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Sample Calculator

PacBio
August 01, 2013

Sample Calculator

PacBio

August 01, 2013
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  1. FIND MEANING IN COMPLEXITY © Copyright 2013 by Pacific Biosciences

    of California, Inc. All rights reserved. Primer and Binding Calculator
  2. SMRTbell™ Template Preparation and Binding Steps Fragment DNA and Concentration

    DNA Damage Repair Repair Ends Ligate Adapters Purify Templates Primer Annealing Bind Polymerase Anneal Primer Bind Polymerase
  3. Calculator Versions Stand-Alone Version Instrument Version Recommended +++ (highly recommended)

    + How to Update https://github.com/PacificBiosciences/Sa mplePrepCalculator http://pap01- "InstrumentName"/Calculator Advantages Most up-to-date version Only updated with instrument SW Residence desktop/local hard drive Pap01/Blade Center Calculation Data Saved locally in your browser Saved locally in your browser 3
  4. Stand-Alone Calculator Requirements Windows Mac Firefox yes yes Chrome yes

    yes Safari yes yes 4 Firefox® Browser: HTML file must be saved in the same folder to retain old calculations Chrome®/Safari® Browser: Ability to find old calculations even if HTML file is stored in different folders Note: All 3 browsers should work on all platforms including iPad®, iPhone®, Android® devices, but not tested Web Browsers
  5. Annealing and Binding Calculator Main Page 5 Template Preparation Run

    Design Polymerase Binding Instrument Run Primary Analysis Secondary Analysis Tertiary Analysis Template Preparation Run Design Polymerase Binding Instrument Run Primary Analysis Secondary Analysis Tertiary Analysis Guides a user to: • Anneal primer to SMRTbell™ templates • Bind polymerase • Dilute samples for loading on the instrument • Bind polymerase-bound libraries to MagBeads
  6. Sample Preparation Compute Options Sample preparation compute options: • Volume

    to Use • # of SMRT® Cells • Loading Titration Tool tips are available for most of the options and input/output parameters in single sample view
  7. Enter Sample Information – Sample Name, Volume to Use, DNA

    Concentration and Insert Size 1. Enter the Sample Name 2. Enter the Volume of SMRTbell™ Library volume you plan to use 3. Enter the Concentration (ng/μL) of SMRTbell Template before annealing 4. Enter Insert Size (bp)
  8. Enter Sample Information – Details 5. Select Magnetic Beads (Yes/No)

    6. Select Binding Kit 7. Select Preparation Protocol : • Select ‘Small scale’ for DNA SMRTbell™ template concentrations less than 62 nM • Conversion Calculator provides (ng/µL ↔ nM) 6. DNA Control Complex (Yes/No) 7. Complex Reuse (Yes/No) • For diffusion loading only 8. Non-Standard Reaction (Yes/No) • Use this option if library sample is limiting
  9. Optional Parameter Settings 9. Use Default values for these options

    unless preparing for a custom sequencing reaction • Concentration On Plate • DNA Control Complex: Template Ratio • Polymerase:Template Ratio
  10. Setup Primer Annealing Reaction 10. Dilute the sequencing primer from

    5000 nM to 1000 nM in Elution Buffer provided by PacBio 11. Prepare annealing reaction using the instructions provided by the calculator
  11. Dilute Polymerase for Binding Reaction 12. Dilute the DNA polymerase

    to the required working concentration (50 nM)
  12. Bind Polymerase to Templates 13. Two options : • Use

    the entire annealed SMRTbell™ template • Enter the # of SMRT® Cells you plan to use
  13. Using Binding Complex for Sequencing • Two steps • Dilute

    complex per the desired loading concentration. Add DNA control if required • Bind complex to MagBead for loading
  14. Diluting the DNA Control Dilute the DNA Control Complex stock

    solution using the Bead Binding Buffer provided in the MagBead Binding Kit to prepare an intermediate working solution
  15. Diluting the Sample Complex Dilute the polymerase-bound DNA sample using

    the Bead Binding Buffer and add the diluted DNA Control Complex from the previous step
  16. Binding Complex to MagBead Prepare MagBead and bind diluted complex

    to washed MagBead. After binding, wash and load the sample on to instrument
  17. Loading Titration Compute Option 18 • Enter the desired titration

    experiment concentrations for up to four SMRT® Cells • For titrations, start with the default loading recommendation in the optional section. Include titration points for 1/2x and 2x of the default loading recommendation.
  18. Loading Titration Reaction Setup 19 • Loading Titration section shows

    how to set up the required sequencing reactions at each loading concentration
  19. Summary of Web-Based Calculator Features • Single sample view •

    List view • Print view Layout – 3 views are available • Calculator output is computed based on choosing one of the following options: 1) ‘Volume to Use’; 2) ‘# of SMRT Cells’; and 3)’Loading Titration’ • Sample information is entered one sample at a time in the single sample view • The information for multiple samples can be displayed in the print view. • Information for previously entered samples stored in database; can be edited at any time Sample information: • Loading titration: 1-4 target concentration points • Magnetic Beads: Yes or No • Binding Kit: P4 of C2 or XL Chemistry • Preparation protocol: Small or large scale • Complex Reuse option: Yes or No • Non-standard Reaction Setup: Yes or No Workflow options: • Custom on-plate sample concentration • Custom DNA control complex:template ratio • Custom polymerase:template ratio • Non-standard reaction setup option: Allows use of samples with low input DNA concentrations and small volumes Custom options:
  20. Primer Annealing Recommendations - P4 Binding Kit Small Scale Large

    Scale MagBead Diffusion MagBead Diffusion 250 NA 20x NA 2x 500 NA 20x NA 2x 1000 20x 20x 5x 5x 2000 20x 20x 10x 10x 5000 20x 20x 5x 5x 10000 20x 20x 5x 5x 24 The calculator automatically applies the above primer to template ratios
  21. Recommended P4 Polymerase to Template Ratios for Binding P4 Binding

    Kit Small Scale Large Scale MagBead Diffusion MagBead Diffusion 250 NA 2:1 NA 2:1 500 NA 2:1 NA 2:1 1000 10:1 3:1 2:1 3:1 2000 10:1 3:1 3:1 3:1 5000 10:1 3:1 3:1 3:1 10000 10:1 3:1 3:1 3:1 25 • The calculator automatically applies the above polymerase to template ratios • Values can be modified, but not recommended
  22. Loading Recommendations P4 Binding Kit (On-plate concentration, pM) Insert (bp)

    Small scale Large scale MagBead 1000 15.0 15.0 2000 15.0 15.0 5000 15.0 15.0 10000 15.0 15.0 Diffusion 250 112.5 112.5 500 300 187.5 1000 150 97.5 2000 180 150 5000 150 120 10000 225 150 26 • Calculator automatically applies the above on-plate concentrations • Highly recommend performing loading titration experiments to achieve optimal results
  23. PacBio® Recommends the Following: • When scaling up your sequencing

    runs, it is highly recommended to perform loading titrations – Use the default loading recommendation as one of your titration points – Avoid under-loading or over-loading of your samples • Use DNA Controls – Helps isolate issues associated with the samples and the instrument – Use the RS Dashboard to view DNA Control Metrics • Sequence samples within three days of binding 27
  24. Maximizing Yield Through Loading Titration 28 0 20000 40000 60000

    80000 100000 120000 140000 p0 p1 p2 15 pM on SMRT Cell 50 pM on SMRT Cell 150 pM on SMRT Cell 2 kb Lambda Library (P4), 55 minute movies
  25. Overloading Increases Yield of Mapped Read, But Reduces Read Length

    and Accuracy 29 Diffusion-loaded 2 kb lambda library
  26. Loading Conditions Affect Read Quality (RQ) and Accuracy Accuracy Mode

    = 92% (post-mapping) Accuracy Mode = 83% (post-mapping) RQ Mode = 0.91 (primary analysis) RQ Mode = 0.80 (primary analysis) Overloading shifts mode to the left Sample Loading Conc = 80 pM Sample Loading Conc = 300 pM 10 kb E.coli sequenced with previous version of polymerase (C2) and C2 chemistry
  27. Pacific Biosciences, the Pacific Biosciences logo, PacBio, SMRT, and SMRTbell

    are trademarks of Pacific Biosciences in the United States and/or other countries. All other trademarks are the sole property of their respective owners.