3 rd Generation Sequencing Technologies. Roger E. Bumgarner

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1 3 rd Generation Sequencing Technologies Roger E. Bumgarner

2 Brief review First generation sequencing technologies Sanger and Maxim Gilbert methods Used either chemical or enzymatic methods to generate a nested set of DNA fragments Used electrophoretic methods to separate the fragments Required lots of DNA (100 s of ng to 1ug) so it typically involved cloning and/or PCR. Limited throughput meant that other methods for ordering DNA clones (e.g. physical mapping) was often necessary prior to shotgun sequencing of smaller fragments.

3 Other technologies out there IBM is working on reading DNA directly in silicon pores. If synthetic pores with chemical modifications can be constructed from nonprotein structures, this may turn out to be a more robust instrument to sequence DNA (e.g. more stable over time and more reusable). Many others are working on STM methods of sequencing but there s been little demonstrate success in this area and I suspect it will be hard to compete with pore based methods.

4 So what s next? Single molecule sequencing Advantages Few or no enzymes involved in preparation of the DNA Reduces cost, time and potential biases/errors In some systems, no enzymes involved in reading the DNA. Can often read RNA directly with the same system/method. Some single molecule systems allow the direct identification of nucleotide modifications.

5 The 1 st method for single molecule sequencing The earliest concept came from the Los Alamos labs in the late 1980 s/early 1990 s. See High-speed DNA sequencing: an approach based upon fluorescence detection of single molecules., Jett JH et.al. J Biomol Struct Dyn Oct;7(2): The plan involved: Generating a strand of DNA in which every nucleotide was fluorescently labeled and attaching the labeled DNA to a substrate in a flow cell (or capillary) Cleaving the labeled nucleotides off with an exonuclease Using LIF in a small volume to detect the individual nucleotides as they were released

6 Issues with this method It was difficult to get enzymes to fully label a strand with fluorescent nucleotides. The exonucleases were inefficient with labeled nucleotides The detection of single molecule fluorescence had high error rates in capillaries due to stochastic nature of photodestruction of the fluors and background fluorescence. No method to re-read the same strand as the process was destructive.

7 Currently or soon to be available single molecule systems Helicos True Single Molecule Sequencing (tsms TM ) system Single base, reversible dye terminator extension reactions Pacific BioSciences Single Molecule Real Time (SMRT TM ) sequencing Dyes that are phospholinked to the nucleotide, very sensitive fluorescent detection in zero mode waveguides Oxford Nanopores GridION and MinION systems Direct reading of unlabeled DNA by threading it through a nanopore

8 Helicos Methodology See -Harris et.al., Science 4 April 2008:Vol no. 5872, pp

9 Sequencing the other strand (repeatedly)

10 Helicos specifications 600M 1B useable reads per run 35bp average read length, 21-35Gbp/run Raw error rates Substitution 0.2%, Insertion 1.5%, Deletion 3.0% Run time 8 days Imaging system can read 1Gbp/hr., actual throughput is about Mbp/hr.

11 Pacific Biosciences Single molecule sequencing Enabling technologies Zero mode waveguide (very low detection volumes, high sensitivity) Passivation of the aluminum wave guide surface using polyphosphonate chemistry to prevent polymerase binding randomly Tethered polymerase Phospho linked fluors

12 Construction of Zero Mode Wave Guides Holes in the coating Solution phase Fused Silica (e.g. quartz) Substrate Aluminum coating Laser Illumination

13 A device containing about 2M Zero Mode Wave Guides M. J. Levene et al., Science 299, (2003) Published by AAAS

14 Zero Mode Wave Guides Published by AAAS M. J. Levene et al., Science 299, (2003) Illumination comes through the silica, the light is evanescent and illuminates only a very small volume.

15 Enzymes are attached inside the waveguide in limiting dilution (one enzyme/waveguide) M. J. Levene et al., Science 299, (2003) Published by AAAS

16 A key enabling technology

17 Fluorescent Signal in the Detection Volume is a Function of diffusion rate Observation of single nucleotide incorporation Published by AAAS M. J. Levene et al., Science 299, (2003)

18 Putting it all together CFc

19 Advantages and interesting applications of PacBio technology The single molecule real-time nature of the technology allows one to observe kinetics of the enzyme in real time. Enzyme kinetics are dependent on nucleotide modifications Alternate enzyme/substrate combination can be used to: Sequence RNA directly Study other enzymes

20 Enzyme kinetics is affected by strand modifications See: Direct detection of DNA methylation during single-molecule, real-time sequencing, Flusberg, et.al. Nature Methods 7, (2010)

21 Pac Bio Specifications Read lengths 3000bp average, can be much longer Limited by photo destruction of the enzyme Can use strobe methods to get paired reads separated by long distances Reads per SMRT cell approx k, each ZMW reads at a rate of a few bp/s Total instrument throughput is about 3Gbp/day Consumable costs approx. $1.40/1MBP (about 10x more than Illumina) Raw error rates of about 10-20%. Can be improved with circular consensus sequencing.

22 Circular Consensus Sequencing With bp inserts, the enzyme can traverse the loop 4-6 times giving 8-12x coverage of the same sequence.

23 Nanopore based methods Basic idea: Can we thread DNA through a small hole and measure some physical property that is related to the nucleotide that is in the hole? Players: Oxford Nanopore. IBM (in stealth mode at present) + others

24 Oxford Nanopore nanopore Silicon chip Lipid bi-layer

25 Structure of the Protein Nanopore

26 Current/dwell time is nucleotide specific (initially demonstrated with free nucleotides) See - Continuous base identification for single-molecule nanopore DNA sequencing Clarke J., et.al.,nat. Nanotechnology, 4: (2009)

27 Methyl G gives a different pattern

28 Two Modes of Sequencing with Nanopores Exonuclease sequencing Using a processive enzyme to cleave individual nucleotides from a DNA strand and pass them through a protein nanopore. Oxford Nanopore has a commercialisation agreement with Illumina for this method. Strand sequencing Identifying individual nucleotides on a DNA strand as it passes intact through a protein nanopore.

29 Nuclease based sequencing Initially the most well developed method at Oxford Big down side destroys the DNA as it is being read Not possible to re-read the same sequence Hence not possible to correct errors in individual reads

30 Direct reading DNA sequence is read directly by passing (or forcing) a ssdna through the pore non-destructive, hairpins allow reading of both strands

31 Link out to Oxford Video

32 Oxford Nanopore s System Announced at the ABGT meeting on Feb. 17 th, 2012 Single disposable device costing $ nanopores in the device to be released in Q3/Q4 of this year. Read rates of 100 s of kb/s, read lengths of 10 s of kbp. Claim that multiple nodes will be combined to produce sequence at $25-40/Gbp THIS YEAR! E.g. $1000 human genome at 30x coverage with the next year or so. 4% error rate at present, claim that can be reduced to 0.1-1%

33 Big claims but So far, the company has only released data demonstrating that they can read 5.4kb in one read. No instruments are in the field yet. Historically, it takes more time than expected to get from initial success to a marketable product. But we ll see.

34 A widening gap between the cost of generating the data and the cost of storing/moving the data Fall 2012?

35 So what does all this mean for you? Genome sequencing is become democratized soon any lab will be able to do this at reasonable cost for any genome without a large upfront expenditure in equipment. $1000/genome is less than the cost of a prenatal test Soon all genetic diagnostics will be replaced with genome sequencing Soon we will simply sequence children at birth Cancer genomes will be sequenced regularly for diagnostic purposes Human pathogen genomes will also be sequenced regularly for diagnostic purposes

36 Time Magazine Cover, Jan 17 th, Maybe they meant now?

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