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Some Proteomics Firms Exploring Next-Generation Sequencing for Biomarker Research

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By Adam Bonislawski

As instrument vendors like Illumina, Roche, and Life Technologies continue to drive adoption of next-generation sequencing for genetic studies and gene-based diagnostics, the technology has also begun to draw interest as a potential tool for proteomics research and protein biomarker-based tests.

Anticipating that NGS will become a key clinical tool in coming years, several proteomics firms and researchers are currently investigating it as a read-out platform for protein biomarker detection assays that employ nucleotides as part of their capture agents.

Specifically, they hope to take advantage of the technology's precision and multiplexing ability and eventually combine it with DNA and RNA-based measurements for use in clinical diagnostics.

"When we started thinking about this a few years ago, the question was, 'When will next-gen sequencing really make it into the clinic?" said Koen Kas, founder and chief scientific officer of Belgian protein biomarker firm Pronota. "And in the last few months we have seen such a dramatic increase in clinical applications on next-gen sequencing devices that I'm convinced it's almost time to see next-gen DNA sequencing applications as part of the clinical routine."

If it turns out that NGS does become widely adopted for clinical use, protein-biomarker assays that can be read by them could enable researchers to develop DNA, RNA, and proteomic-based diagnostics that run on the same instrument Kas told Proteomonitor.

He cited prenatal testing as an example for the potential for such a platform. Several companies, including Sequenom, LifeCodexx, Fluidigm, and Artemis Health, are developing NGS-based tests for Trisomy 21, the chromosomal defect linked to Downs syndrome.

"But if you think about the next step — 'What are the other things that you want to screen for in pregnant women?' — not everything you want to measure in a pregnant woman can be measured at the DNA level," said Kas. "It might be handy to measure something at the protein level. So I see [protein biomarker measurements] in the clinic combined with DNA and RNA-based measurements to give a comprehensive analysis in one single device."

While Pronota has typically used mass spec for its protein biomarker work, the company has also been investigating using aptamers as protein-affinity probes. These short, single-stranded oligonucleotides act as capture agents for proteins of interest and, after the capture step, can be read by NGS to determine the number of proteins present in a sample.

Last June the company announced that it completed a proof-of-concept study for a protein biomarker diagnostic platform using next-generation sequencing (PM 06/04/2010). In December, a team including Kas and several other Pronota researchers published a paper in Analytical Chemistry describing the use of an Illumina GAII machine to measure serum levels of endogenous immunoglobulin E captured by aptamers.

According to Kas, the key technological barrier to such an approach is generating aptamers against enough proteins to make the technique broadly useful. Currently in the public domain there are aptamers for only several hundred proteins compared to antibodies to around 2,000 proteins.

Proteomics firm SomaLogic, however, has developed roughly 1,100 proprietary somamers — a type of modified aptamer — demonstrating that building sufficiently large aptamer libraries is possible, Kas suggested.

NGS is potentially applicable to other DNA-based protein detection platforms, as well. At Sweden's Uppsala University, researcher Ulf Landegren is looking at NGS for reading proteins detected via proximity ligation assays, a technique that uses pairs of antibodies attached to unique DNA sequences to capture proteins of interest.

When the antibodies bind to their targets, the attached DNA strands are brought into proximity and ligate, forming a new DNA amplicon that is then quantified, with the amount of DNA corresponding to the quantity of target protein.

Landegren is a founder and board member of Olink Bioscience, which sells the PLA technology under the name DuoLink. He recently submitted a study for publication describing his lab's efforts to adapt the technique for use with NGS.

Currently, PLA's approach uses real-time PCR to quantify the DNA amplicons formed when target proteins are captured, but NGS offers potential advantages in terms of precision and multiplexing, Landegren suggested.

"One of the principal advantages of next-generation sequencing is that you're counting molecules, you're not measuring levels of reactions," he told ProteoMonitor. "When we detect a protein, we get a ligation product which we can then amplify, and then with [NGS] we can count how many times we see a particular ligation product."

NGS could also allow for easy pooling of samples, he said, enabling, for instance, higher-throughput protein biomarker validation workflows.

"We can do multiplex assays in several different patient samples, tag them differently, pool them, and then just read them out in parallel and count how many times we see the proteins," said Landegren.

In the recently submitted study, Landegren's team used a Life Technologies SOLiD next-gen sequencer and an Illumina Genome Analyzer to detect levels of 36 different protein biomarkers for various cancers, inflammatory disorders, and cardiovascular diseases.

In the future, he said, he plans to apply the approach to an expanded menu of proteins.

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Watching With Interest

While both Illumina and Life Technologies declined to be interviewed for this article, there are indications that these vendors are following with interest efforts to use NGS for proteomics.

In February, as part of its European Ion Torrent Personal Genome Machine Sequencer Grants Program, Life Technologies awarded Landegren an Ion Torrent sequencer to support his PLA work. Life Tech has licensed the PLA technology from Olink and offers it as part of Applied Biosystems' TaqMan product line.

Last year, Illumina purchased for an undisclosed amount antibody firm Affomix, which had similarly been exploring the use of NGS to read proteins, in this case after detecting them using antibodies with attached oliginucleotide backbones (PM 06/04/2010).

Thus far, Pronota's NGS work has been done independently, but the company "has started a number of discussions with developers of [NGS] equipment," Kas said, declining to name any firms.

"It makes great sense [for vendors] to expand the use of their devices beyond just DNA and RNA applications," he said.

Sequencing vendors "at some point have to turn their machines into a workhorse for something that’s done all the time, not just once when you sequence your individual genome," SomaLogic CEO Larry Gold told ProteoMonitor. "Proteomics [testing] is something that people will do in their lives 100 times, and genomics is something they'll probably do once, unless they get cancer and then they'll probably do some cancer genomics.

"So there's an enormous reason the deep sequencing guys want to move towards assays," he added. "It's the difference between an annuity and winning the lottery."

However, Gold sounded a note of skepticism regarding the usefulness of NGS in proteomics, suggesting that the dynamic range of samples like serum could make it difficult to measure low-abundance proteins with good precision.

In order to achieve acceptable CVs, NGS platforms will likely need to measure at least 1,000 copies of the target analyte, he said. In samples with low-dynamic range, that doesn't pose a significant problem.

In broad biomarker-discovery screens measuring levels of hundreds of different proteins in blood, however, the large dynamic range means that measuring 1,000 copies of low-abundance proteins will require sequencing billions of copies of DNA linked to higher-abundance proteins.

"It's not obvious that for big plexes [NGS] has an advantage over [DNA] hybridization" techniques like those used by SomaLogic in its biomarker discovery assays, Gold said.

Kas agreed that next-gen sequencing will probably be most useful for fairly targeted protein validation work as opposed to biomarker-discovery assays. He noted that Pronota uses mass spec to perform its differential-expression analyses and chooses proteins with which to make aptamers based on those that look most promising.

For his part, Landegren conceded that his team has struggled with precision in its NGS work, in part due to difficulties measuring low-abundance analytes.

"Currently we're not doing so well," he said. "This is early days, and we have a lot of sources of variation that we haven't yet completely removed. One obvious source [of variation] is that some molecules are not so abundant, so if you only count a few representations of those molecules then there will be Poisson noise in the system, so then you can't hope for very good precision. There are also other sources in the way the assay is done in terms of capturing molecules.

"I don't think we can claim that our precision is very impressive at this point," he added. "It's slightly better than using other methods like PCR, but only slightly."

Nonetheless, Kas said, he believes NGS is coming to proteomics – so much so that Pronota has no plans to develop its aptamer-based system for read-out on a conventional microarray.

"If [NGS keeps] developing at the speed we've seen in the last few years, I believe that all of what we currently run on [custom] microarray platforms will in the near future be run on [NGS] platforms," he said. "So we didn't even bother to develop [Pronota's aptamer-based system] on a microarray."


Have topics you'd like to see covered in ProteoMonitor? Contact the editor at abonislawski [at] genomeweb [.] com.

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