Rubicon Overview

Rubicon Genomics is a pre-analytical platform company that has successfully developed and commercialized a unified approach for significantly improving the performance of DNA and RNA analytical instruments for research and diagnostics. The Rubicon platforms are a set of unique PCR-based processes that pre-amplify and standardize nucleic acids before genetic, expression or methylation analyses, thereby solving the common problems of insufficient or variable quantity and quality of the DNA/RNA. With standardized, pre-amplified nucleic acids the analytical instruments yield more accurate and reproducible results, and can be used to test for an unlimited number of analytes. These simple, low-cost pre-analytical solutions are proven to increase sensitivity, specificity and robustness of virtually all analytical platforms without modification of the platforms themselves. The performance of qPCR, microarray, and next-gen sequencing instruments are improved even for the most challenging samples such as single cells, plasma, serum, urine, circulating tumor cells and biopsies. Researchers are able to acquire more information from more samples, companies develop products faster, and clinicians better able to diagnose and manage disease using genetic, epigenetic and expression biomarkers.

The Rubicon pre-analytical processes have been used for the testing of many hundreds of thousands of samples in thousands of research laboratories, major pharmaceutical and diagnostics companies, and large international genomics initiatives. The results of those projects are numerous research publications, discovery of more than 50 genetic, epigenetic, and expression biomarkers, as well as a creation of a diverse set of diagnostic tests developed or in development. Rubicon is committed to playing a key role in nucleic acid research and clinical testing by improving the performance of the major research and diagnostic platforms, especially to enable more sensitive, specific, and robust clinical tests for cancer and other diseases.

Major Limitations of Nucleic Acid Analysis Platforms

DNA and RNA analysis platforms require that the sample nucleic acids meet specific requirements for quantity, quality, and purity. The platforms perform best when all samples have the same properties, sub-optimally when samples have variable properties even within the allowed range for the parameters, and fail for samples that fall outside the allowed range for the parameters. As a result, researchers sometimes have sample drop outs and incorrect results. Clinical applications have low tolerance for such problems and therefore testing must be limited to larger samples and fewer analytes in order that failures and false results do not occur. This precludes effective testing from plasma, serum, single cells, and many surgical and biopsy specimens. For over five years, customers and collaborators have employed Rubicon to process DNA and RNA to enable an expanding list of specific research and clinical applications to overcome practical limitations in nucleic acid quantity, quality, or purity.

Generic research and clinical examples of instrument-independent limitations include:

These generic limitations are important to all types of testing (mutations, genotypes, gene amplification/deletion/fusion, expression, and methylation) on all types of analytical platform (qPCR, microarrays, next-gen sequencing, and mismatch detection). Clearly, elimination of these limitations would enable new research and clinical applications. Although there are many pre-amplification methods that have been developed over the last 20 years to mitigate these limitations for specific applications, in the last three years the Rubicon platforms have been chosen by major government agencies, diagnostic companies, and institutions to give better sensitivity, representation and robustness as well as lower random background than other pre-analytical platforms such as MDA, LM-PCR and DOP PCR. In addition, Rubicon has the only platforms that are universally applicable across the full range of genetic, epigenetic, and expression analyses and instrumentation.

Rubicon Platforms as Universal Solutions to Major Analytical Limitations

Rubicon has successfully commercialized a series of PCR-based amplification platforms and stand-alone processes. Each platform synthesizes a molecular library that is amplified more than 1,000-fold to produce a standardized (constant concentration, molecular weight and purity) reamplifiable DNA library with minimal locus/allele bias and background. The company’s GenomePlex™ platform is used to amplify and standardize total DNA for genetic analysis, its TransPlex™ platform amplifies and standardizes RNA sequences for expression analysis, and its MethylPlex™ platform selectively amplifies and standardizes the methylated portion of DNA for methylation analysis. These platforms increase the analytical sensitivity, specificity, reproducibility, and robustness of nucleic acid analyses for research and clinical applications. These improvements have been proven across virtually all the analytical platforms for analyzing genotypes, mutations, deletions and amplifications, aneuploidies, gene expression, presence of infectious agents, and DNA methylation. In fact, the ability to amplify and reamplify nucleic acids without introducing bias or background makes multi-analyte or genome-wide profiling routine even with very difficult clinical samples such as single cells, plasma/serum, and formalin-fixed tissue. Rubicon believes that all diagnostic and research platforms (including qPCR, microarray, and next-gen sequencing instruments) and most existing diagnostic assays can be significantly enhanced through the application of the Rubicon proprietary pre-analytical platforms.

In addition, Rubicon has an extensive portfolio of patented “derivative” pre-analytical processes that were originally developed for the platforms, but also have significant value as independent pre-analytical processes in their own right. Derivatives enable important improvements in conventional locus-specific qPCR, whole genome amplification of bisulfite converted DNA, simplification of dye labeling, novel hot-start PCR, complexity reduction for microarrays and next-gen sequencing, and single-tube preparation of molecules for next-gen sequencing

For research applications, these pre-analytical platforms and processes are simple and reproducible methods to achieve the highest quality results using very small samples. For diagnostics and pharmacogenomics, they enable discovery and validation of better biomarkers, shorter product development cycles, and patient tests that have higher performance.

Rubicon Products

Rubicon has commercialized its pre-analytical products in multiple forms:

Research Products

Rubicon’s strategy is to initially market its products in kit form to the research genetics and expression markets while providing premium pre-analytical and analytical services to pharmaceutical companies and large institutions. The GenomePlex™ and TransPlex™ product lines have been exclusively licensed to Sigma-Aldrich for sale in the worldwide research market, specifically excluding all clinical applications. This approach has generated commercial validation of both methods, generated revenue and stimulated second-party development of advanced diagnostic applications. Simultaneously, the company has developed and commercialized the MethylPlex pre-analytical and analytical platforms for quantifying DNA methylation and is presently using this platform for in-house research as well as in fee-for-service projects with other companies. It is the company’s intention to market the MethylPlex platform in kit format to the research market for discovery and testing of methylation biomarkers.

Diagnostic Products

Rubicon is bringing its GenomePlex, TransPlex, and MethylPlex pre-analytical platforms to the diagnostic markets for novel biomarker discovery and validation, and ultimately for clinical testing, using qPCR, microarrays or next-gen sequencing.

To improve existing diagnostic tests for genetic and expression abnormalities, Rubicon is collaborating with specific diagnostic companies to improve clinical tests for cancer and other diseases. These diagnostic companies currently purchase or will purchase customized diagnostic GenomePlex or TransPlex kits from Rubicon that will be incorporated into their final tests to improve these DNA and RNA tests.

Technology

Rubicon’s pre-analytical platforms substantially improve nucleic acids for downstream analysis by standardizing the quality and quantity of the nucleic acids, increasing the amount of nucleic acid that can be analyzed, and reducing the background from unwanted sequences. These improvements enable qPCR, microarray, and next-gen sequencing platforms to achieve higher sensitivity, specificity, and robustness. This has been validated for genotyping, aCGH, sequencing, and methylation analysis in research and diagnostic applications.

GenomePlex, TransPlex, and MethylPlex pre-analytical platforms are schematically displayed above. After conventional isolation of the DNA or RNA, the molecules are converted to amplifiable libraries consisting of total DNA, cDNA, or methylated DNA sequences. Each type of library is then amplified >1,000-fold using a universal-primer PCR reaction to produce DNA or cDNA of standardized concentration, molecular weight and quality, ready to be loaded onto any of the microarray, qPCR, or next-gen sequencing platforms for analysis. Any impurities such as oligonucleotides, unwanted salts, and PCR inhibitors are diluted >1,000-fold in the process. As a result of nucleic acid pre-amplification and standardization, the analytical platforms are operated under ideal conditions to produce more consistent results than could be produced from the unamplified DNA or RNA. For non-invasive testing, as little as 2 mL of plasma is sufficient for detection of an unlimited number of mutation or methylation biomarkers. For specialized analysis of specific parts of the genome, a proprietary method of sequence enrichment is used to reduce the sequence complexity of the amplified materials by ~100 times, which is especially important in microarray and next-gen sequencing applications. Synthesis, amplification, and enrichment of these libraries is rapid, inexpensive, and automatable on existing general-purpose instruments.

GenomePlex is a patented method that employs quasi-random, non-self-complementary primers to make libraries for amplifying total genomic DNA with high fidelity. Highly representative and reproducible amplification can be achieved on intact or degraded DNA, as found in most clinical samples such as plasma, serum, urine, formalin-fixed, and necrotic frozen tissue. GenomePlex can also amplify DNA from single cells and single isolated chromosomes. GenomePlex is more robust and reproducible than any of the other total DNA amplification products that have been brought to market over the years. In fact, the National Cancer Institute has chosen GenomePlex as the best method to amplify the cancer and normal DNA samples for the Cancer Genome Atlas Project. Other organizations, such as GSK, Genome Institute of Singapore, Sanger Institute, Johns Hopkins University, have published results showing that the GenomePlex amplified DNA performs exceptionally well for genotyping, sequencing, and gene copy number analysis using qPCR and microarray platforms.

TransPlex is a patented method that uses quasi-random primers to make libraries for amplifying total cellular or viral RNA with high fidelity. Highly representative and reproducible amplification can be achieve from intact RNA, or degraded RNA as found in most clinical samples. TransPlex is very robust and reproducible and seems to be the only effective method to amplify RNA from fixed tissue. Numerous diagnostic companies and universities have chosen TransPlex as the standard pre-analytical platform for discovery of novel expression biomarkers for cancer.

MethylPlex represents several patented methods for selective amplification of the methylated portions of any genome utilizing very efficient quasi-random primers or ligation. MethylPlex is unique and elegant, and results in increasing the amount of DNA that is specific for the presence and status of the tumor. MethylPlex is achieved in a simple, enzymatic process that completely avoids the bisulfite conversion chemical reactions used in other methylation assays. MethylPlex enables the discovery and validation of diagnostic biomarkers faster and more accurately than bisulfite methods and is the only method capable of measuring multiple methylated DNA analytes from a single sample of plasma or serum. MethylPlex has been chosen by multiple diagnostic companies to discover new cancer biomarkers and will be used as a pre-analytical step in patient tests. Not only is MethylPlex processing simple, rapid, and inexpensive, but it has also been automated on sophisticated diagnostic platforms.

Platform Combinations are useful for achieving maximal multiplexing among genetic, epigenetic, and expression assays. For example, total DNA and RNA can be amplified in a single reaction in a single tube for purposes of single-array detection of DNA and RNA viruses (currently under evaluation by the Navy and by USDA). Another example is the combination of methylation and gene copy number profiling on the same arrays. In principle, all three amplified libraries from a single patient samples can be combined and hybridized to a single array for simultaneous genetic, epigenetic, and expression analysis.

Derivative Rubicon Technologies are patented processes that have value independent of the amplification platforms. These include:

Summary

Rubicon fills a novel and key role in nucleic acid testing. In response to the known problems in nucleic acid analysis caused by variations in DNA or RNA quantity and quality, Rubicon has developed elegant and effective pre-analytical processes for amplification, standardization and enrichment of nucleic acids for research and clinical applications. These tools are currently used in thousands of laboratories to improve genetic, epigenetic and expression research using the major analytical platforms., These pre-analytical processes will continue to greatly accelerate the discovery of new disease genes and biomarkers, as well produce the next generation of more sensitive and robust patient tests.