project, the EdgeSeq scheme was tested in conjunction with Life Technologies' Ion Torrent and Illumina's HiSeq 2000 and MiSeq instruments.
The HTG Molecular researchers have not identified specific sequence read lengths or error profiles that are more or less compatible with EdgeSeq. At the moment, they believe the approach will be compatible with a range of high-throughput sequencing instruments.
"Obviously as we get deeper into our development efforts we'll probably uncover those things," Johnson said. "But through our initial phase I feasibility study, we were able to get the quality of results that we were looking for on both platforms."
Over the course of phase II, the team plans to take a look at a wide range of tissue and sample types, he noted, with a particular focus on formalin-fixed, paraffin-embedded tumor samples and other samples of interest in oncology settings.
Because the nuclease protection probe approach does not involve RNA or DNA extraction steps, Johnson noted, it can be used to assess very small quantities of genetic material — both in research and clinical settings.
The minimum amount of input RNA needed is not yet known, since the researchers often start from a small sample scraping, which is tricky to quantify. During the ongoing process of EdgeSeq development, though, they hope to more completely characterize the lower limit for sample input.
"One of the issues there is that we don't actually extract RNA," noted Debrah Thompson, the HTG Molecular researcher leading the EdgeSeq program. "So giving a nanogram, picogram, [or] femtogram amount is a little bit difficult."
Based on results so far, they believe the method will be sensitive enough to look at expression or targeted variant profiles in miniscule tissue or fine needle aspiration samples.
"Biopsies are getting smaller and smaller, with less tissue, yet people are trying to do more with it," Johnson noted. "So we're trying to help solve that problem by being able to multiplex … multiple genes and multiple samples together."
"We believe we'll dramatically reduce the amount of material required," he said, "thus allowing more experimentation to take place or, in the event of diagnostic applications … broader testing to be done with the biopsy."
The group anticipates completing phase II of the project, which is already underway, by mid-2014. The phase I study was supported by a $600,000 grant from SBIR and the company will have the option of applying for an additional $587,000 from SBIR to support the fourth year of the project, if necessary.
For more than a decade, HTG Molecular has been developing technologies — most of them array-based — that center on its nuclease protection assay approach and related analytical methodology. This summer, for example, the firm announced plans to file for diagnostic use of its array-based Edge System in the US, Canada, and the European Union (see BioArray News 7/2/2013).
That system centers on two instruments, Johnson explained: a processor for conducting the nuclease protection chemistry — from the initial probe hybridization step through to the completed sample preparation — and a propriety reader that recognizes chemiluminescence signals from the probe in an array format.
The nature of the chemistry makes it possible to analyze genetic material without extraction, amplification, and, in the case of RNA, reverse transcription steps. Johnson noted that such a "lysis-only" protocol has advantages related to sample fidelity and small sample sizes.
"We believe the fact that we avoid a lot of the complications associated with extraction-based techniques will give us a very critical advantage, from the perspective of the data fidelity as well as the amount of initial material required," he said.
For instance, the existing nuclease protection probe approach has already shown promise for assessing FFPE samples, company representatives say. The company is now attempting to marry that chemistry to next-generation sequencing as part of the EdgeSeq project.
The approach "gives us access to the power of next-generation sequencing of a wide dynamic range," Modur noted. "The level of sensitivity for gene expression is quite unparalleled for multiplexing and expression."
To that end, the researchers have been developing methods for multiplexing at both the sequence and sample levels with the goal of being able to simultaneously quantify many genes or RNAs in many samples on the sequencer.
Thompson noted that the group has been able to look at as many as 2,500 genes in a single assay nuclease protection probe assay, though it believes that this is not the upper limit for sequence multiplexing.
Each of the samples is also multiplexed on the sequencing instrument using barcoding methods. To date, the researchers have barcoded roughly 200 samples to run together on the Illumina HiSeq 2000. They are now considering the optimal balance between sequence and sample multiplexing.
The sample prep process associated with EdgeSeq is distinct from that used in conventional next-generation sequencing instrument library preparation kits, Thompson said.
Rather than isolating DNA or RNA and feeding into such kits, the idea is to apply the same type of extraction-free sample preparation protocol that HTG Molecular uses for other forms of its Edge methodology, before slapping the appropriate adaptors on during the last stages of the sample prep process.
"What we do is add our lysis buffer to a sample, add these probes from the reaction, do some PCR, and we're ready to sequence," Thompson explained.
"The workflow is actually very simple and it doesn't use any of the kits from either company," she said. "What we do is add the adaptors in the final PCR step. And that's really why we're agnostic — we can add whatever adaptors are needed for a platform."
Although the bioinformatics side of EdgeSeq is expected to be fairly straightforward, Thompson noted that the HTG Molecular researchers eventually intend to put together corresponding software for users.
HTG Molecular has not yet determined the estimated cost per sample or cost per kit for using EdgeSeq. Johnson said the appropriate price should become clearer as phase II of the project progresses, since the team anticipates ongoing cost analysis on the approach as it develops.
He did not disclose whether HTG Molecular is currently collaborating with any of the firms marketing next-generation sequencing instruments.