A Novel Application of Pacific Biosciences SMRT Technology. Steven T. Lott, PhD, MB(ASCP) CR

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1 A Novel Application of Pacific Biosciences SMRT Technology Steven T. Lott, PhD, MB(ASCP) CR

2 Agenda for Today Technology Overview Technology Applications

3 DNA Polymerase as a Sequencing Engine ZMW with DNA polymerase ZMW with DNA polymerase and phospholinked nucleotides

4 A New Concept for Labeled Nucleotides Base-linked (2 nd Generation) Phospholinked (PacBio) Cleavage by DNA polymerase Fluorophore stays in DNA Inhibits enzyme Creates background light Fluor naturally cleaved off by polymerase DNA synthesized is uninterrupted Eliminates steric hindrance and noise

5 Processive Synthesis with Phospholinked Nucleotides Step 1: Fluorescent phospholinked labeled nucleotides are introduced into the ZMW. Step 2: The base being incorporated is held in the detection volume for tens of milliseconds, producing a bright flash of light. Step 3: The phosphate chain is cleaved, releasing the attached dye molecule. Step 4-5: The process repeats.

6 Real-Time Detection of ZMWs in a Multiplex Array 75,000 ZMWs monitored simultaneously Tens of thousands of polymerase molecules monitored immobilized in the bottom of the ZMWs* 1 bases incorporated per second

7 Sample Preparation Workflow Sample Preparation Building of the SMRTbell TM DNA Sample Fragment DNA Repair Ends Ligate Adapters Purify DNA Binding

8 Sample Preparation Workflow Genomic DNA Resequencing, de novo sequencing, metagenomics Resequencing, de novo sequencing, metagenomics WGA DNA Sample WGA Cancer Genomics FFPE (< 600 bp) Targeted Sequencing, Resequencing Targeted Enrichment Products Fragment DNA Repair Ends cdna Gene Expression, Transcriptome Profiling Ligate Adapters Purify DNA Binding

9 Single Molecule Prep Keeps Bias Low 2G SMRT

10 Flexibility of Sequencing with Multiple Protocols Sequence Standard Sample Preparation Large insert sizes Generates one pass on each molecule sequenced Strobe Very large insert sizes Generates distributed reads on each molecule sequenced Circular Consensus Small insert sizes Generates multiple passes on each molecule sequenced

11 Agenda for Today Technology Overview SMRT Applications

12 SMRT technology will enable comprehensive profiling by providing real time measurements RNA DNA PROTEIN animations by: wehi.edu.au

13 One of the first applications to leverage the power of SMRT was the real-time disease weather map

14 Fast Time to Result In collaboration with NYDOH and JCVI

15 The capability of SMRT sequencing to sequence the same molecule repeatedly leads to unprecedented accuracy Example: Influenza, H1N1 (A/New York/1682/2009), Fragment 7 (1,027 bp) 3 consecutive subreads: 12 kb combined read (4+ complete SMRTbell laps) 2.7 kb 4.1 kb 5.2 kb Full-length genomic segment consensus sequence: 100% accurate Example section:

16 Detection of Viral Pathogens from Inanimate Surfaces High-traffic surfaces at Pacific Biosciences: Front door handle Common laboratory bench top Break room refrigerator door handle Slide projector remote control Lavatory toilet flush handle Lavatory door handle Laboratory telephone handle Cubicle desk surface Money Sampled every week for a period of one month

17 Pacific Biosciences Volunteers Anonymous donors, submitting nasopharyngeal swabs every two weeks over ~2.5 months PACIFIC BIOSCIENCES CONFIDENTIAL

18 Detection of Viral Pathogens Influenza on Surfaces Multiple strains identified H1N1 H3N2 H2N2, H5N1 (rarely) Some samples with multiple strains present Some strains only occurred over single sampling period H5N1 1 H2N2 2 front door 1 fridge door 2 projector remote 1 projector remote 2 restroom door 1 restroom door 2 toilet flush handle 1 desk 2 desk 3 desk 8 $1* $1* Sampling period H1N1 H1N1/H3N2 H1N1/H3N2 H5N1 H1N1/H3N2 H5N1 H1N1 H2N2 H1N1/H2N2 H1N1/H3N2 H1N1 H1N1 H1N1/H3N2 H1N1/H3N2 H1N1/H3N2 H1N1/H3N2 H1N1 H1N1/H3N2 H1N1/H3N2 H1N1 H1N1 H3N2/H1N1 H1N1/H3N2

19 Rapid identification of the Haiti cholera outbreak strain World Health Organization 19

20 Typical Cholera Outbreak Patterns (Given by WHO in 2009) No outbreaks seen in nearly 100 years 20

21 Timeline Initiating the project (6-7 Nov) Sample prep and sequencing (8-12 Nov) Analysis (13-15 Nov) Writing the paper (15-19 Nov) Provisional acceptance (20-24 Nov) Formal acceptance (25 Nov 1 Dec) Paper Published!! (9 Dec) 6 Nov: Matt calls 7 Nov: PacBio decides to go 8 Nov: Matt s group cultures samples 10 Nov: Matt s group sends DNA 11 Nov: Sequencing begins 12 Nov: Sequencing of 5 genomes complete Data QC Assembly and variation detection Building phylogenetic trees Annotating structure variation regions PacBio first draft (16 Nov) Refined with editor input (18 Nov) Submitted to NEJM (19 Nov) Reviews received 22 Nov Decision indicating intent to publish 24 Nov Multiple revisions with editor (25-30 Nov) Official acceptance (1 Dec) 21

22 Single nucleotide variations unambiguously positioned the Haitian cholera strain next to South Asia strains Groups solidly within South Asia group, NOT Latin America group 22

23 What is the functional relevance of a complete genome: Resolving the cholera toxin region Cholera toxin prophage Adjacency to other elements define ability, for example to produce virions Knowing order of these elements in this region is critical to understanding infectivity, virulence and other parameters related to public health threat

24 Biological Hydrogen Production A potential transportation fuel for the future At present a major commodity chemical Ammonia production Hydrocracking of crude oil 2008 market for hydrogen = 10 billion kg projected market for hydrogen = 20 billion kg. An appealing aspect of hydrogen is that it can be produced by many different routes. Today, 98% of the world s hydrogen comes from fossil fuels.

25 R. palustris as an Efficient Path to Hydrogen Production Photosynthesis Rhodopseudomonas (image from ORNL) Carbon metabolism Hydrogenproduction involves hundreds of proteins in a web of molecular interactions We are seeking to understand this network to enhance hydrogen production

26 Collaboration with Carrie Harwood/UW: Characterize a population of strains (125 isolates from the wild) of Rhodopseudomonas Whole Genome sequencing: To reveal genetic variation between strains. Will serve as scaffold on which to map transcriptional activity. Digital, sequence-based transcriptome profiling: To reveal transcriptional output of strains grown under different hydrogen-producing conditions. Measure H 2 and nitrogenase activities: To reveal physiological potential of strains INTEGRATE!!! Probabilistic causal networks that drive production of hydrogen Experimentally validate computationally derived networks

27 Beyond the long reads and flexible configurations for a run lies the time dimension: Getting back to real time all the time Kinetic variation at a given site observed over multiple reads covering that site can be directly observed In R. pal. and other alpha-proteobacteria, the A residue in a GATC context can be methylated by the DNA adenine methylase enzyme (for gene regulation) Fluorescence intensity (a.u.) T C G A T C A A m A Time (s) RNA Fluorescence intensity (a.u.) G C T C G A TC A AG T A C A A DNA PROTEIN Time (s)

28 Beyond Long Reads and the A s, C s, G s, and T s by Detecting Kinetic Variation Regions that Affect Phenotype R pal genome covered by many reads Analyze R. pal seq data for kinetic variation 400 Fluorescence intensity (a.u.) Regions detected with little variation Vs. regions with a lot of variation Fluorescence intensity (a.u.) Time (s) Ratio of Rates 4000 bases Ratio of Rates Time (s) Genome Coordinate Genome Coordinate

29 Genes associated with kinetic variation that in turn associate with hydrogen production Regions detected with little variation Results: Ratio of Rates Ratio of Rates 4000 bases Regions detected with a lot of variation 1500 bases Thousands of sites detected with significant variation (1% FDR) AT residues in GATC context were > 12-fold enriched for kinetic variation! Regions of increased and decreased variation found Even without validation these data can be used as a covariate in QTL analyses (i.e., is kinetic variation region associated with H2 production) Genome Coordinate Nitrogenase genes

30 An Entire Network Identified that Affects Nitrogenase Activity and then Hydrogen Production as a Result

31 Structural differences differences in energy production Portion of the R. pal genome: Structural differences emerge in comparing these strains; structural differences drive the hydrogen network Structure of DX-1 Strain Structure of BIS3 Strain Expression level of key H2 x2.2 network drivers (log ratio) BIS3 genotype DX-1 genotype Small H2 output Structural QTL Genotype x2.1 in the H2 Network of Genes Big H2 output

32 If we want to succeed at this kind of game, we need the power of SMRT to provide a more accurate picture Long reads to assemble de novo genomes (not using whole genome sequencing for SNP detection) Long reads to fully characterize the transcriptome (fusion products, etc) Kinetic information to inform on the full epigenome Circular consensus to get at the most highly accurate base calls possible Quick time to results for maximal impact

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