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Ultra-High Throughput DNA Sequencing on the 454/Roche GS-FLX

Ultra-High Throughput DNA Sequencing on the 454/Roche GS-FLX. Methods, Automation, Applications Graham Wiley Roe Lab. 100,000,000. 454/Roche GS-FLX. A Brief History of Automated DNA Sequencing Instruments. 454-GS20. 64,000,000. ABI 3730. ABI 370/377. ABI 3700. 2007.

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Ultra-High Throughput DNA Sequencing on the 454/Roche GS-FLX

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  1. Ultra-High Throughput DNA Sequencing on the 454/Roche GS-FLX Methods, Automation, Applications Graham Wiley Roe Lab

  2. 100,000,000 454/Roche GS-FLX A Brief History of Automated DNA Sequencing Instruments 454-GS20 64,000,000 ABI 3730 ABI 370/377 ABI 3700 2007

  3. 454 GSFLX Sequencer • Pico-scale sequencing reactions • 2 Core Techniques: • Emulsion PCR • Pyrosequencing

  4. Emulsion PCR • Micro-reactors • Water-in-oil emulsion generates millions of micelles. • Each micelle contains all reagents/templates for a PCR reaction. • ~10 Million individual PCR reactions in a single tube.

  5. Emulsion PCR

  6. 44 μm Load Beads into 454 Plate Load Enzyme Beads Load beads into PicoTiterPlate Centrifugation

  7. PP i Pyrosequencing DNA Bead dTTP • Polymerase adds • nucleotide (dNTP) (1) Polymerase A A T C G G C A T G C T A A A A G T C A T APS Annealed Primer (2) • Pyrophosphate • is released (PPi) Sulfurylase Luciferase ATP (3) • Sulfurylase creates ATP • from PPi and APS Enzyme Bead (5) luciferin (4) CCD camera detects bursts of light • Luciferase hydrolyses ATP • to oxidize luciferin and • produce light Light + oxy luciferin

  8. Pyrosequencing Output

  9. Base Calling via Flowgram TTCTGCGAA

  10. Types of Libraries • 454/Roche • Shotgun • Random 250+bp reads • Paired-End • 25-50bp ends of a circularized DNA molecule • Amplicon • PCR product for SNP discovery • Roe Lab • Paired-End/Shotgun • Best of both worlds

  11. Nebulization DNA End Repair 5’ 3’ 3’ 5’ 5’ 3’ Adaptor Ligation (A&B) 3’ 5’ A B DNA End Repair 5’ 3’ A B 3’ 5’ Library Quantification on Caliper 454 Shotgun Library Preparation Protocol Overview

  12. Shear to 2-4 Kbp fragments on the Hydroshear Quantitate on Caliper AMS-90 DNA End Repair & Linker Ligation Cleave the Terminal Linkers with EcoR1 Ligate to Circularized the DNA Shear to ~500 bp fragments in the Nebulizer 454 Paired End/Shotgun DNA Preparation Protocol Overview

  13. Quantitate on Caliper AMS-90 DNA End Repair, Adaptor Ligation, Adapter End Repair Amplification (emPCR) Pyrosequencing on 454/Roche GS-FLX 454 Paired End/Shotgun DNA Preparation Protocol Overview (cont)

  14. 454 Paired-End/Shotgun • Separate Sequences • Linker? • Left and Right? • ~3-5% • Triple Assembly • 100 flows, 84 flows, 62 flows • Convert Paired Ends for Exgap • *.454f and *.454r

  15. Automation of the Library Preparation Steps • Why automate? • Time • Reproducibility • What are the obstacles? • Reaction Cleanup • Qiagen Minelute centrifuge columns are difficult to automate, so replace those steps with • Agencourt SPRI magnetic beads and add a magnetic station to the Zymark SciClone bed • Enzyme Stability and Storage • Build an enzyme cooling station on the Zymark SciClone bed

  16. SPRI Bead Technology • Solid Phase Reversible Immobilization • Carboxyl coated magnetic particles suspended in a solution of 10% PEG and 1.25M NaCl • Reversibly binds DNA • Hawkins, et al. (1994) DNA purification and isolation using a solid-phase. Nucleic Acids Research, 22(21):4543-4544 http://www.agencourt.com/products/spri_reagents/ampure/

  17. DNA Purification through the Qiagen Minelute Columns vs Agencourt SPRI Magnetic Beads Agencourt SPRI magnetic beads Qiagen Minelute centrifuge column Both procedures give an almost similar yield but the yield is slightly better with the SPRI beads and the automation of the SPRI bead prep is somewhat easier to achieve

  18. 96 well Magnetic Plate for Purification of the SPRI Beads

  19. Enzyme Chilling Station

  20. Enzyme Mixes Waste EtOH Buffers Magnet Zymark SciClone Deck Arrangement Shaker Shaker Sample SPRI Beads Shaker

  21. Adding SPRI Beads on the SciClone

  22. Magnetically Separating SPRI Beads on the SciClone

  23. Washing SPRI Beads on the SciClone

  24. Applications • Whole Genome Sequencing • Sample Pools • BACs • Viruses • EST Libraries • Bacterial Communities

  25. Optimization of the DNA to Bead Ratios used in emPCR??? Xanthamonas campestris • ~5.0 MB genome • 47.9 MB total sequence • ~10 x coverage

  26. Xanthamonas campestris

  27. Heterosigma akashiwo Virus • 270-300 Kb Genome • 13Mb Sequenced • ~ 43x Coverage Contig Size Total Number Total Length % Of Cumulative 0 - 1 kb 1087 355308 55.1% 1 - 2 kb 26 33630 5.2% 2 - 3 kb 3 7983 1.2% 3 - 4 kb 1 3712 0.6% 4 - 5 kb 1 4533 0.7% 5 - 10 kb 1 8207 1.3% 10 - 20 kb 3 46888 7.3% 20 - 30 kb 1 27216 4.2% 30 - 40 kb 2 73763 11.4% 40 - 50 kb 2 83744 13.0% 50 - 100 kb 0 0 0.0% >100 kb 0 0 0.0% Cumulative 1127 644984 Consed_Err/10KB = 1186.29 Cumulative>1 kb 40 289676 Consed_Err/10KB = 213.7 Cumulative>2 kb 14 256046 Consed_Err/10KB = 30.07

  28. Pool 3 X Pool B Current BAC Pooling Strategy • 10x10 Grid of 100 BAC clones • 1-fold coverage of each pool of 10 150Kb BACs is 1.5 Mb • 1 quarter 454/Roche GS-FLX picotiter plate give ~13Mb or 10-fold cov. • 5 full picotiter plate runs are required for 20-fold coverage of each individual BAC at the horizontal/vertical intersect. • $12k/run = ~$600/BAC • Additional ABI 3730 runs are needed for each pool to aid in deconvolution at ~ $1000 for each of the 20 pools and an additional ~800/BAC or$1400 total cost per BAC

  29. Future Tagged BAC Pooling Strategy • 24 uniquely tagged individual shotgun libraries would be pooled and sequenced on one full 454/Roche GS-FLX picotiter plate • 24 150 Kb BACs would require 3.6 Mb for 1 x sequence coverage • With >75 Mb of DNA sequence obtained per full plate, >20x coverage is obtained for each of the 24 pooled BACs • 96 BACs would therefore require 4 full plate runs on the 454/Roche GS-FLX • At $12k/run = ~$500 per BAC for >20-fold shotgun coverage and no ABI 3630 runs are needed to deconvolute the individual BACs as each BAC is individually tagged

  30. Conclusions Graham- these are not conclusions, conclusions are of the form, we observed this and it means the following!! • New techniques lead to more useful data • It is possible to automate 454 preparation protocols • 454 pyrosequencing technology is incredibly powerful • Massive amounts of data • Technique advances continue to lower costs

  31. Acknowledgments

  32. Qiagen Minelute vs Agencourt SPRI • 50ul nebulized DNA • 20ul control • 15ul through Qiagen Minelute centrifuge column (final volume 25ul) • 15ul through Agencourt SPRI beads (final volume 25ul) Control

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