Digital PCR Analysis Software version 3

Transcription

1 Digital PCR Analysis Software version 3 User Guide Fluidigm Corporation. All rights reserved. DRAFT August 6, :27 pm, Digital PCR_Title.fm

2 Limited License and Disclaimer for Fluidigm Systems with Access Array IFCs, Dynamic Array IFCs and Digital Array IFCs Except as expressly set forth herein, no right to copy, modify, distribute, make derivative works of, publicly display, make, have made, offer to sell, sell, use, or import a Fluidigm system or any other product is conveyed or implied with the purchase of a Fluidigm system (including the BioMark system, RT1 System, EP1 system, FC1 Cycler, or any components thereof), Access Array IFCs, Dynamic Array IFCs and Digital Array IFCs (integrated fluidic circuits/microfluidic chips with or without a carrier), IFC controller, software, reagents, or any other items provided hereunder. This limited license permits only the use by the buyer of the particular product(s), in accordance with the written instructions provided therewith in the User Guide, that the buyer purchases from Fluidigm or its authorized representative(s). Except to the extent expressly approved in writing by Fluidigm or its authorized representative(s), the purchase of any Fluidigm product(s) does not by itself convey or imply the right to use such product(s) in combination with any other product(s). In particular, (i) no right to make, have made, or distribute other instruments, Access Array IFCs, Dynamic Array IFCs and Digital Array IFCs, software, or reagents is conveyed or implied by the purchase of the Fluidigm system, (ii) no right to make, have made, import, distribute, or use the Fluidigm system is conveyed or implied by the purchase of instruments, software, reagents, or Access Array IFCs, Dynamic Array IFCs and Digital Array IFCs from Fluidigm or otherwise, and (iii) except as expressly provided in the User Guide, the buyer may not use and no right is conveyed to use the Fluidigm system in combination with instruments, software, reagents, or dynamic arrays and digital arrays unless all component parts have been purchased from Fluidigm or its authorized representative(s). For example, purchase of a Fluidigm system and/or the IFC controller conveys no right or license to patents covering the Access Array IFCs, Dynamic Array IFCs and Digital Array IFCs or their manufacture, such as 5,376,252, 6,408,878, 6,645,432, 6,719,868, 6,793,753, 6,929,030; and EP Patent Nos and Fluidigm Access Array IFCs, Dynamic Array IFCs and Digital Array IFCs delivered hereunder are licensed for one (1) time use and may not be reused, refurbished, or resold. All Fluidigm products are licensed to the buyer for research use only. The products do not have FDA or other similar regulatory body approval. The buyer may not use the Fluidigm system, any component parts thereof, or any other Fluidigm products in any setting requiring FDA or similar regulatory approval or exploit the products in any manner not expressly authorized in writing by Fluidigm in advance. No other licenses are granted, expressed or implied. Please refer to the Fluidigm website at for updated license terms. Fluidigm, the Fluidigm logo, BioMark, RT1, EP1, FC1, Access Array, Dynamic Array, and Digital Array are trademarks or registered trademarks of Fluidigm Corporation in the U.S. and/or other countries. All other trademarks are the sole property of their respective owners. Recommended Reagents Fluidigm recommends that you use only TaqMan dual-labeled probes and/or other licensed PCR assay reagents from authorized sources. If you have any questions regarding whether you have a license to use particular reagents in PCR systems, you should contact the appropriate licensor and obtain clarification and their permission if necessary. For example, certain probes and their use may be covered by one or more patents held by Applied Biosystems and/or Roche Molecular Systems, which may be contacted at the Director of Licensing at Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California or the Licensing Department, Roche Molecular Systems, Inc., 1145 Atlantic Avenue, Alameda, California Fluidigm Product Patent Notice Fluidigm products including IFCs (integrated fluidic circuits/microfluidic chips with or without a carrier) such as Access Array IFCs, Dynamic Array IFCs and Digital Array IFCs, the IFC controller, FC1 Cycler and the Fluidigm system (BioMark system, EP1 system, RT1 system, readers, thermal cycler, etc.) and methods for reading and controlling the Access Array IFCs, Dynamic Array IFCs and Digital Array IFCs and/or their use and manufacture may be covered by one or more of the following patents and patent applications owned by Fluidigm Corporation and/ or sold under license from California Institute of Technology, Gyros AB, and other entities: U.S. Patent Nos. 5,376,252, 6,408,878, 6,645,432, 6,719,868, 6,793,753, 6,929,030, 7,195,670, 7,216,671, 7,307,802, 7,323,143, 7,476,363, 7,588,672, 7,704,735 7,749,737; EP Patent Nos and ; and additional issued and pending patents in the U.S. and other countries.

3 Please refer to the User Guide for certain license terms for Fluidigm system products and the Fluidigm website at for updates. [Version 4.2] Every effort has been made to avoid errors in the text, diagrams, illustrations, figures, and screen captures. However, Fluidigm assumes no responsibility for any errors that may appear in this publication. It is Fluidigm s policy to improve products as new techniques and components become available. Therefore, Fluidigm reserves the right to change specifications at any time. No right to modify, copy, use, or distribute Fluidigm software is provided except in conjunction with the instrument delivered hereunder and only by the end user receiving such instrument. In cases where this software and the associated instrument are beta test systems NO WARRANTIES ARE PROVIDED, EXPRESSED OR IMPLIED. ALL WARRANTIES, INCLUDING THE IMPLIED WARRANTIES OF FITNESS FOR PURPOSE, MERCHANTABILITY, AND NON-INFRINGMENT ARE EXPRESSLY DISCLAIMED. By continuing the installation process, user agrees to these terms. Please refer to the full text of the software license agreement supplied with the installation media for this application. Contacting Fluidigm For Technical Support, send to: TechSupport@fluidigm.com Phone in United States: FLUIDLINE ( ) Outside the United States: On the Internet: Fluidigm Corporation 7000 Shoreline Court, Suite 100 South San Francisco, CA PN , Rev. D

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5 Contents Chapter 1 System Overview Digital PCR Overview Quantifying Target Nucleic Acid Absolute Quantitation Quantifying Samples for DNA Sequencing Copy Number Variation Efficient Drug Metabolism Loss of Heterozygosity New Applications System Components Digital Array IFCs System Process Overview Digital Array IFC Pipetting Map Digital Array IFC Pipetting Map What You Need for Experiments Reagents Supported Detection Reagents PCR Master Mixes Sample Requirements DNA Quality DNA Storage Preparing the Fluidigm Reaction Mix for a Digital Array IFC Preparing the Fluidigm Reaction Mix for a Digital Array IFC Loading the Digital Array IFC Loading the Digital Array IFC Chapter 2 Using the Digital PCR Analysis Software Launching the Digital PCR Analysis Software Opening an Existing Chip Run Finding Corners Manually Forced Manual Corner Find Setting Up Sample Information Using the Dispense Map Editor Using Sample Mapping Setting Up Assay (Detector) Information Digital PCR Analysis Software User Guide v

6 Using Sample and Detector Set Up Real-time Applications (for BioMark system only) Using the Task Pane Overview of Chip Explorer Viewing Chip Run Results Using Panel Summary Creating a New Chip Run from an Analyzed Run Create a New chip Run Convert a Real-time Run to an End Point Run Chapter 3 Performing End Point Analysis Runs Introduction Overview of Chip Explorer Secondary View for Panel Summary Secondary Views for Panel Details Using the Secondary View Toolbar Viewing Chip Run Results Using Panel Summary Go to Panel Results Table Image View (Panel Details only) Heat Map: Hit Color View Using the Task Pane Exporting User-defined Threshold Settings Chapter 4 Generating Reports and Exporting Data Using the Tool bar Options Feature Output Chip Color Scheme No Call Color Selection Application Template Generating Reports Chip Run Preparation Report Chip Analysis Report Exporting Chip Run Data Exporting Data Appendix A vi Digital PCR on the Array Workflow Digital PCR Workflow Priming the Digital Array IFC Preparing Sample Pre-Mix and Samples Loading the Chip Using the Data Collection Software Digital PCR Analysis Software User Guide

7 Using the Digital PCR Analysis Software Appendix B Digital PCR on the Workflow Digital PCR Workflow Priming the Digital Array IFC Preparing Sample Pre-Mix and Samples Loading the Chip Using the Data Collection Software Using the Digital PCR Analysis Software Appendix C Copy Number Variation Advanced Development Protocol Digital Array IFC Copy Number Variation Advanced Development Protocol Introduction Calculation Determining DNA Copy Number References Index Digital PCR Analysis Software User Guide vii

8 viii Digital PCR Analysis Software User Guide

9 System Overview 1 1 In this chapter: Digital PCR Overview Quantifying Target Nucleic Acid Absolute Quantitation Copy Number Variation Efficient Drug Metabolism Loss of Heterozygosity New Applications System Components Digital Array IFCs Digital Array IFC Pipetting Map What You Need for Experiments Reagents Supported Detection Reagents PCR Master Mixes Sample Requirements DNA Quality DNA Storage Preparing the Fluidigm Reaction Mix for a Digital Array IFC Preparing the Fluidigm Reaction Mix for a Digital Array IFC Loading the Digital Array IFC Loading the Digital Array IFC Digital PCR Analysis Software User Guide 9

10 System Overview Digital PCR Overview Digital polymerase chain reaction (dpcr) is a technique that allows researchers to quantify nucleic acid sequences that are present in a DNA sample. Digital PCR typically relies on standard PCR techniques but increases their sensitivity by dividing a sample into hundreds of smaller reactions and performing a PCR assay on each such sample. Digital Array IFCs (integrated fluidic circuits) run on the BioMark /EP1 system are being used to quantify target nucleic acid sequences and DNA sequences, and study copy number variation (CNV), drug metabolism, the loss of heterozygosity, and rare mutations. Quantifying Target Nucleic Acid Absolute Quantitation A BioMark/EP1 system with Digital Array IFCs can be used to quantify target nucleic acid sequences. The Digital Array IFC accepts serial dilutions of sample mixed with PCR reagents and finely partitions each dilution within separate, onchip grids of reaction chambers. A signal is produced only in chambers containing copies of the target sequence, the positives in each chamber grid are easily counted and evaluated for statistical significance. Thus, the Digital Array IFC provides a practical means to determine absolute quantitation by the limiting dilution method. Quantifying Samples for DNA Sequencing Fluidigm offers a sample quantification kit, called the SlingShot Kit, that can be used with the or the Digital Array IFCs to increase the efficiency of next-generation sequencing. SlingShot Kit uses digital PCR assays to detect only amplifiable molecules within the sample mixture. This absolute measure eliminates the need for mass-based calibrators or reference samples. In addition, because of digital PCR sensitivity, only minimal sample concentrations are required to achieve quantitative results. See Advanced Development Protocols ADP Library Absolute Quantitation Using the Digital Array IFC (PN ) or ADP 22 Illumina Library Absolute Quatitation Using the Digital Array IFC (PN ). Copy Number Variation The Digital Array IFC allows simultaneous quantification of both the gene of interest and a reference gene by using a different fluorescent dye for each assay. The ratio of the signal is the relative copy number of the gene of interest in the sample. See Appendix C: Copy Number Variation Standard Operating Procedure for more detail on copy number variation. 10 Digital PCR Analysis Software User Guide

11 Digital PCR Overview Efficient Drug Metabolism The system enables highly efficient drug metabolism studies by allowing accurate copy number analysis using Digital Array IFCs. See Application Note Efficient Drug Metabolism Studies Using the BioMark System, part number MRKT00081, for more information. Loss of Heterozygosity Loss of Heterozygosity (LOH) refers to a change from state of heterozygosity in a normal genome to a homozygous state in a paired tumor genome. Research shows the loss of an entire X chromosome is involved in numerous cancers. See Application Note Enabling Loss of Heterozygosity Studies using Fluidigm Digital Array IFCs, part number MRKT00082, for more information. New Applications Fluidigm is continuing to develop new applications for the Digital Array IFC. For upto-date application information, please visit the Fluidigm website: Digital PCR Analysis Software User Guide 11

12 System Overview System Components BioMark System EP1 Reader IFC Controller MX FC1 Cycler Digital Array IFCs The Digital Array IFC is an efficient solution for absolute digital PCR. The key to this efficiency is the matrix of channels, chambers, and integrated valves finely patterned into layers of silicone. This PDMS material is gas permeable, allowing the blindfill of fluids into valve-delimited chambers. For the Digital Array IFC, the valves partition samples into 765 chambers in each of the 12 panels (765 chambers x 12 panels = 9,180 reactions per chip). For the Digital Array IFC, the valves partition samples into 770 chambers in each of the 48 panels (770 chambers x 48 panels = 36,960 reactions per chip). 12 Digital PCR Analysis Software User Guide

13 Digital Array IFCs Digital Array IFC Sample Inlets Elastomeric Device Hydration Inlets Hydration Inlet Do NOT use Digital Array IFC Sample Inlets Do NOT use Hydration Inlet Elastomeric Device Digital PCR Analysis Software User Guide 13

14 System Overview System Process Overview The simplicity of running experiments is illustrated in the process below. For more detail, see Digital PCR Workflow Quick Reference Card, PN , or the Digital PCR Workflow Quick Reference Card, PN Prime the Digital Array IFC. 2 Pipette the sample mixtures into the Digital Array IFC. 3 Load sample-assay mix using the IFC Controller MX. 4 Run experiment on the BioMark system or EP1 reader Digital Array IFC Pipetting Map Containment Accumulator Hydration Inlet A1 Do NOT use panels of the elastomeric device Sample Inlets Do NOT use Hydration Inlet 14 Digital PCR Analysis Software User Guide

15 Digital Array IFC Pipetting Map Digital Array IFC Pipetting Map A1 notch Sample Inlets Add 10 µl of 1X GE Sample Loading Reagent into all 16 hydration inlets What You Need for Experiments This section describes the materials that you need to perform your experiments including reagents we support and sample requirements. In addition, you need the following: BioMark System or EP1 Reader and FC1 Cycler IFC Controller MX or Digital Array IFC Control line fluid syringes Reagents Store at 4 C 2X TaqMan Gene Expression Master Mix (Applied Biosystems, PN ) 20X GE Sample Loading Reagent (Fluidigm, PN ) 20X TaqMan Assays Store at room temperature DNA-free, DNase-free, RNase-free water Digital PCR Analysis Software User Guide 15

16 System Overview Supported Detection Reagents We support the following detection reagents 1 with the system. Probe Types FAM-MGB VIC-MGB FAM-TAMRA FAM-non fluorescent quencher 2 Additional Probe Types Fluidigm does not support other probe types at this time. However, additional probe types may be run with the System using the following guidelines: Fluorophores With... Excitation Wavelengths Emission Wavelengths between 465 and 505 nm And between 500 and 550 nm between 510 and 550 nm And between 540 and 600 nm PCR Master Mixes The protocol described in this quick start guide uses Gene Expression Master Mix (Applied Biosystems, PN ). If you choose to use another master mix, you may have to alter the protocol described in this manual. Contact Fluidigm Technical Support for additional information. IMPORTANT: You must use a passive reference (ROX). 1. Fluidigm recommends that you use onlytaqman probes and/or other licensed PCR assay reagents from authorized sources. If you have any questions regarding whether you have a license to use particular reagents in PCR systems, you should contact the appropriate licensor and obtain clarification and their permission if necessary. For example, certain probes and their use may be covered by one or more patents held by Applied Biosystems and/or Roche Molecular Systems, which may be contacted at the Director of Licensing at Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California or the Licensing Department, Roche Molecular Systems, Inc., 1145 Atlantic Avenue, Alameda, California Contact Fluidigm technical support to discuss your non-fluorescent quencher requirements. 16 Digital PCR Analysis Software User Guide

17 Sample Requirements Sample Requirements DNA Quality DNA should have an 260:280 ratio between 1.5 and 1.8. Prior to use on a chip, monitor the integrity of your DNA on a system such as the Agilent 2100 bioanalyzer. DNA Storage Avoid multiple freeze-thaw cycles by storing DNA at 4 C. For longer storage, aliquots may be stored at -20 C. Digital PCR Analysis Software User Guide 17

18 System Overview Preparing the Fluidigm Reaction Mix for a Digital Array IFC To make the reaction mix, combine the following, in a DNA-free hood: Component Volume per Panel (µl) Volume per Chip (µl) (equals 13 reactions) Combine the following using a sterile tube for each chip to be loaded: TaqMan Gene Expression Master Mix (Applied BioSystems, PN ) 20x GE Sample Loading Reagent (Fluidigm, PN ) DNA-free water X assays DNA 0.8 Total µl NOTE: Adjust the DNA and water volumes as needed. 1 In a DNA-free hood, combine the TaqMan Gene Expression Master Mix, 20x GE Sample Loading Reagent, DNA-free water and 20X assay in a 1.5 ml sterile tube enough volume to fill the entire chip. 7.2 µl of this Sample Pre- Mix can then be aliquoted for each sample (12 total). 2 Remove these aliquots from the DNA-free hood and add 0.8 µl of DNA to each, making the total volume 8 µl in each aliquot. 18 Digital PCR Analysis Software User Guide

19 Preparing the Fluidigm Reaction Mix for a Digital Array IFC Preparing the Fluidigm Reaction Mix for a Digital Array IFC To make the reaction mix, combine the following, in a DNA-free hood: Component Volume per Panel (µl) Volume per Chip (µl) (equals 48 reactions) Combine the following using a sterile tube for each chip to be loaded: TaqMan Gene Expression Master Mix (Applied BioSystems, PN ) 20x GE Sample Loading Reagent (Fluidigm, PN ) DNA-free water X gene-specific assays DNA 1.2 Total µl NOTE: Adjust the DNA and water volumes as needed. 1 In a DNA-free hood, combine the TaqMan Gene Expression Master Mix, 20x GE Sample Loading Reagent, DNA-free water and 20X assays in a sterile tube enough volume to fill the entire chip. 2.8 µl of this Sample Pre-Mix can then be aliquoted for each sample (48 total). 2 Remove these aliquots from the DNA-free hood and add 1.2 µl of DNA to each, making the total volume 4 µl in each aliquot. Digital PCR Analysis Software User Guide 19

20 System Overview Loading the Digital Array IFC 1 Place the IFC inot the IFC Controller MX and run the Prime (115x) script. 2 When the Prime (115x) script has finished, remove the primed chip from the IFC controller. 3 Pipette 8 µl sample mix into the Samples inlets on the chip. 4 Pipette 10 µl H 2 O into the hydration inlets. 5 Return the chip to the IFC controller. 6 Using the IFC controller software, run the Load (115x) script to load the samples into the chip. 7 When the Load (115x) script is complete, remove the chip from the IFC controller. 8 Peel the blue protective film from the underside of the loaded chip. 9 Remove any dust particles or debris from the chip surface. 10 Place the chip into the BioMark System or FC1 Cycler. For more detail about the Digital Array IFC, please see Appendix A. Loading the Digital Array IFC 1 Place the IFC into the IFC Controller MX and run the Prime (148x) script. 2 When the Prime (148x) script has finished, remove the primed chip from the IFC controller. 3 Pipette 10 µl of 1X GE Sample Loading Reagent into all hydration inlets on the chip. 4 Pipette 4 µl of sample mix into the sample inlets on the chip. 5 Return the chip to the IFC controller. 6 Using the IFC controller software, run the Load (148x) script to load the samples into the chip. 7 When the Load (148x) script is complete, remove the chip from the IFC controller. 8 Peel the blue protective film from the underside of the loaded chip. 9 Remove any dust particles or debris from the chip surface. 10 Place the chip into the BioMark System or FC1 Cycler. For more detail about the Digital Array IFC, please see Appendix B. 20 Digital PCR Analysis Software User Guide

21 Using the Digital PCR Analysis Software 2 2 In this chapter: Launching the Digital PCR Analysis Software Opening an Existing Chip Run Finding Corners Manually Using the Dispense Map Editor Using Sample Mapping Setting Up Assay (Detector) Information Using Sample and Detector Set Up Real-time Applications (for BioMark system only) Using the Task Pane Overview of Chip Explorer Viewing Chip Run Results Using Panel Summary Viewing Chip Run Results Using Panel Summary Convert a Real-time Run to an End Point Run Digital PCR Analysis Software User Guide 21

22 Using the Digital PCR Analysis Software Launching the Digital PCR Analysis Software NOTE: The Digital PCR Analysis software supports more colors than the standard configuration of the instrument. For explanation purposes, up to four colors are shown in this manual. For additional information on system configuration options, please contact Fluidigm Technical Support. 1 Double-click the Digital PCR Analysis software icon on the desktop to launch the software. Opening an Existing Chip Run 1 Click opening an existing chip run in the Chip Run Summary pane or, click Open a Chip Run in the Task pane or, File > Open. 22 Digital PCR Analysis Software User Guide

23 Opening an Existing Chip Run 2 Double-click the chip run file (.bml extension). A progress dialog box opens. The chip run file opens in the Chip Explorer pane. 3 Click Panel Summary. Digital PCR Analysis Software User Guide 23

24 Using the Digital PCR Analysis Software 4 If you change any parameters, click Analyze. NOTE: A reminder to analyze the chip run appears on the lower left status bar. IMPORTANT: You must click Analyze in the task pane each time you change parameters. A reminder dialog box opens, as well as a reminder in red text, if you fail to click Analyze after each change. The first time a chip is analyzed, the chamber-finding algorithm locates the chamber boundaries of each captured image. Thus, this is the most lengthy step in the entire analysis procedure. Continue with Setting Up Sample Information on page Digital PCR Analysis Software User Guide

25 Finding Corners Manually Finding Corners Manually Fluidigm requires putting passive reference dye (ROX) into all reactions on the chip. Failing to add ROX to all cells may require you to manually find the corners. The following steps describe this process. If this occurs, you can manually set the corners and then analyze the chip. 1 Click OK. 2 Zoom in to see the corner cells. NOTE: If you cannot see the four corner cells, adjust the Contrast slider. (If an insufficient amount of ROX dye was used in setting up the chip corners, it will be difficult to see the corner cells.) 3 Move the red cross hairs to each of the four corner cells. Contrast slider Red cross hairs 4 Make sure each cross hair is on the outer edges of each corner cell (see below). Upper left corner Upper right corner Lower left corner Lower right corner Digital PCR Analysis Software User Guide 25

26 Using the Digital PCR Analysis Software 5 Click Done. NOTE: If no ROX is present, the corner cells are very dark. You may have to count the number of rows and columns to make sure you are placing cross hairs correctly. Forced Manual Corner Find If the automated manual corner find results are not satisfactory, you can perform a forced manual corner find by pressing the Ctrl key and simultaneously clicking on the Analyze button. This only works for a chip run that has not been processed. Setting Up Sample Information The Digital Array IFC requires dispensing of a sample and detector mix into the chip inlets either directly or via an SBS well plate. In the Digital PCR Analysis software you can annotate this information in the combined view Sample and Detector Setup or in the separate views Sample Setup and Detector Setup. Modifying the dispensing setup in one view applies the changes to all of the views. Set up your sample information to match the actual sample wells you used in your test. 1 Click Sample Setup in the Chip Explorer pane. 2 Click New. Alternatively, click Import to open a saved sample list (.plt or.csv extensions). If the sample plate has been set up previously, an alert dialog box opens: 3 Click Yes. The Sample Plate Setup Wizard opens. Container type drop-down menu options: SBS Plate: the sample and detector mixes are dispensed into the chip from a source SBS well plate. If you select SBS Plate (for use with Digital Array IFCs only), you have two plate choices: SBS 96 SBS Digital PCR Analysis Software User Guide

27 Setting Up Sample Information Sample Inlets: the sample and detector mixes are dispensed directly into the chip inlets. 4 Select container type and format. 5 Click OK. 6 Click the Sample Mapping button (located on the Task pane) to open the Sample Mapping File dialog box if dispensing is from a source SBS plate. 7 From the Sample Mapping File dialog box, select either the Left or Right dispense map. NOTE: If a dispensing map does not exist for the method of dispensing the chip run requires, you can create a new dispensing map using the Dispensing Map Editor. See Using the Dispense Map Editor on page Highlight the cells you wish to name by using one of the following methods: Click and drag Press Ctrl key while clicking individual cells Click the corner square to select all cells 9 Click the Editor button to open the Sample Editor. Digital PCR Analysis Software User Guide 27

28 Using the Digital PCR Analysis Software 10 Select Unknown, NTC, Blank or Control in the Sample Type drop-down menu. 11 Enter a Sample Name. 12 Enter a Sample Relative Concentration. 13 Click Update. The highlighted cells reflect the changes you made. NOTE: The new parameters are not saved when you click Update. To save, click File > Save. If you attempt to exit the application without saving, a warning dialog box opens, as shown below. Using the Dispense Map Editor Use the Dispense Map Editor to set dispensing map parameters and to record custom load maps for future use. After recording your loading sequence, you can save it and play it back anytime. 1 Click Tools > Dispense Map Editor. 2 Click File > New in the Dispense Map Editor. The New Dispense Map dialog box opens. 28 Digital PCR Analysis Software User Guide

29 Using the Dispense Map Editor 3 Complete the New Dispensing Map dialog box using the following example as a guide. Unique experiment name or chip barcode. Relevant characteristics of the experiment (optional). (148x) 4 Click OK to open the new dispense map in the Dispense Map Editor. Source Plate - graphical representation of the plate from which the samples and/or assays are pipetted. Target Plate - graphical representation of the plate into which the samples/assays are pipetted. Dispense Map - This table shows you where the samples and assays are on the chip. Digital PCR Analysis Software User Guide 29

30 Using the Digital PCR Analysis Software 5 Click the Begin Editing button in the Recording Control pane. a Click the first cell from the Source Plate. Then, click the location in the Target Plate. b Continue clicking appropriate cells until your custom loading map has been recorded. NOTE: When you click Begin Editing, the dispensing pane becomes inactive. 6 Click Stop Editing. Using Sample Mapping After setting up the sample plate, view and/or record the loading pattern in the Sample Mapping Viewer. 1 Click Mapping Viewer in the chip explorer pane. The dispensing map opens. 30 Digital PCR Analysis Software User Guide

31 Setting Up Assay (Detector) Information 2 Click a cell in the Source Plate to see where it loads on the Target Plate. Setting Up Assay (Detector) Information The Digital Array IFC requires dispensing of a sample and detector mix into the chip inlets either directly or via an SBS well plate. In the Digital PCR Analysis software, you can annotate this information in the combined view Sample and Detector Setup or in separate views Sample Setup and Detector Setup. Modifying the dispensing setup in one view applies the changes to all of the views. Set up your assay information to match the actual assay wells you used in your test. 1 Click Detector Setup in the Chip Explorer pane. 2 Click New. Alternatively, click Import to open a saved detector list (.plt or.csv extensions). If the plate has been set up previously, an alert dialog box opens: 3 Click Yes. The Detector (Assay) Plate Setup Wizard opens. 4 Select a container type and a container format in the drop-down menus. 5 Click OK. Digital PCR Analysis Software User Guide 31

32 Using the Digital PCR Analysis Software 6 Click the Detector (Assay) Mapping button to open the Detector (Assay) Mapping file dialog box. 7 From the Sample Mapping file, select either Left or Right dispense map. 8 Highlight the cells that you are going to name using one of the following: Click and drag. Press Ctrl while clicking individual cells. Click the corner of a square to select all cells. 9 Click Editor to launch the Assay Editor. 10 In the Assay Editor, type: Assay name Assay type 11 Click Update. Using Sample and Detector Set Up Alternately, you can use the Sample and Detector Setup option and annotate both at the same time. 1 Click on Sample and Detector Setup in the Chip Explorer Pane. 2 Click Editor. The Sample & Detector Editor window appears. 32 Digital PCR Analysis Software User Guide

33 Using Sample and Detector Set Up In this example, we change the sample type to NTC. The first detector type to Test. And the second detector type to Blank. Click Update to see the results, shown below. Type unique names for sample and detectors An experimental sample. No Template Control (negative control): everything included except the sample; to show that a positive result cannot be obtained when the sample is left out. An unused position. (Deionized water in the chamber.) A standard against which other conditions are compared Perform this procedure for each of the 12 panels. 3 Click Update after annotating samples and detectors in the Sample and Detector Editor. Changes are reflected in the Sample and Detector Setup pane, shown above. Alternately, use the right-click menu to copy and paste the complete setup information from one panel to another panel as shown below. Digital PCR Analysis Software User Guide 33

34 Using the Digital PCR Analysis Software 1. Highlight the panel to be copied and then click Copy from the Right-click menu. 2. Highlight the panel to be pasted into and then click Paste from the Right-click menu. Both panels now have the same sample and detector information. 34 Digital PCR Analysis Software User Guide

35 Real-time Applications (for BioMark system only) Real-time Applications (for BioMark system only) Using the Task Pane Various analysis parameters are set in the Task pane. Use the following as a guide. Analyze You must click Analyze each time you change a parameter in the software. Quality Threshold This function allows the user to set the pass/fail cutoff threshold for expression curves. The curve processing algorithm generates a quality score for each curve based on specific curve fit and quality metrics on a scale of Baseline Correction Linear Use when the amplification is low, producing higher C t values. Linear baseline correction flattens the baseline. Constant Shows the data without baseline correction. Linear (derivative) An additional method of baseline correction with a more robust handling of nonlinear baselines and their impact on C t estimates. C t Threshold Method Auto (Global) This option allows you to set the threshold for the entire chip. Auto (Panel) This option allows you to set the threshold for each panel on the chip independently. User (Global) This option allows you to manually adjust the threshold for each probe type when searching for the C t rise in the slope. The value you supply is applied to the entire chip. User (Detectors) This option allows you to manually adjust the threshold for each probe type in each panel when searching for the C t rise in the slope. Target Ct Range 1. Click the Target C t Range button next to the probe type name. 2. Click one or several panels. Selected panels open in the graph. 3. Change the minimum and/or maximum values: Type the values in the Min and/or Max text boxes. OR, Use the sliders to adjust values. 4. Click OK to close window. 5. Click Analyze to apply changes. Digital PCR Analysis Software User Guide 35

36 Using the Digital PCR Analysis Software Overview of Chip Explorer The Chip Explorer allows you to select Panel Summary Views, Panel Details or individual panel views. The Panel Summary view allows you to view a panel based breakdown of the data, including analysis group calculations. The Panel Details view allows you to view the chamber information for the entire chip (for viewing individual calls, values and curve data). The Individual Panel views (Panel 01...Panel 12) allow you to view the same views as the Panel Details view, but condensed down to show chambers for the selected panel. The secondary view has a tool bar, status bar and view tabs. For each probe type you set up for the chip run a tab is created (black text), and for each analysis group you define in the Application Template a tab is created (blue text).if you have not selected or defined the application template, the default tab labeled All Group will appear in blue text. (See example below.) 36 Digital PCR Analysis Software User Guide

37 Real-time Applications (for BioMark system only) Secondary Views for Panel Summary When viewing individual probe types in Summary view, you see real-time curves for the selected panel. Viewing Chip Run Results Using Panel Summary In the Panel Summary pane, you can examine your data in two views: Results Table (default when you click Panel Summary in the Chip Explorer pane) Heat Map View Each view is described in this section. Click Panel Summary and then click the Results Table drop-down menu for data view options. Examine data in each of these views. Digital PCR Analysis Software User Guide 37

38 Using the Digital PCR Analysis Software Results Table Examine data by panel. 1. Click Panel Summary and then click a panel of interest. 2. Click a row to display its amplification curves. 3. Click the tabs to view specific detector amplification curves for an entire panel. Each row represents one of the panels. 38 Digital PCR Analysis Software User Guide

39 Real-time Applications (for BioMark system only) Image View (Panel Details View only) Tool bar Views Access three options for viewing data from this drop-down menu. Show Selected Cell Selected cell(s) move to the top of the pane. Choose Image Choose from 1-50 images. Manual Contrast Manually adjust the contrast using the sliders. Auto Contrast The software automatically adjusts the contrast. Toggle Overlay Turns the red grid on and off as needed. Here is an example of the Image with the overlay toggled off. Toggle Location Map Fit Fit the view to the width of the active pane. 100% Increases the image view 100% from the default. Zoom Click to zoom in. Click to zoom out. Alternately, use the scroll wheel on your mouse to zoom in or out. Select the Dye Select the dye or dye-combination used with this chip run. Digital PCR Analysis Software User Guide 39

40 Using the Digital PCR Analysis Software Location Map Image View (in Panel Details view) has a small map that locates a highlighted panel or cell within the framework of the entire chip. Use the graphics below as a guide to its use. 1. Click the Location Map icon on the toolbar 2. Click a panel of interest to highlight it in green. 3. Click anywhere in the gray area and then roll your mouse wheel or, repeatedly click the zoomin button until you have the magnification you want. As you zoom in and the 12-panel view gets larger, click and drag the blue rectangle on the Location Map to the cells of interest within the panel. Heat Map: Hit Color View Hit Color selected Hold cursor over a cell to display cell information 40 Digital PCR Analysis Software User Guide

41 Real-time Applications (for BioMark system only) Heat Map: Spectrum View Change Hit Color view to Spectrum view to see the C t distribution range on the chip. 1. Click the Legend 2. Click Spectrum from the drop-down menu. button to open the legend. 3. Refer to the legend for C t values. Capture Heat Map Image If you want to save an image of a Heat Map view, go to the Edit menu and select either Copy View Image or Save View Image. Copy View Image copies the entire view to the clipboard regardless of the view you have displayed on your monitor screen. Save View Image prompts you to save the image of the view to disk in a JPG, PNG, BMP, GIF or TIFF format. User Configurable and Persistent Y-Scale On the secondary tool bar, you can select Full Range, Auto Range or Manual Range to adjust the visible percentage of the secondary graphs. Digital PCR Analysis Software User Guide 41

42 Using the Digital PCR Analysis Software Manual Range allows the user to use a slider bar to adjust the graphs (only for real-time chip runs). Creating a New Chip Run from an Analyzed Run If you want to create a new chip run from a previously analyzed run, follow the steps below. 1 Open an analyzed and annotated chip run (.bml file extension). 2 Click File > New From Current Chip Run. The New From Current Chip Run dialog box opens. 3 Open an unanalyzed and annotated chip run to create a run based on the analyzed run. 4 Type a name for the new chip run or, type the barcode number and then check Use Barcode as Chip Run Name. 5 Click Browse to navigate to a desired save location. 6 Click OK. 42 Digital PCR Analysis Software User Guide

43 Create a New chip Run Create a New chip Run To create a new chip run, follow the steps in the wizard: 1 Select Create a New Chip Run... from the launch page. 2 Follow the steps in the Chip Run Setup Wizard. S NOTE: If you select an application template when setting up your new chip run, you cannot modify the number or order of probe types. Convert a Real-time Run to an End Point Run A real-time chip run can be converted to an end point chip run. To do so: 1 Open the real-time chip run that you want to convert to end point. 2 Go to File > Save as End Point Chip Run. The software automatically converts the run to an end point run. The software creates a separate file named End Point within the real-time run s original folder. The new end point chip run is saved within a subfolder of the chip run folder. Digital PCR Analysis Software User Guide 43

44 Using the Digital PCR Analysis Software 44 Digital PCR Analysis Software User Guide

45 Performing End Point Analysis Runs 3 3 In this chapter: Introduction Overview of Chip Explorer Secondary View for Panel Summary Secondary Views for Panel Details Using the Secondary View Toolbar Viewing Chip Run Results Using Panel Summary Results Table Image View (Panel Details only) Heat Map: Hit Color View Heat Map: Hit Color View Using the Task Pane Digital PCR Analysis Software User Guide 45

46 Performing End Point Analysis Runs Introduction When you analyze an end-point chip run with the Digital PCR Analysis Software, the software reconfigures itself to display fewer graphs and screens than when you analyze a real-time chip run (as described in Chapter 2). Overview of Chip Explorer For End-point runs, the Chip Explorer allows you to select Panel Summary Views and Panel Details views. The Panel Summary view allows you to view a panel based breakdown of the data, including analysis group calculations. The Panel Details view allows you to view the chamber information for the entire chip or for individual panels. When you select either Panel Summary or Panel Details, you will see a primary view area and a secondary view area. The primary view areas on the top are switchable (click Results Table drop down and you can change between Results Table and Heat Map. The primary views function differently, depending on whether you are in Panel Summary or Panel Details. The secondary view has a tool bar, status bar and view tabs. For each probe type you set up for the chip run a tab is created (black text), and for each analysis group you define in the Application Template a tab is created (blue text). If you have not selected or defined the application template, the default tab labeled All Group appears in blue text. (See example below.) 46 Digital PCR Analysis Software User Guide

47 Overview of Chip Explorer Secondary View for Panel Summary When viewing an end point run, the individual probe type (FAM-MGB in the example below), you see a histogram for the normalized intensity values for the selected panel. The values that are above the normalized intensity threshold for the selected probe type (from tab) and panel (from primary view) are represented in the defined hit color for the probe type (see graphic below). When viewing an analysis group in Summary view, the histogram is represented by overlaying the histograms for each probe type in the group. Values over the threshold for each probe type are colored by their hit color. Secondary Views for Panel Details When viewing a specific probe type (the selected tab) in the detailed view for an end point run, the secondary view is populated with a histogram without hit coloring. The histogram shows the entire chip for the probe type selected (see graphic below). When viewing an analysis group (All tab selected), the histogram displays the values for all the probes for the entire chip. (See graphic below.) Digital PCR Analysis Software User Guide 47

48 Performing End Point Analysis Runs Using the Secondary View Toolbar The Normalized Intensity Histogram displays an overview of the threshold values for a particular panel. You can manipulate the histogram by using the Log Graph, Full/Auto Range, Pass, Fail, No Call, Clear and Clear All buttons. (See descriptions below.) Expand/collapse pin Switch between two graph views Full or Auto Range Pass or Fail a chamber Change status to No Call Clear one or Clear All user calls Copy to Clipboard, Print, Axis Settings View individual or all detectors in the histogram Viewing Chip Run Results Using Panel Summary In the Panel Summary pane, you can examine your data in two views: Results Table (default when you click Panel Summary in the Chip Explorer pane) Heat Map View Each view is described in the following text. 48 Digital PCR Analysis Software User Guide

49 Viewing Chip Run Results Using Panel Summary Click Panel Summary and then click the Results Table drop-down menu for data view options. Examine data in each of these views. Go to Panel In Summary View, Heat Map View or Image View, you can right-click on the panel of interest and a context sensitive menu appears. Click Go to Panel to retrieve the data for a specific panel of interest. Digital PCR Analysis Software User Guide 49

50 Performing End Point Analysis Runs Results Table Examine data by panel. 1. Click Panel Summary and then click a panel of interest. 2. Click a row to display its normalized intensity. 3. Click the tabs to view specific detector normalized intensity for an entire panel. Each row represents one of the 12 panels. Image View (Panel Details only) You can examine data by detector and image number. 1. Click Panel Details. 2. Click Image View from the drop-down menu. 3. Click detectors from the drop-down menu. 4. Click an image (1-50) from the dropdown menu. 50 Digital PCR Analysis Software User Guide

51 Viewing Chip Run Results Using Panel Summary Image View Tool bar Views Access three options for viewing data from this drop-down menu. Show Selected Cell Selected cell(s) move to the top of the pane. Choose Image Choose a specific cycle image. Manual Contrast Manually adjust the contrast using the sliders. Auto Contrast The software automatically adjusts the contrast. Toggle Overlay Turns the red grid on and off as needed. Here is an example of the Image with the overlay toggled off. Toggle Location Map Fit Fit the view to the width of the active pane. 100% Increases the image view 100% from the default. Zoom Click to zoom in. Click to zoom out. Alternately, use the scroll wheel on your mouse to zoom in or out. Select the Dye Select the dye or dye-combination used with this chip run. Digital PCR Analysis Software User Guide 51

52 Performing End Point Analysis Runs Location Map Image View (in Panel Details view) has a small map that locates a highlighted panel or cell within the framework of the entire chip. Use the graphics below as a guide to its use. 1. Click the Location Map icon on the toolbar 2. Click a panel of interest to highlight it in green. 3. Click anywhere in the gray area and then roll your mouse wheel or, repeatedly click the zoomin button until you have the magnification you want. As you zoom in and the 12-panel view gets larger, click and drag the blue rectangle on the Location Map to the cells of interest within the panel. 52 Digital PCR Analysis Software User Guide

53 Viewing Chip Run Results Using Panel Summary Heat Map: Hit Color View Click a panel to display data in the panes below Capture Heat Map Image If you want to save an image of a Heat Map view, go to the Edit menu and select either Copy View Image or Save View Image. Copy View Image copies the entire view to the clipboard regardless of the view you have displayed on your monitor screen. Save View Image prompts you to save the image of the view to disk in a JPG, PNG, BMP, GIF or TIFF format. Digital PCR Analysis Software User Guide 53

54 Performing End Point Analysis Runs Using the Task Pane Various analysis parameters are set in the Task pane. Use the following as a guide. Analyze You must click Analyze each time you change a parameter in the software. Quality Threshold Method This function allows you to set the pass/fail cutoff threshold for chamber quality on a scale of Intensity Threshold Method Auto (Global) This option allows you to set the threshold for the entire chip. Auto (Panel) This option allows yo uto set the threshold for each panel. (See User defined options below.) User (Global) This option allows you to set the threshold for each probe type. User (Panel) This option allows you to set the threshold for each probe type for each panel. If you check Initialize with Auto and then click the Analyze button, you can set the user threshold for each panel and each detector to their Auto (Panel) values. Exporting User-defined Threshold Settings For copying user-defined threshold settings from one chip run to another, you can right-click the threshold table and load or save the settings.. 54 Digital PCR Analysis Software User Guide

55 Using the Task Pane To load the table: 1 Right-click the threshold table. 2 Select Load Table. A Load Threshold Table dialog box appears. 3 Select a location to retrieve a load file from. 4 Enter the name of the file. 5 Click Open. To save the Threshold Table: 1 Right-click the threshold table. 2 Select Save Table. A Save Threshold Table dialog box appears. 3 Select a folder location to save the file in. Digital PCR Analysis Software User Guide 55

56 Performing End Point Analysis Runs 4 Name the file. 5 Click Save. 56 Digital PCR Analysis Software User Guide

57 Generating Reports and Exporting Data 4 4 In this chapter: Using the Tool bar Options Feature Output Chip Color Scheme Application Template Generating Reports Chip Run Preparation Report Chip Analysis Report Exporting Chip Run Data Exporting Data Digital PCR Analysis Software User Guide 57

58 Generating Reports and Exporting Data Using the Tool bar Options Feature Set export and report parameters using Options. When you export data later, they reflect the options you choose here. Output Results Export 1 Click Tools > Options. The Options dialog box opens. 2 Click Results Export. Choose a destination from the Default Folder drop-down menu. The browse icon is activated when you choose User Specified Folder. Select Use Post Export Command if you want to browse to a batch file. 3 Use the default export destination or browse to another location. 4 Click Apply. 5 (Optional) If you are not changing the Report or Chip options, click OK. Report 1 Click Tools > Options. The Options dialog box opens. 2 Click Report. 3 Use the default export destination or browse to another location. 58 Digital PCR Analysis Software User Guide

59 Using the Tool bar Options Feature 4 In the Graphs and Images panes, check the features you want shown in your exported data. 5 Click Apply. (Optional) If you are not changing the Chip options, click OK. Chip Color Scheme 1 Click Tools > Options. The Options dialog box opens. 2 Click Color Scheme. 3 Click the drop-down menus to change colors. When you click on a color, the color palette opens. Click Define Custom Colors for more colors 4 (Optional) In the Color Scheme options window: a Click Load Scheme to load a previously saved color scheme (file has.xml extension). Saved schemes are in program files > fluidigm > DigitalAnalysis > Bin folder. b Click Save Scheme to save the current color scheme for later use (file has.xml extension). c Click Default Scheme to clear current color scheme and return to the default. Digital PCR Analysis Software User Guide 59

60 Generating Reports and Exporting Data No Call Color Selection You can also select a color for No Call cells for real-time chip runs only. 1 Go to Tools > Options. 2 Select Color Scheme from the Options dialog box. 3 Click the drop-down arrow in the No Call column to view the selection of colors. 4 Select a color and click OK on the Color dialog box. 5 Click Apply on the Options dialog box. Application Template An Application Template allows you to define the relationships of probe types in a chip run. Once you have defined an application template for a chip run, you can save it to disk for use in other chip runs. You can load the application template into a chip run that has been imaged or use it as a starting point for a new chip run in the new chip run wizard. Each application template has a probe type footprint that is a set of all probe types used in the analysis groups for that template. This footprint is used to match the template with the probe types in a chip run when you load a template or define the probe types for setting up a chip run. If you try to load a template with a probe type footprint that does not match the chip run currently open, then the software will warn you that it cannot load the template for this chip run. To access the application template option: 1 Click Tools > Options. The Options dialog box opens. 2 Click Application Template. 60 Digital PCR Analysis Software User Guide

61 Using the Tool bar Options Feature The plus sign icon allows you to add an application group. The Edit icon allows you to change the selected application group. The X icon allows you to delete the selected application group. List of templates Allows you to create a new template. Allows you to save a template after loading, editing or creating it. Allows you to browse to a template and load it. 3 Click the plus sign or edit sign and the Analysis Group Editor launches. The Analysis Group Editor allows you to specify the relationship between two or more probe types. This relationship defines the operations the application is to perform on the hit data for each panel. Each operation selected in the Operations checklist adds the appropriate column to the Summary tables and exported data. Many operations are limited to two probe type analysis groups. The operations are: Union - Selecting this option allows the total number of hits in both probe types to be displayed in a Union column in the summary table. Intersection hits are subtracted from the total number. Intersection - Selecting this option allows the number of hits common to both probe types to be displayed in an Intersection column in the summary table. Est. Ratio (#1/#2) - Selecting this option allows the estimated ratio between the distribution estimated counts of each probe type to be displayed in an Est. Ratio column in the summary table. Est. R-95L (#1/#2) - Selecting this option allows the lower estimated count of the 95% confidence range to be displayed in an Est. R-95L column in the summary table. (See graph below.) Est. R-95U (#1/#2) - Selecting this option allows the upper estimated count of the 95% confidence range to be displayed in an Est. R-95U column in the summary table. (See graph below.) Digital PCR Analysis Software User Guide 61

62 Generating Reports and Exporting Data Est. Diff (#1/#2) - Selecting this option allows the estimated difference in the counts for each probe type to be displayed in an Est. Diff column in the summary table. Est. D-95L (#1/#2) - Selecting this option allows the lower estimated count of the 95% confidence range to be displayed in an Est. D-95L column in the summary table. (See graph below.) Est. D-95U (#1/#2) - Selecting this option allows the upper estimated count of the 95% confidence range to be displayed in an Est. D-95U column in the summary table. (See graph below.) 62 Digital PCR Analysis Software User Guide

63 Generating Reports Generating Reports Two different reports are available depending on the type of chip run being opened. Chip Run Preparation Report This report is available if you open an unanalyzed chip run file or, if you create a new chip run file. The report shows dispense mapping information, and is saved as an.htm file. 1 Click Report > Chip Preparation Report to open the report in the preview window. 2 Select a file type from the Export drop-down list to save the Chip Run Report as a file. 3 Click Save. Digital PCR Analysis Software User Guide 63

64 Generating Reports and Exporting Data Chip Analysis Report This report is available if you open an analyzed chip run or, when an unanalyzed chip run is analyzed. The report shows analysis data, and is saved as an.htm file. 1 Click Report > Chip Run Analysis Report. The Chip Run Report opens in a preview window. 2 Select a file type from the Export drop-down list to save the Chip Run Report as a file. 64 Digital PCR Analysis Software User Guide

65 Exporting Chip Run Data 3 Click Save to save the Chip Run Analysis Report as a.htm file. The report opens automatically after you save it. An example of a Chip Run Analysis Report is shown below. Exporting Chip Run Data Exporting Data Export chip run data in a comma separated value format (.csv) that can be viewed in a program like Microsoft Excel. You can export data from: Summary Table View Detailed Table View Call Map View Digital PCR Analysis Software User Guide 65

66 Generating Reports and Exporting Data 1 Click File > Export. The chip barcode is the default name for the exported file. 2 Select the appropriate type of view from the Save as type drop-down menu. 3 Click Save. 4 To open the saved data, navigate to the.csv file. 5 Double-click the.csv file to open it in a program such as Microsoft Excel. An example of exported Summary Table Results data in a.csv file, opened in an Excel spreadsheet. 66 Digital PCR Analysis Software User Guide

67 Exporting Chip Run Data Digital PCR Analysis Software User Guide 67

68 Generating Reports and Exporting Data 68 Digital PCR Analysis Software User Guide

69 Digital PCR on the Array Workflow A A In this Appendix: Digital PCR Workflow Priming the Digital Array IFC Preparing Sample Pre-Mix and Samples Loading the Chip Using the Data Collection Software Using the Digital PCR Analysis Software Digital PCR Analysis Software User Guide 69

70 Digital PCR on the Array Workflow Digital PCR Workflow Below is a workflow that you can use with the Digital Array IFC. Priming the Digital Array IFC CAUTION! Use the chip within 24 hours of opening the package. Due to different accumulator volumes, use only syringes with 300 µl of control line fluid. Control line fluid on the chip or in the inlets makes the chip unusable. Load the chip within 60 minutes of priming. 1 Inject control line fluid into the accumulator on the chip. 2 Place the chip into the IFC Controller MX. Then, using the IFC controller software, run the Prime (115x) script to prime the control line fluid into the chip. Preparing Sample Pre-Mix and Samples 1 Combine the components in the table below to make the Sample Pre-Mix and the final Sample Mixture (scale up appropriately for multiple runs). Component Volume per Inlet (µl) Volume per Inlet with Overage (µl) Volume per Chip (µl) TaqMan Gene Expression Master Mix (Applied BioSystems, PN *) 20x GE Sample Loading Reagent (Fluidigm, PN ) X gene-specific assays ** DNA-free water DNA Total * TaqMan Universal PCR Master Mix (Applied Biosystems, PN ) may be substituted. Fluidigm recommends using TaqMan Gene Expression Master Mix for the Digital Array IFC. ** The 20X assay can be removed from the Sample Pre-Mix and added separately if different assays are to be used on the same chip. 2 In a DNA-free hood, combine the TaqMan Gene Expression Master Mix, 20x GE Sample Loading Reagent, DNA-free water and 20X assay in a 1.5 ml sterile tube enough volume to fill the entire chip. Nine (9) µl of this Sample Pre-Mix can then be aliquoted for each sample (12 total). 70 Digital PCR Analysis Software User Guide

71 Digital PCR Workflow 3 Remove these aliquots from the DNA-free hood and add 1 µl of DNA to each, making a total volume of 10 µl in each aliquot. Loading the Chip IMPORTANT: Vortex thoroughly and centrifuge all sample solutions before pipetting into the chip inlets. Failure to do so may result in a decrease in data quality. CAUTION! While pipetting, do not go past the first stop on the pipette. Doing so may introduce air bubbles into the inlets. 1 When the Prime (115x) script has finished, remove the primed chip from the IFC controller MX. 2 Pipette 8 µl of DNA-free water into each H inlet. Digital Array IFC Pipetting Map Add 8 µl of DNA-free water Do Not Use Pipette samples into inlets 1 12 A1 CAUTION! While pipetting, do not go past the first stop on the pipette doing so may introduce air bubbles into the inlets. 3 Pipette 8 µl sample mix into the Sample inlets on the chip. Digital PCR Analysis Software User Guide 71

72 Digital PCR on the Array Workflow 4 Return the chip to the IFC controller MX. 5 Using the IFC controller MX software, run the Load (115x) script to load the samples into the chip. 6 When the Load (115x) script is complete, remove the chip from the IFC Controller MX. 7 Remove any dust particles or debris from the chip surface. You are now ready for your chip run. CAUTION! Start the run on the instrument within four (4) hours of loading the samples. Using the Data Collection Software IMPORTANT: Be sure to select all probe types present in your experiment. Data are not collected on unspecified probes. 1 Double-click the Data Collection Software icon on the desktop to launch the software. 2 Click Start a New Run. 3 Check the status bar to verify that the lamp and the camera are ready. Make sure both are green before proceeding. 4 Place the loaded chip into the instrument. 5 Click Load. 6 Verify chip barcode and chip type. a Choose project settings (if applicable). b Click Next. 7 Chip Run file: a Choose New or Predefined. b Choose a file location for data storage. c Click Next. 8 Application, Reference, Probes: a Select Application Type Digital PCR. b Select Passive Reference (ROX). c Select Assay Single probe, Two probes, or More than two probes. d Select probe types. e Click Next. 9 Click Browse to find the appropriate thermal protocol file dpcr Standard v1.pcl. 72 Digital PCR Analysis Software User Guide

73 Digital PCR Workflow 10 Confirm Auto Exposure is selected. 11 Verify the chip run information. 12 Click Start Run. NOTE: To run this protocol as an end-point run and use the Fluidigm Stand- Alone Thermal Cycler or the Fluidigm FC1 Cycler, refer to the Fluidigm Stand- Alone Thermal Cycler Usage Quick Reference (PN ) or the Fluidigm FC1 Cycler Usage Quick Reference (PN ), respectively. Using the Digital PCR Analysis Software IMPORTANT: Be sure to click Analyze each time you change a parameter in the software (changing the target C t range, for example). 1 Double-click the Digital PCR Analysis software icon on the desktop to launch the software. 2 Click Open a Chip Run. 3 Double-click a chiprun.bml file to open it in the software. 4 Click Sample and Detector Setup in the Chip Explorer pane. 5 Click New or Import. 6 Highlight the wells and then annotate them. 7 Click Editor in the Sample and Detector Setup pane. 8 Choose Sample Type from the drop-down menu in the Editor. 9 Type a name for the sample. 10 Choose Detector Type from the drop-down menu in the Editor. 11 Type a name for the detector. 12 Click Update to see the changes reflected in the highlighted wells. 13 Click Panel Summary in the Chip Explorer pane. 14 Click Analyze in the Task pane. 15 Click Panel Summary or Panel Details. 16 Choose a view from the drop-down menu: Results Table Image View Heat Map View. Digital PCR Analysis Software User Guide 73

74 Digital PCR on the Array Workflow 74 Digital PCR Analysis Software User Guide

75 Digital PCR on the WorkflowB B In this Appendix: Digital PCR Workflow Priming the Digital Array IFC Preparing Sample Pre-Mix and Samples Loading the Chip Using the Data Collection Software Using the Digital PCR Analysis Software Digital PCR Analysis Software User Guide 75

76 Digital PCR on the Workflow Digital PCR Workflow Below is a workflow that you can use with the Digital Array IFC. Priming the Digital Array IFC CAUTION! Use the chip within 24 hours of opening the package. Due to different accumulator volumes, use only syringes with 300 µl of control line fluid. Control line fluid on the chip or in the inlets makes the chip unusable. Load the chip within 60 minutes of priming. 1 Inject control line fluid into each accumulator on the chip. 2 Remove and discard the blue protective film from the bottom of the chip. 3 Place the chip into the IFC Controller MX, then run the Prime (148x) script to prime the control line fluid into the chip. Preparing Sample Pre-Mix and Samples 1 Combine the components in the table below to make the Sample Pre-Mix and the final Sample Mixture (scale up appropriately for multiple runs). Component Volume per Inlet (µl) Volume per Inlet with Overage (µl) Volume per Chip (µl) TaqMan Gene Expression Master Mix (Applied BioSystems, PN *) 20X GE Sample Loading Reagent (Fluidigm, PN ) X gene-specific assays ** DNA-free water** DNA 1.2 Total * TaqMan Universal PCR Master Mix (Applied Biosystems, PN ) may be substituted. Fluidigm recommends using TaqMan Gene Expression Master Mix for the Digital Array IFC. ** The 20X assay can be removed from the Sample Pre-Mix and added separately if different assays are to be used on the same chip. 76 Digital PCR Analysis Software User Guide

77 Digital PCR Workflow 2 In a DNA-free hood, combine the TaqMan Gene Expression Master Mix, GE Sample Loading Reagent, DNA-free water and 20X assay(s) in a sterile tube enough volume to fill the entire chip. 4.2 µl of this Sample Pre-Mix can then be aliquoted for each sample (48 total). 3 Remove these aliquots from the DNA-free hood and add 1.8 µl of DNA to each, making a total volume of 6 µl in each aliquot. NOTE: For a Copy Number Variation application, substitute RNase P for the DNA-free water. Loading the Chip IMPORTANT: Vortex thoroughly and centrifuge all sample solutions before pipetting into the chip inlets. Failure to do so may result in a decrease in data quality. CAUTION! While pipetting, do not go past the first stop on the pipette. Doing so may introduce air bubbles into the inlets. 1 When the Prime (148x) script has finished, remove the primed chip from the IFC controller MX. Digital PCR Analysis Software User Guide 77

78 Digital PCR on the Workflow 2 Pipette 10 µl of 1X GE Sample Loading Reagent into all hydration inlets. into sample inlets A1 notch Add 1X GE Sample Loading Reagent into all 16 hydration inlet 3 Pipette 4 µl sample mix into the Sample inlets on the chip. 4 Return the chip to the IFC Controller MX. 5 Using the IFC Controller MX software, run the Load (148x) script to load the samples into the chip. 6 When the Load (148x) script is complete, remove the chip from the IFC controller. 7 Remove any dust particles or debris from the chip surface. You are now ready for your chip run. CAUTION! Start the run on the instrument within four (4) hours of loading the samples. Using the Data Collection Software IMPORTANT: Be sure to select all probe types present in your experiment. Data are not collected on unspecified probes. 1 Double-click the Data Collection Software icon on the desktop to lauch the software. 2 Click Start a New Run. 78 Digital PCR Analysis Software User Guide