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Signal Generation and Imaging

Photons Detected by Camera. 1600K field of addressable wells. PicoTiterPlate Wells. Photons Generated. Reagent Flow. Sequencing By Synthesis. Signal Generation and Imaging. Spectral Instruments Series 800 Camera with Fairchild Imaging LM485 CCD (4096x4096,

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Signal Generation and Imaging

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  1. Photons Detected by Camera 1600K field of addressable wells PicoTiterPlate Wells PhotonsGenerated Reagent Flow Sequencing By Synthesis Signal Generation and Imaging • Spectral Instruments Series 800 Camera with Fairchild Imaging LM485 CCD (4096x4096, • 15 μm pixels), directly bonded to a 1:1 imaging fiber bundle; cooled to -25 °C • PTP in direct contact with imaging fiber bundle (no alignment or focusing issues); NA ~ 0.75 • Full-frame imaging mode; read-out during wash (dark portion of flow cycle) Proprietary

  2. Image Processing Raw data - series of images T C G A T dNTP Base Addition Data converted into flowgrams” Proprietary

  3. TACG Flow Order 4-mer 3-mer TTCTGCGAA 2-mer 1-mer Signal-Processing & Basecalling Image Data → Signal processing Pipeline → Flowgrams → Quality Filtering →HQ Reads →Basecalling → HQ Bases → Mapping & Assembly Key sequence = TCAG for well identification and calibration Proprietary

  4. Mapping in Flowgram Space Reference Chromosome Flowgram …,1, 3, 1, 0, 0, 2, 2, 0, 0, 1, 2, 3, 0, 1, 0… Fragment Flowgrams (RN) …, 1.00, 3.14, 0.15, 0.20, 0.21, 1.84, 1.95,… • Re-sequencing and find variants to the reference genome Proprietary

  5. Fragment Flowgrams (RN) De Novo Assembly in Flowgram Space Overlap to form contigs • Draft sequences of new genomes (species that have not been sequenced before) Proprietary

  6. Potential Sources of Error Non-DNA Well DNA Well True signal False signal Flow PPi Non-DNA Well DNA Well False signal True signal Optical Cross-Talk • Light penetrates to adjacent fibers; leads to signal contamination Chemical Cross-Talk • PPi / ATP produced in DNA well appears in down-stream non-DNA wells due to • convection / diffusion; leads to signal contamination Proprietary

  7. Original Filtered (51x51) kernel 2D intensity contour Pixel Pixel Signal-Processing Solution Filtering removes signal cross-contamination Proprietary

  8. Typical GS20 Run Results • 42 Flow Cycles • ~ 30MB per run • ~ 300,000 reads • ~ 100 bp per read • > 50% wells error-free • ~ 1 % individual read error Proprietary

  9. 2 -GTGCGCGCGCGGGACTAATCCCGGTTCGCGCGTCGGGCATGACACGCAAC- Consensus Accuracy > 99.99+% when 10x over-sampling Example Ref / assembled genome: Read #: FlowSignal Read 1: 2.52 Read 2: 1.95 Read 3 2.11 Read 4: 1.53 Read 5: 1.32 Read 6: 2.14 Read 7: 2.06 Read 8: 1.85 Read 9: 2.21 Read 10: 2.17 Consensus (mean): 1.99 10 reads aligned to this position Proprietary

  10. De Novo Assembly Results • 4 runs of GS20 (E. Coli 4,639,675 bp) • Each data point represents 1/2 GS20 run 1 run 2 runs 3 runs 4 runs Proprietary

  11. Genome Sequencer FLX Recently Launched Next Generation Sequencing Platform in Q1, 2007 in collaboration with Roche Diagnostics • Improved fluidics for faster reagent delivery • Firmware control of reagent delivery & camera timing • On-board reagent dilution • Optimized biochemistry • Improved algorithms with corrections for • Crosstalk (for higher densities) • Signal droop & Phasing • Numerical filter for improved rejection of low quality reads • At least 400K (200K) reads of avg. 250 bp (100 bp) • 8 hours (5 hours) run time • Single read avg accuracy >99.5% (99%) over 200 (100) bases • Consensus read accuracy > 99.99% • Avg yield from single run ~ 100 Mb (20-30 Mb) (GS20 performance) Proprietary

  12. Fluidics Modification – Air Plug Insertion Camera cover De- bubbler G A C Air Concentration Profile with & w/o air bubble air plug in tube mM t air bubbles removed at debubbler Proprietary

  13. Whole Genome Sequencing Results from GS FLX E. Coli (50% GC) C. jejuni (35%GC) T. thermophilus (71% GC) Proprietary

  14. E. coli run #1 E. coli run #2 E. coli run #3 E. coli run #4 T. thermophilus C. jejuni Reported in Nature 2005 GS20 Q2 2006 Observed Individual Read Accuracy • All blind-filtered reads (no reference genome required) Proprietary

  15. Newbler™ Assembly Results from GS FLX Proprietary

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