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Pacific Biosciences of California, Inc. Message Board

  • dragan7777777777 dragan7777777777 Nov 3, 2010 6:19 AM Flag

    DD-2......

    Sequencing-by-Synthesis

    http://www.pacificbiosciences.com/assets/files/pacbio_technology_backgrounder.pdf


    Single Molecule Real Time (SMRT™) DNA Sequencing

    Long Reads, Fast Results, and More Informative Data at Lower Overall Cost

    Single Molecule Real Time (SMRT™) DNA sequencing technology enables, for the first time, the observation of natural DNA synthesis by a DNA polymerase as it occurs. The approach is based on eavesdropping on a single DNA polymerase molecule working in a continuous, processive manner. Distinguished by long reads, fast time to results, more informative data, and lower overall costs, SMRT DNA sequencing promises to be a transformative technology that will enable a new paradigm in genomic analysis.

    SMRT technology is built upon three key innovations that overcome major challenges facing the field of DNA sequencing:


     The SMRT Cell, which enables single molecule, real-time observation of individual fluorophores against a dense background of labeled nucleotides while maintaining a high signal-to-noise ratio.


     Phospholinked nucleotides, which enable long readlengths by producing a completely natural DNA strand through fast, accurate, and processive DNA synthesis, and


     A novel detection platform that enables single molecule, real-time detection as well as flexibility in run configurations and applications.


    SMRT Technology At-a-Glance

    DNA sequencing is performed on SMRT Cells, each containing thousands of zero-mode waveguides (ZMWs).

    Utilizing the latest geometries available in semiconductor manufacturing, a ZMW is a hole, tens of nanometers in diameter, fabricated in a 100nm metal film deposited on a silicon dioxide substrate. Each ZMW becomes a nanophotonic visualization chamber providing a detection volume of just 20 zeptoliters (10-21 liters). At this volume, the activity of a single molecule can be detected amongst a background of thousands of labeled nucleotides.


    Figure 1. Problem of background interference

    For proper functioning, DNA polymerase requires a high concentration of labeled nucleotides, which creates a fluorescent background thousands of times brighter than the signal of a single incorporation event.

    This creates a high background noise level, against which it is not possible to detect individual fluorophores. However, polymerases require this high concentration level, without which the speed, accuracy, and processivity of the enzyme all suffer.

    Sequencing approaches that circumvent this problem by step-wise addition of base-labeled nucleotides followed by washing, scanning, and removal of the label, severely limit the capabilities of the polymerase, making it significantly slower and drastically reducing readlength. The need for washing between bases also dramatically increases the consumption of reagents.

    Pacific Biosciences has addressed this problem with
    the SMRT Cell, which contains thousands of zero-mode
    waveguides (ZMWs) (Figure 2). The ZMW provides the
    world’s smallest detection volume, representing a 1000-
    fold improvement over existing single-molecule detection technology. Because the detection volume is so dramatically reduced, a single incorporation event can be observed against the background created by the high concentration of fluorescently labeled nucleotides. This makes possible robust real-time observation of a single molecule of DNA polymerase as it synthesizes DNA.

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    • RainDance?....

    • Oh you must mean Ilumina's, SBS? Unlike PacBio who are millions in the RED, ILMN is making money.

      Third quarter 2010 results:
      Revenue of $237.3 million, a 50% increase over the $158.4 million reported in the third quarter of 2009.

      PacBio stock will end up in single digits, then I may buy unless someone buys them out first.



      Illumina’s sequencing by synthesis (SBS) technology is the most successful and widely-adopted next-generation sequencing platform worldwide. SBS technology supports massively parallel sequencing using a proprietary reversible terminator-based method that enables detection of single bases as they are incorporated into growing DNA strands. A fluorescently-labeled terminator is imaged as each dNTP is added and then cleaved to allow incorporation of the next base. Since all four reversible terminator-bound dNTPs are present during each sequencing cycle, natural competition minimizes incorporation bias. The end result is true base-by-base sequencing that enables the industry’s most accurate data for a broad range of applications. See a video of SBS technology in action


      Applications

      SBS technology supports both single read and paired-end libraries. It is the only platform that offers a short-insert paired-end capability for high-resolution sequencing as well as long-insert paired-end reads using the same robust chemistry for efficient sequence assembly, de novo sequencing, large-scale structural variation detection, and more. The combination of short inserts and longer reads increase the ability to fully characterize any genome. A wide array of available sample preparation methods serve to enable diverse applications, including: whole-genome and candidate region resequencing, transcriptome analysis, small RNA discovery, methylation profiling, and genome-wide protein-nucleic acid interaction analysis.


      Workflow

      With Illumina Sequencing, researchers get the simplest and- fastest workflow available. Sample libraries are prepared in just a few hours using a range of ready-to-use kits. With the cBot system, clonal clusters of DNA library fragments are automatically generated on planar, optically transparent substrates called flow cells. In approximately four hours, up to 96 multiplexed samples can be isothermally amplified in the flow cell’s eight channels. This self-contained system minimizes handling errors and contamination concerns, eliminating the need for ePCR, robotics, or clean rooms. Walk-away automation for DNA sequencing can be initiated with less than ten minutes of hands-on time using any of the Illumina sequencing systems. This streamlined workflow generates meaningful data quickly and efficiently, while reducing project time lines and costs.

 
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