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Apr 15, 2010 (Vol. 30, No. 8)

Third-Generation Sequencing Debuts

Single-Molecule Detection Solutions Push the Technique to New Levels

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    Pacific Biosciences’ sequencing process uses SMRT Cells, each of which contains thousands of zero-mode waveguides (ZMW). A single DNA polymerase molecule is attached to the bottom of each ZMW, which provides a real-time observation window of a single molecule of DNA polymerase as it synthesizes DNA.

    Third-generation (gen-3), single-molecule sequencing technology is not only about making quantifiable enhancements to second-generation (gen-2) capabilities, but also about improving data quality and expanding the types of data produced. “It is becoming clear that we will need information about the structure of genomes and other types of information to put the whole picture together,” said Stephen Turner, Ph.D., founder and CTO of Pacific Biosciences. This will include information about a variety of epigenetic modifications.

    In Dr. Turner’s view, one of the advantages of the company’s technology is that “it unifies the previously separate fields of genomic and epigenomic research.” At present, research in the fields of developmental biology and oncology, in particular, would greatly benefit from this synergy, observed Dr. Turner.

    The Single Molecule Real Time (SMRT™) sequencing technology that Pacific Biosciences unveiled at the recent “Advances in Genome Biology and Technology” (“AGBT”) conference generates both DNA sequence and epigenomic information directly from the real-time sequencing of genomic DNA. Single-molecule sensitivity enables faster results and longer read lengths, Dr. Turner asserted.

    “Read length is extremely important for understanding the structure of the genome,” he added. “It is, perhaps, the dominant force driving the transition from microarrays to sequencing applications in personalized medicine.” Being able to detect and understand changes in the structure of individual genomes as they relate to disease—the presence of nucleotide duplications, inversions, deletions, etc.—may reveal a lot more about how people differ than single-base changes can.

    SMRT DNA sequencing technology eavesdrops on the actions of a single DNA polymerase molecule as it works its way along a DNA template synthesizing a new complementary strand. “The technology allows us to see the kinetics of every base incorporation,” Dr. Turner said. In addition to generating sequence data, the process provides direct epigenetic information based on the effects that various base modifications have on the kinetics of the sequencing reaction.

    Within about two years, the company plans to offer an application that will enable direct RNA sequencing in real time on the SMRT system without the need to convert RNA to cDNA. This application will provide insights into the “epigenetics of RNA,” said Dr. Turner, citing an example presented at the conference in which RNA sequencing using SMRT technology could distinguish pseudo-uridine from its native analog.

    The company is already disseminating its vision for version 2 of the SMRT DNA sequencing system, scheduled for release in 2014. It will offer higher sensitivity and faster resolution, “will be highly portable and will be able to sequence a human genome in 15 minutes for less than $100,” added Dr. Turner.

    With single-molecule sequencing (SMS), “the technology will no longer be a limitation to the application of genomic data for clinical decision making,” said Patrice Milos, Ph.D., vp and CSO at Helicos BioSciences, noting, however, that a clear understanding of how to apply this information in a medical setting is still, in general, emerging.

    At the “AGBT” meeting, Dr. Milos offered a vision of genome biology that will be achievable through a combination of sequence and quantitative data. It will enable a range of applications including chromatin profiling by direct sequencing of immunoprecipitated DNA, direct RNA sequencing, small RNA quantitation, digital gene expression, copy number variation assessment, and epigenetic analysis.

    Helicos has pioneered gen-3 sequencing with its True Single Molecule Sequencing (tSMS™) technology, a sequencing-by-synthesis technique. tSMS technology allows researchers to analyze nearly a billion molecules at the single-molecule level in one experiment, according to Dr. Milos. In addition to sequencing DNA, it can be used for direct sequencing of RNA without the need for a cDNA intermediate, thereby eliminating cDNA synthesis-based artifacts.

    Life Technologies recently introduced its new gen-3 system that performs SMS directly on the surface of a 10 nm quantum dot nanocrystal. This method is “complementary to and not competitive with” second-generation methods, said Joseph Beechem, Ph.D., CTO at Life Technologies.

    Gen-2 technology is well suited for genomic discovery and unraveling the genetic basis of disease. Over the next three years, Dr. Beechem predicts that as many as one million human genomes will be sequenced using these methods, giving researchers the information needed to link specific genes with disease predisposition and a range of human disorders.

    “Gen-3 sequencing technology will then make it possible to start applying these discoveries in the clinic to guide medical decisions.” Dr. Beechem emphasized the need for long, continuous read lengths to enable some medical applications, such as haplotype phasing in which an allele of interest is identified as belonging to either the maternal or paternal DNA strand. This has important implications for predicting immunological responses, drug metabolism/sensitivity, cancer progression/metastasis, genomic structural variations, and other medically relevant genetic effects.

    In his presentation at “AGBT”, Dr. Beechem described how the quantum-dot SMS technology enables several new sequencing capabilities. Conceptually, it is as though a movie camera (i.e., the quantum dot) were mounted on a DNA polymerase molecule, filming the DNA sequencing reaction in real time along a single DNA strand as it would occur in nature. All of the action being captured on film—the simultaneous, parallel sequencing of about 150,000 DNA strands—takes place in one field of view of the microscope. 

    “Since the sequencing engine is a ‘replaceable’ reagent, continuously tunable long reads and tunable accuracies can be realized by simply washing-in new sequencers to replace the older ones as they ‘wear out’ inside the microscope,” Dr. Beechem explained. The detection field tracks with the moving polymerase so sequence data is collected in real time.

    “It follows the polymerase wherever it goes, allowing sequencing reactions to be performed in a wide variety of formats in addition to standard arrays (for example,  flowing channels, tissue slices, sample surfaces). Each genomic DNA template is sequenced multiple times. The technology relies on Qdot® nanocrystals composed of inorganic materials (semiconductors) that provide 100-fold greater absorbance than a typical organic dye and generate about a 200X stronger fluorescent signal,” said Dr. Beechem.

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