Importance of Next-generation Sequencing Electron Tunelling Commercial Application Conclusion Bibliography Next-generation Sequencing: Electron tunelling Bc. Zuzana Ruttkayová Faculty of Informatics, Masaryk University in Brno 26 April 2012 Bc. Zuzana Ruttkayová Next-generation Sequencing: Electron tunelling Importance of Next-generation Sequencing Electron Tunelling Commercial Application Conclusion Bibliography Contents 1 Importance of Next-generation Sequencing 2 Electron Tunelling Nanopore sequencing Scanning tunneling microscope 3 Commercial Application 4 Conclusion Bc. Zuzana Ruttkayová Next-generation Sequencing: Electron tunelling Importance of Next-generation Sequencing Electron Tunelling Commercial Application Conclusion Bibliography Contents 1 Importance of Next-generation Sequencing 2 Electron Tunelling Nanopore sequencing Scanning tunneling microscope 3 Commercial Application 4 Conclusion Bc. Zuzana Ruttkayová Next-generation Sequencing: Electron tunelling Importance of Next-generation Sequencing Electron Tunelling Commercial Application Conclusion Bibliography Contents 1 Importance of Next-generation Sequencing 2 Electron Tunelling Nanopore sequencing Scanning tunneling microscope 3 Commercial Application 4 Conclusion Bc. Zuzana Ruttkayová Next-generation Sequencing: Electron tunelling Importance of Next-generation Sequencing Electron Tunelling Commercial Application Conclusion Bibliography Contents 1 Importance of Next-generation Sequencing 2 Electron Tunelling Nanopore sequencing Scanning tunneling microscope 3 Commercial Application 4 Conclusion Bc. Zuzana Ruttkayová Next-generation Sequencing: Electron tunelling Importance of Next-generation Sequencing Electron Tunelling Commercial Application Conclusion Bibliography Contents 1 Importance of Next-generation Sequencing 2 Electron Tunelling Nanopore sequencing Scanning tunneling microscope 3 Commercial Application 4 Conclusion Bc. Zuzana Ruttkayová Next-generation Sequencing: Electron tunelling Importance of Next-generation Sequencing Electron Tunelling Commercial Application Conclusion Bibliography Importance of Next-generation Sequencing Main goals – to build a nanopore based instrument for sequencing a mammalian genome for less than $1,000 Requirements: 1 High-speed sequential identification of the DNA’s nucleotides; 2 Very long, indefinite length reads; 3 The requisite sequence coverage (genomic DNA from < 106 cells) – no amplification, minimal preparative steps. Bc. Zuzana Ruttkayová Next-generation Sequencing: Electron tunelling Importance of Next-generation Sequencing Electron Tunelling Commercial Application Conclusion Bibliography Importance of Next-generation Sequencing Main goals – to build a nanopore based instrument for sequencing a mammalian genome for less than $1,000 Requirements: 1 High-speed sequential identification of the DNA’s nucleotides; 2 Very long, indefinite length reads; 3 The requisite sequence coverage (genomic DNA from < 106 cells) – no amplification, minimal preparative steps. Bc. Zuzana Ruttkayová Next-generation Sequencing: Electron tunelling Importance of Next-generation Sequencing Electron Tunelling Commercial Application Conclusion Bibliography Importance of Next-generation Sequencing Main goals – to build a nanopore based instrument for sequencing a mammalian genome for less than $1,000 Requirements: 1 High-speed sequential identification of the DNA’s nucleotides; 2 Very long, indefinite length reads; 3 The requisite sequence coverage (genomic DNA from < 106 cells) – no amplification, minimal preparative steps. Bc. Zuzana Ruttkayová Next-generation Sequencing: Electron tunelling Importance of Next-generation Sequencing Electron Tunelling Commercial Application Conclusion Bibliography Nanopore sequencing Scanning tunneling microscope Contents 1 Importance of Next-generation Sequencing 2 Electron Tunelling Nanopore sequencing Scanning tunneling microscope 3 Commercial Application 4 Conclusion Bc. Zuzana Ruttkayová Next-generation Sequencing: Electron tunelling Importance of Next-generation Sequencing Electron Tunelling Commercial Application Conclusion Bibliography Nanopore sequencing Scanning tunneling microscope Electron Tunelling Definition A quantum mechanical effect in which particles have a finite probability of crossing an energy barrier, such as the energy needed to break a bond with another particle, even though the particle’s energy is less than the energy barrier. Electron tunelling in proteins: donors and acceptors are held at fixed distances and orientations electron flow control factors are less well understood uncertainties in the relative orientations and structures Bc. Zuzana Ruttkayová Next-generation Sequencing: Electron tunelling Importance of Next-generation Sequencing Electron Tunelling Commercial Application Conclusion Bibliography Nanopore sequencing Scanning tunneling microscope Nanopore sequencing – the method under develop since 1995 – individual strands of DNA pass through nanoscopic holes (pores) "Strand sequencing": intact DNA polymers pass through a protein nanopore, sequencing in real time as the DNA translocates the pore. "Exonuclease sequencing": individual nucleotides pass through a protein nanopore, aided by a processive exonuclease enzyme. Bc. Zuzana Ruttkayová Next-generation Sequencing: Electron tunelling Importance of Next-generation Sequencing Electron Tunelling Commercial Application Conclusion Bibliography Nanopore sequencing Scanning tunneling microscope Nanopore sequencing Nanopore – is a nano-scale hole, which can be: Biological: a pore-forming protein in a membrane (lipid bilayer) Solid-state: in synthetic materials (silicon nitride, graphene) Hybrid: a pore-forming protein set in synthetic material Bc. Zuzana Ruttkayová Next-generation Sequencing: Electron tunelling Importance of Next-generation Sequencing Electron Tunelling Commercial Application Conclusion Bibliography Nanopore sequencing Scanning tunneling microscope Nanopore sequencing "Exonuclease sequencing" Bc. Zuzana Ruttkayová Next-generation Sequencing: Electron tunelling Importance of Next-generation Sequencing Electron Tunelling Commercial Application Conclusion Bibliography Nanopore sequencing Scanning tunneling microscope "Exonuclease sequencing" Results Bc. Zuzana Ruttkayová Next-generation Sequencing: Electron tunelling Importance of Next-generation Sequencing Electron Tunelling Commercial Application Conclusion Bibliography Nanopore sequencing Scanning tunneling microscope Nanopore sequencing "Strand sequencing" Bc. Zuzana Ruttkayová Next-generation Sequencing: Electron tunelling Importance of Next-generation Sequencing Electron Tunelling Commercial Application Conclusion Bibliography Nanopore sequencing Scanning tunneling microscope Scanning tunneling microscope STM – an electron microscope that shows 3D images of a sample – works best with conducting materials, but is possible to fix organic molecules on a surface and study their structures Method: a delicate electrode tip (very close to the DNA sample) This tip is fitted with a particular nucleotide and brought in contact with its complementary mate ⇒ the hydrogen bonds bind the bases and they attach to each other. sensing chemicals are attached to one end of the electrode and the sensor sample to the other end when the junctions spontaneously self-assemble, you get a specific signal Bc. Zuzana Ruttkayová Next-generation Sequencing: Electron tunelling Importance of Next-generation Sequencing Electron Tunelling Commercial Application Conclusion Bibliography Nanopore sequencing Scanning tunneling microscope Scanning tunneling microscope A–T pair: two hydrogen bonds C–G pair: three hydrogen bonds Bc. Zuzana Ruttkayová Next-generation Sequencing: Electron tunelling Importance of Next-generation Sequencing Electron Tunelling Commercial Application Conclusion Bibliography Contents 1 Importance of Next-generation Sequencing 2 Electron Tunelling Nanopore sequencing Scanning tunneling microscope 3 Commercial Application 4 Conclusion Bc. Zuzana Ruttkayová Next-generation Sequencing: Electron tunelling Importance of Next-generation Sequencing Electron Tunelling Commercial Application Conclusion Bibliography Commercial Application Oxford Nanopore Industries – commercial nanopore sequencer – 17.02. 2012 - presented first DNA sequence data – raw error read rate: 4% – array chip containing 2 000 nanopores – real time sequencing – has the potential to reach the $1,000 genome in under an hour by 2013 Bc. Zuzana Ruttkayová Next-generation Sequencing: Electron tunelling Importance of Next-generation Sequencing Electron Tunelling Commercial Application Conclusion Bibliography Contents 1 Importance of Next-generation Sequencing 2 Electron Tunelling Nanopore sequencing Scanning tunneling microscope 3 Commercial Application 4 Conclusion Bc. Zuzana Ruttkayová Next-generation Sequencing: Electron tunelling Importance of Next-generation Sequencing Electron Tunelling Commercial Application Conclusion Bibliography Conclusion faster-than-expected or more significant ramp for new products market growth uncertainties unstable funding environment in large-scale genotyping projects are declined, methodological frameworks are more affordable Bc. Zuzana Ruttkayová Next-generation Sequencing: Electron tunelling Importance of Next-generation Sequencing Electron Tunelling Commercial Application Conclusion Bibliography Bibliography The Harvard Nanopore Group. Project Nanopore. Taken from: Nanopore sequencing. [24.April 2012] http://www.mcb.harvard.edu/branton/projects-NanoporeSequencing.htm T. Kawai DNA sequencing and detection with nanoprobes. Taken from: DNA Sequencing by Electron Tunneling: STM and Gating Nanopore. [24.April 2012] http://www.cecam.org/workshop-4-822.html?presentation_id=8006 M. L. Metzker. Sequencing technologies d’˙z˝ the next generation. Taken from: Nature Reviews Genetics 11, 31-46 (January 2010) [24.April 2012] http://www.nature.com/nrg/journal/v11/n1/full/nrg2626.html A. Dubrow New Gene Sequencing Method Could Reduce Cost, Increase Speed. 2010. [25.April 2012] http://jmcb.oxfordjournals.org/content/early/2010/09/07/jmcb.mjq024.long Bc. Zuzana Ruttkayová Next-generation Sequencing: Electron tunelling Importance of Next-generation Sequencing Electron Tunelling Commercial Application Conclusion Bibliography Bibliography Oxford NANOPORE Technologies. [25.April 2012] http://www.nanoporetech.com/ Towards the 15-minute genome. 2011. [25.April 2012] http://www.economist.com/node/18304268 The Scanning Tunneling Microscope 2012. [25.April 2012] http://www.nobelprize.org/educational/physics/microscopes/scanning/index.html http://www.biodesign.asu.edu/research/projects/the-cheapgenome/scientific-detail [25.April 2012] http://www.biodesign.asu.edu/research/projects/the-cheap- genome/scientific-detail Speed-Reading DNA Inches Closer 2009. http://www.technologyreview.com/biomedicine/22217/ Bc. Zuzana Ruttkayová Next-generation Sequencing: Electron tunelling Importance of Next-generation Sequencing Electron Tunelling Commercial Application Conclusion Bibliography Bibliography About the figures Nanopore. Taken from: New Gene Sequencing Method Could Reduce Cost, Increase Speed. 2010. [25.April 2012] http://www.livescience.com/6720-gene-sequencing-method-reduce- cost-increase-speed.html Protein nanopores. Taken from: Protein nanopores. 2012. [25.April 2012] http://www.nanoporetech.com/technology/analytes-and-applications- dna-rna-proteins/dna-an-introduction-to-nanopore-sequencing Solid-state nanopores. Taken from: Future generations of nanopores: solid-state. 2012. [25.April 2012] http://www.nanoporetech.com/technology/introduction-to-nanopore- sensing/solid-state-nanopores Bc. Zuzana Ruttkayová Next-generation Sequencing: Electron tunelling Importance of Next-generation Sequencing Electron Tunelling Commercial Application Conclusion Bibliography Bibliography About the figures "Exonuclease sequencing" 1-4. Taken from: Nanopore Sequencing to Identify DNA Bases. 2009. [25.April 2012] http://www.technologyreview.com/biomedicine/22220/page4/#photo "Exonuclease sequencing" (results) 1-3. Taken form: DNA exonuclease sequencing. 2012. [25.April 2012] http://www.nanoporetech.com/technology/analytes-and-applications- dna-rna-proteins/dna-exonuclease-sequencing"Strand sequencing". Taken from: Future generations of nanopores: solid-state. 2012. [25.April 2012] http://www.nanoporetech.com/technology/introduction-to-nanopore- sensing/solid-state-nanopores Bc. Zuzana Ruttkayová Next-generation Sequencing: Electron tunelling Importance of Next-generation Sequencing Electron Tunelling Commercial Application Conclusion Bibliography Bibliography About the figures Scanning tunneling microscope. Taken from: More about the science. [25.April 2012] http://www.biodesign.asu.edu/research/projects/the-cheap- genome/scientific-detail MinION. Taken from: Why a Portable DNA Device Could Yield Better Data. 2012. [25.April 2012] http://www.technologyreview.com/biomedicine/39786/ Bc. Zuzana Ruttkayová Next-generation Sequencing: Electron tunelling