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Small Molecule Real Time Sequencing
The document provides an overview of the next generation sequencing (NGS) single molecule real-time (SMRT) method, which utilizes DNA polymerase and fluorescent probes for sequencing. It outlines the step-by-step process, including the immobilization of DNA templates and polymerase, introduction of phospho-linked nucleotides, and detection of signals emitted during base incorporation. The method operates in parallel across thousands of zero-mode waveguide (ZMW) chambers to facilitate rapid sequencing.
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Small Molecule Real Time Sequencing
- 1. Next Generation Sequencing SingleMolecule Real Time Method
- 2. Overview • Uses actualDNA polymerase to sequence • Enzymatic speed • fluorescent probe not to the nucleotide
- 3. Step 1 • Acomplex of a DNA template and active polymerase is immobilized at the bottom of the ZMW Image found at http://esciencecentral.org/ebooks/applications-of-molecular-genetics/dna-sequencing-sanger-and-nextgeneration-sequencing.php
- 4. Step 2 • Phospho-linkednucleotides are introduced into the chamber of the ZMW. Image found at http://esciencecentral.org/ebooks/applications-of-molecular-genetics/dna-sequencing-sanger-and-nextgeneration-sequencing.php Image found at http://www.pacificbiosciences.com/products/smrt-technology/
- 5. Step 3 • Eachof the four nucleotides is labeled with a different colored fluorophore and loaded. Image found at http://esciencecentral.org/ebooks/applications-of-molecular-genetics/dna-sequencing-sanger-and-nextgeneration-sequencing.php Image found at http://www.umich.edu/~caparray/products/ngs/pacbio
- 6. Step 4 • Asa base is held in the detection volume, a light pulse is produced. Image found at http://esciencecentral.org/ebooks/applications-of-molecular-genetics/dna-sequencing-sanger-and-nextgeneration-sequencing.php
- 7. Step 5 • Nucleotidesheld by the polymerase prior to incorporation emit an extended signal that identifies the base being incorporated. Image found at http://www.omixon.com/the-basics-of-next-generation-sequencing/ Image found at http://esciencecentral.org/ebooks/applications-of-molecular-genetics/dna-sequencing-sanger-and-nextgeneration-sequencing.php
- 8. Step 6 • Thisprocess occurs in parallel in up to thousands of ZMWs that make up the SMRT cell. Image found at http://esciencecentral.org/ebooks/applications-of-molecular-genetics/dna-sequencing-sanger-and-nextgeneration-sequencing.php
- 9. Work Cited • AmbryGenetics. Making Sense of NextGent Sequencing. Kelly Gonzalez, MS, CGC, and Senior Manager of Clinical Genomics. Available at http://www.ambrygen.com/sites/default/files/pdfs/NERRG_4-10- 12_Making_Sense_of_NetGen_Sequencing_KG(3).pdf. Access verified May 21, 2014. • Omixon. Allen Van Deynze, 2010; Solanaceae Coordinated Agricultural Project. Next Generation Sequencing. Available at http://www.omixon.com/the-basics-of-next- generation-sequencing/. Access verified May 21, 2014. • DNA Sequencing, Sanger and Next-Generation Sequencing. Omics eBooks Group. (2014). Yao Fei, Genome Institute of Singapore. Available at http://esciencecentral.org/ebooks/applications-of-molecular-genetics/dna- sequencing-sanger-and-nextgeneration-sequencing.php Access verified Aug 5, 2014. • SMRT TECHNOLOGY. Pacific Biosciences. (2014). Available at http://www.pacificbiosciences.com/products/smrt-technology/ Access verified Aug 5, 2014. • PacBio RS II Sequencing. Microarray Core Facility. (2012). Available at http://www.umich.edu/~caparray/products/ngs/pacbio/ Access verified Aug 5, 2014. • DNA SEQUENCING/GENOMICS: Toward personalized medicine: 3G DNA sequencing. (March 2010). Stephen Turner. BioOptics. Available at http://www.bioopticsworld.com/articles/print/volume-3/issue-3/features/dna- sequencing-genomics.html Access verified Aug 5, 2014.
- 10. Recommended Videos SMRT TECHNOLOGY http://www.pacificbiosciences.com/produc ts/smrt-technology/ Introductionto SMRT Sequencing http://article.wn.com/view/2014/05/14/Sin gle_Molecule_RealTime_SMRT_DNA_S equencing_Technology_From_g/
- 11. Next Generation Sequencing IonTorrent Method
Editor's Notes
- #2 This video gives an overview on how Single Molecule Real Time Sequencing, another type of Next Generational Sequencing, works.
- #3 Single Molecule Real Time sequencing by the Pacific Biosciences company uses a completely different method. Very unique in that this method uses the actual DNA polymerase to sequence. The way they can do this is that they have these little mouths where DNA sequences come in where the DNA polymerase is attached and then will start sequencing at enzymatic speed. This enzymatic speed is much faster, the Next Generation Sequencing takes days to weeks to do a run whereas sequencing at this enzymatic rate allows sequencing to be finished in minutes to weeks. 10bp/sec Another difference is that they added the fluorescent probe not to the nucleotide but to the phosphorous base instead of the base itself like in Illumina and True Single Molecule Sequencing, which makes it a smaller molecule and allows it to go through the sequencing much more efficiently.
- #4 The DNA Template library consists of double stranded fragments of DNA from about 250bp to 20Kbp in length with single stranded end adapters. Primers are then annealed to the end adapters and bound with polymerases. The end adapters make the double stranded DNA fragments into circular templates on which the polymerase will continue to run until it becomes inactive or until the end of the observation period. A read containing multiple passes around the circular template can be condensed into a higher accuracy consensus sequence.
- #5 With an active polymerase immobilized at the bottom of each ZMW, nucleotides diffuse into the ZMW chamber. In order to detect incorporation events and identify the base, each of the four nucleotides A, C, G and T are labeled with a different fluorescent dye having a distinct emission spectrum. Since the excitation illumination is directed to the bottom of the ZMW, nucleotides held by the polymerase prior to incorporation emit an extended signal that identifies the base being incorporated.
- #6 The SMRT Cell contains 150,000 wells called zero mode waveguides or ZMWs. A minimum of 40,000 ZMW's will contain readable templates and produce sequencing data. Each ZMW is visualized by a laser beamlet to record additions of fluorophore-tagged bases to a DNA template by a polymerase attached to the bottom of the ZMW. Each SMRT Cell is loaded with one sample and up to 12 SMRT cells may be run within a 24 hour day.
- #7 As the DNA polymerase incorporates complementary nucleotides, each base is held within the detection volume for tens of milliseconds, orders of magnitude longer than the diffusion time. During this time, the polymerase-bound fluorophore emits fluorescent light, the color of which corresponds to the base identity. Then, as part of the incorporation cycle, the polymerase cleaves the bond connecting the nucleotide to the fluorophore, allowing the fluorophore to quickly diffuse out of the detection volume.
- #8 Nucleotides held by the polymerase prior to incorporation emit an extended signal that identifies the base being incorporated. This processive synthesis with phospholinked nucleotides takes place in a series of steps: First the Fluorescent phospholinked labeled nucleotides are introduced into the ZMW, Then the base being incorporated is held in the detection volume for tens of milliseconds, producing a bright flash of light, after that the phosphate chain is cleaved, releasing the attached dye molecule, and then the process repeats. Because of this particular way the label is attached, the DNA synthesized by the polymerase is completely natural and unmodified, leaving the polymerase without any memory of having processed an unnatural nucleotide. Inhibition of enzymatic activity, which is commonly observed with other types of labeled nucleotides, is thereby avoided.
- #9 This process occurs in parallel in up to thousands of ZMWs that make up the SMRT cell. The sensitivity of the ZMW-based single-molecule detection technology is ~1,000 times greater than that of existing microscopes. This enables it to discriminate signals against background noise while reading the individual bases of DNA as close as possible to the speed in which they are synthesized in nature. A prototype instrument enables recording of labeled nucleotide incorporations by single polymerase enzymes in real-time and high multiplex. In this highly parallel optics system, a detected flash of light is separated into a spatial array–from which the identity of the incorporated base is determined.
- #10 This is the Work Cited for this video.
- #11 These are recommended videos in order to further understand how Small Molecule Real Time sequencing works. SMRT TECHNOLOGY http://www.pacificbiosciences.com/products/smrt-technology/ Introduction to SMRT Sequencing http://article.wn.com/view/2014/05/14/Single_Molecule_RealTime_SMRT_DNA_Sequencing_Technology_From_g/
- #12 The next video will give an overview on another type of Next Generational Sequencing called Ion Torrent.