FIND MEANING IN COMPLEXITY © Copyright 2013 by Pacific Biosciences of California, Inc. All rights reserved. Primer and Binding Calculator 

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 

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 

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 

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 

Sample Calculator Main Page 6 Create a new calculation View and edit saved calculations 

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 

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) 

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 

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 

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 

Dilute Polymerase for Binding Reaction 12. Dilute the DNA polymerase to the required working concentration (50 nM) 

Bind Polymerase to Templates 13. Two options : • Use the entire annealed SMRTbell™ template • Enter the # of SMRT® Cells you plan to use 

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 

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 

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 

Binding Complex to MagBead Prepare MagBead and bind diluted complex to washed MagBead. After binding, wash and load the sample on to instrument 

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. 

Loading Titration Reaction Setup 19 • Loading Titration section shows how to set up the required sequencing reactions at each loading concentration 

Print Calculations 20 To print, select the sample name and click the Print button 

Calculator v2.0: Print Options 21 Format your print-outs by selecting these options. 

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: 

Annealing, Binding and Loading Recommendations 

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 

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 

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 

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 

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 

Overloading Increases Yield of Mapped Read, But Reduces Read Length and Accuracy 29 Diffusion-loaded 2 kb lambda library 

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 

Where to Find More Information 31 http://www.smrtcommunity.com/SampleNet/Sample-Prep 

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