QX ONETM Software
Regulatory Edition
User Guide
Version 1.4

QX ONE Software Regulatory Edition

QX ONE Software
Regulatory Edition
User Guide
Version 1.4

For Research Use Only

Bio-Rad Technical Support Department
The Bio-Rad Technical Support department in the U.S. is open Monday through Friday, 5:00 AM to 5:00 PM,
Pacific time.
Phone: 1-800-424-6723, option 2
Email: Support@bio-rad.com (U.S./Canada Only)
For technical assistance outside the U.S. and Canada, contact your local technical support office or click theContact Us link at bio-rad.com.

Legal Notices
No part of this publication may be reproduced or transmitted in any form or by any means, electronic ormechanical, including photocopy, recording, or any information storage or retrieval system, without permission
in writing from Bio-Rad Laboratories, Inc.
Bio-Rad reserves the right to modify its products and services at any time. This guide is subject to change
without notice. Although prepared to ensure accuracy, Bio-Rad assumes no liability for errors or omissions, orfor any damage resulting from the application or use of this information.
BIO-RAD, DDPCR, DROPLET DIGITAL, DROPLET DIGITAL PCR, QUANTASOFT, and QX ONE are
trademarks of Bio-Rad Laboratories, Inc. in certain jurisdictions.
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reagents containing EvaGreen Dye for use in real-time PCR, for research purposes only.
Purchase of Digital PCR and/or Single-Cell NGS Sample Preparation products (the “Products”) from
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can be accessed at https://bio-rad.com/en-us/terms-conditions. Unless we expressly state otherwise inadditional Terms and Conditions, no rights are granted for you to distribute or resell the Products. Unless we
expressly state otherwise in additional Terms and Conditions, ​no rights are granted for the development orcommercialization of diagnostic assays for use with the Products without a license from Bio-Rad. It is theuser’s obligation to obtain a commercial license from Bio-Rad for (i) all commercial uses (not just diagnosticuses) and (ii) sale of assays for use on Bio-Rad's ddPCR and ddSEQ instruments. The Products and/or theiruse are covered by U.S.​ and foreign patents and/or pending patent applications owned by or under license to
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All trademarks used herein are the property of their respective owner.
Copyright © 2023 by Bio-Rad Laboratories, Inc. All rights reserved.

Revision History

Revision History
Document
QX ONE Software User Guide,
Regulatory Edition

Date
December
2023

Description of Change
Added new Software Settings tab in
Version 1.4

Version 1.4
DIR 10000148670 Ver D
QX ONE Software User Guide,
Regulatory Edition

January 2023

Update for Version 1.3, as follows:n Update screen shots to show in thesoftware toolbar the percentage of disk

Version v1.3

space available on the touch screen

DIR 10000148670 Ver C

computer hard drive instead of the numberof plates than can be run.
n Add important note regarding foil piercing
on all wells in a plate (including excluded
wells), although droplets are not generated
or read in excluded wells.

QX ONE Software User Guide
Regulatory Edition

December
2021

Update user guides with Version 1.2
enhancements, includingn Positive control-based thresholding

Version 1.2

n Ability to enable the User Management
module (Standard Edition only)n Added user privilege
n Enhanced data recoveryn Access to troubleshooting datan Ability to view an audit log (Regulatory
Edition only)

QX ONE SoftwareUser Guide
Regulatory Edition

October 2020

Add brief descriptions, bullet points andcross-references to the instrument guide inboth software guides

User Guide | iii

iv QX ONE Software, Regulatory Edition

Table of Contents
Front Cover
Revision History

Chapter 1 About QX ONE Software
Signing In, Viewing Privileges, and Changing Preferences

a
iii

12
14

Signing into the Software

15

Viewing User Privileges

16Managing Your Preferences18

Signing Out or Changing Users

19

Instrument Status Bar

20

Tabs to Functional Windows21

Add Plate Window

22

Run Status Window

23

Data Analysis Window

24

Chapter 1 Creating or Modifying Templates

25

System Status Window

26

User Management Window

27

Help Window

28

Linking the User Guides in the Help Window

29

Compatible File Types

30

Chapter 1 Creating or Modifying Templates

31

Creating or Modifying Plate Templates

33

Opening a New Plate Template

36

Opening an Existing Plate Template37

Template Plate Editor Window38

Template Setup — Setting Up Your Wells

41

Template Setup – Excluding Wells

50

Creating and Storing Plate Templates from CSV Files

53

Creating or Editing Thermal Cycling Protocol Templates

55

User Guide | v

Table of Contents

Opening a New Thermal Cycling Protocol

57

Opening an Existing Thermal Cycling Protocol

58

Thermal Cycling Protocol Editor Window

59

Adding Steps to a Protocol Template

60

Adjusting Step Options

61

Deleting Steps from a Protocol Template

62

Creating or Modifying Report Templates

63

Chapter 3 Data Analysis Module Overview

67

Opening a Data File for Analysis

69

Saving a Data File70

Analysis Dashboard

71

Dashboard Options

72

Modifying the Dashboard Display

74

Analysis Display and Output Options

75

Universal Options

76

Expanding or Reducing the Chart

79

Selecting Chart Display Preferences80

Selecting Chart Scale and Label Options

81

Using Chart Menu Options

83

Using Table Menu Options

85

Plate View Windows

88

Plate Editor — Analysis View

88

Plate View

89

2D Plate View

90

Analysis Windows

91

Data Windows

92

Data Table Tab

92

Plate Well Data Table

99

Plate Editor Well Data Table

101

Viewing Run, Lot, and Audit Information

102

Chapter 4 Data Analysis Methodology

103

Amplitude Plot Analysis Options

104

1D Amplitude

110

2D Amplitude

115

vi QX ONE Software, Regulatory Edition

Table of Contents

3D Amplitude

117

Data Visualization Chart Analysis Options

120

Concentration

123

Copy Number

126

Ratio

129

Fractional Abundance

131

Event Counts

133

Chapter 5 Analysis Module Reporting

135

Creating Analysis Reports

135

Viewing the Audit Log

138

Chapter 6 Gene Study Module

139

Gene Study Options

141

Setting up the Gene Study

142

Adding Data Files

144

Removing Data Files

144

Selecting Data Files for Analysis

144

Showing the Plate View

145

Gene Study Reports
Creating a Gene Study Report

Chapter 7 System Utilities

146
147

149

System Settings Tab

150

Preferred Location

151

Shared Settings

152

Software Settings

153

System Log Files

154

Event Log

154

Maintenance Log

156

Maintenance Reports

157

System Data

158

Archiving Raw Data

158

Troubleshooting Data

159

Recovering Data

160

System Calibration

161

User Guide | vii

Table of Contents

Chapter 8 Managing Users

163

Adding Users

164

Adding or Removing User Privileges

166

Removing Users

168

Modifying User Preferences

168

Appendix A Experiment Examples

169

Direct Quantification Multiplexed

170

Setting Up Experiment Parameters

170

Viewing and Adjusting Results

172

Copy Number Variation Triplexed

174

Setting Up Experiment Parameters

174

Viewing and Adjusting Results

176

Drop-Off Assays for Genome Edit Detection

177

Setting Up Experiment Parameters

178

Viewing and Adjusting Results

179

Direct Quantification with the Advanced Classification Method

180

Setting Up Experiment Parameters

180

Viewing and Adjusting Results

182

Appendix B End User License Agreement

183

Appendix B Ordering Information

187

Back Cover

191

viii QX ONE Software, Regulatory Edition

User Guide | 11

Chapter 1 About QX ONE Software

Chapter 1 About QX ONE Software
A Bio-Rad field service engineer installs your software edition on the QX ONE Droplet Digital PCR System
touch screen, which provides the interface to the instrument controls.
Using the software on the touch screen, you cann

Set up customized ddPCR experiments for plate runs in the QX ONE™ Droplet Digital™ PCR(ddPCR™) SystemnCreate and store templates for plate layouts, thermal cycling protocols, and reports.

n

Use the live analysis function during the droplet reading phasenWith the Manage Plate user privilege, cancel runs and reorder plates while they are in the instrument
Inbox

n

Analyze your data files in a variety of charts and tables in the Analysis modulenGenerate reports on your datanRestrict template access to ensure the use of approved templates only

In the Regulatory Edition, you cannProduce system and experiment audit logs to ensure clear audit trails for activities andconsumable/reagent use through the Event Log, Data Archive, and automatic Lot Management
functionsnSafeguard user access with secure logins and traceable user activity

Important: The Regulatory Edition prompts you to log in again and reauthenticate your user
credentials. Enter your user name and password, and then tap Sign in.
For information on using the instrument, refer to the QX ONE Droplet Digital PCR System Instrument
Guide.
The QX ONE guides instruct you to tap options on the touchscreen. If you are using QX ONE Softwarewith a keyboard and mouse, click the options instead.
Tip: You can download and link the software and instrument guides to corresponding buttons in theHelp window. For information, see Linking the User Guides in the Help Window on page 29.
Important: The Regulatory Edition provides additional features to permit operation in compliancewith Title 21 of the U.S. Code of Federal Regulations Part 11 (21 CFR Part 11) within a closed
system. A closed system is defined as “an environment in which system access is controlled by the
persons who are responsible for the content of electronic records that are on the system” (Section
11.3(b) (4)).

12 QX ONE Software, Regulatory Edition

n

The security controls built into the Regulatory Edition must be properly configured and administeredby the software administrators in your organization in order to be secure and in compliance with 21
CFR Part 11.

n

Bio-Rad makes no claim that the Regulatory Edition is CFR-compliant in and of itself, nor does thecompany guarantee compliance for the user. Your organization must establish policies and standard
operating procedures that work in conjunction with the tools provided by Bio-Rad to ensurecompliance with 21 CFR Part 11.

n

The Regulatory Edition prompts you to log in again before executing an auditable action.

LEGENDThe status bar displays information about the instrument and user.
Tabs provide access to the main functional windows.
The main pane displays the details of the selected tab.

If QX ONE Software goes into standby mode after being idle, tap in the lockout screen to sign into thesoftware again and display the last active window.

User Guide | 13

Chapter 1 About QX ONE Software

Signing In, Viewing Privileges, and Changing Preferences
Typically, a system or software administrator sets up users and privileges for QX ONE Software, andcommunicates the information to you.
Note the following:n

You cannot change your user privileges unless you are assigned the Add/Manage Users privilege.
For information, see Viewing User Privileges on page 16.

n

You can change your user preferences, which are set to defaults. For information, see Managing
Your Preferences on page 18.

14 QX ONE Software, Regulatory Edition

Signing In, Viewing Privileges, and Changing Preferences

Signing into the Software
Important: The Regulatory Edition includes audit capabilities. For auditable actions, you areprompted to sign in again before continuing.

To sign in
1.

Tap the touch screen to open the Sign in dialog box.

2.

Enter the user name and password and tap Sign in.

3.

Ensure the Do not show EULA on sign in checkbox is selected and tap I Agree.

Each user must agree to the end user license agreement on initial login in order to user the software.
If you clear the checkbox, the End User License Agreement appears the next time you log into thesoftware, and you must agree again to the terms before the software opens. If you do not agree to theEULA, the application closes immediately.
When you agree to the EULA, the dialog box closes and the application opens. Your user nameappears in the Instrument Status bar.
The Add Plate window opens on the instrument touch screen.
If you are opening the software on a standalone computer, the Data Analysis window opens.

User Guide | 15

Chapter 1 About QX ONE Software

Viewing User Privileges
In the User Management window, your privileges are identified by the selected checkboxes.

To view your assigned privilegesu

Select the User Management tab.

Unless you are assigned the Add/Manage Users privilege, checkboxes for your assigned privileges are
selected but not enabled, as shown in the following graphic. To request changes to your privileges,
contact your software administrator.

16 QX ONE Software, Regulatory Edition

Signing In, Viewing Privileges, and Changing Preferences

Table 1 lists the available privileges, which can be assigned in any combination.
Table 1. User privileges

Privilege

PurposeAdd and manage

Add or remove users, set privileges, and change preferences.

users

Important: Users who are assigned the Add/Manage Users privilege can create
users and update privileges and preferences for any user. Only the superuser canremove this privilege from user accounts.

Create new

Save a design for a plate, thermal cycling protocol, or analysis report as a template.

templates
View data files

See run files that were created from runs executed by other users.

(created by otherusers)
Overwrite

Save the file with a new file name. Files are always saved with the extension for the

existing data fileapplication you are using. For information, see Compatible File Types on page 30.

nameNote: Users without this permission can open files and perform analysis but
cannot create a new file.

System settings

View logs, reprocess data with new calibration values, and change file locations. Allusers can view the file locations.
Important: If your administrator selected the Preferred Locations checkbox in
System Settings, individual user location preferences are overridden.

Maintenance

View the event log and the system data settings.

Note: All users can reprocess data, view system folder settings, as well as themaintenance log and maintenance reports.
System Data

Move raw data from the touch screen computer to free up disk space.
Access troubleshooting data.
Recover data from a temporary file created for a failed run.

Access Module

For manual plate recovery, you can specify the processes to be rerun.

Recovery ToolManage Plate

Cancel runs on the QX ONE Droplet Digital PCR System.
Reorder plates that are still in the instrument Inbox.

User Guide | 17

Chapter 1 About QX ONE Software

Managing Your Preferences
Use the User Management window to modify your personal user preferences. Default folder locations
appear in your preferences, but you can change them. You can also choose to keep all data files andtemplates private. The software prompts you to choose either your personal folder or the shared folder
each time you save a file or template.

To change your preferences
1.

Change any of the following preferences:nEnter a different file path for your data files and templates.

nSelect or clear the check boxes to change your data file and template privacy settings.
Important: The paths and checkboxes are disabled if your administrator has set Preferred
Locations in System Settings.

2.

3.

n

Enter a different system timeout period.

nEnter a different total of completed plates to show in the Run Status window, up to a
maximum of 100.

Do one of the following:n

If you are setting preferences for a new user, tap Add.

n

If you are updating preferences, tap Save.

When the confirmation message appears, tap Yes, and then tap OK.

18 QX ONE Software, Regulatory Edition

Signing In, Viewing Privileges, and Changing Preferences

Signing Out or Changing Users
While the instrument is idle, one user can log out on the touch screen and another user can log in.
Important: If User Management was not enabled during the software installation, the sign in, signout, and change users functions are not available.
Note the following:n

Do not change users while a run is in progress or data might be affected.

n

If the screen is locked and the initial user left unsaved changes, an advisory prompt appears. Click
Save before proceeding or the previous user’s work is lost.

To sign out
1.

Tap the user name link in the upper-right corner and select Sign Out.

2.

Tap Yes to confirm.

To change users
1.

Tap anywhere in the lockout screen to display the Sign in window.

2.

Enter a user name.
n

The domain name appears below the user login fields. If you are on the same domain as theprevious user, you can enter your user name only.

n

If the user is on a different domain, enter the domain name followed by a backslash and the user
name.
<domain name>\<user name> (for example, global\john_smith)

3.

Enter the password and tap Sign in.

User Guide | 19

Chapter 1 About QX ONE Software

Instrument Status Bar
An instrument status bar appears above all windows except the analysis displays, which open in a
separate module.
The status bar on the touch screen displays the information an-d indicators shown below

LEGEND
Software name and version
Instrument name and status information
Number of runs in progress and runs available
Oil and waste levels
Open or close the Inbox or Outbox
Used and free disk space
Storage availability is displayed in GB and by number of plates that you can run before runningout of space on the touch screen computer.
Note: When QX ONE Software detects low disk space, the software displays an advisory
prompt to archive data.
Name of the current user, along with the current date and time
Closes the QX ONE Software application

20 QX ONE Software, Regulatory Edition

Tabs to Functional Windows

Tabs to Functional Windows
Table 2 describes the functional areas and primary windows in QX ONE Software.
Table 2. Window Tabs

Tab

Name

PurposeAdd Platen Add and configure a plate and protocol for a ddPCR run
n Recover a plate from a failed run

Run Statusn In the top pane, view the runs in progressn In the bottom pane, view up to 100 completed runs

Data Analysis

n Access the Data Analysis and Gene Study modules
Note: In QX ONE Software, Gene Study is available on astandalone computer, but not from the touch screen.

Templates

n Set up templates for plates thermal cycling protocols, and reports,and search for existing template files

Systemn View storage locations, logs, and maintenance reports
n Archive data, access troubleshooting information, and recover datafrom a temporary file created for a failed run
Important: Available functionality depends on your assigned
user privileges. Instrument calibration is available only to the
Bio-Rad service engineer.

Users

n View the user privileges you have been assigned, and view andmodify your personal preferencesn Create, edit, or remove QX ONE Software users (if you areassigned the Add/Manage Users privilege)Helpn Access software version information, current calibration values,
End User License Agreement company website, and open source
software license information
Tip: You can also link the user and instrument guides to thelabeled buttons. For information, see Linking the User Guides inthe Help Window on page 29.

User Guide | 21

Chapter 1 About QX ONE Software

Add Plate Window
On the QX ONE Droplet Digital PCR System touch screen, QX ONE Software opens to the Add Plate
window, where you can set up a plate for an experiment and start a ddPCR run. You can also recover aplate that experienced a failure during the run.
Note: If you are using the software on a computer other than the touch screen, the Add Plate window
is not accessible and QX ONE Software displays the Data Analysis window by default.

From the Add Plate window you cann

Add plates in the softwaren

Recover a plate from a failed runnAccess the Plate and Protocol Configuration window to configure your plates and PCR protocolsnRun experiments on your plates and protocols

QX ONE Software enables each button sequentially (Add Plate, Configure Plate, Start Run) when you
complete the prerequisite tasks. If the QX ONE Droplet Digital PCR System experienced a failure during
the run, the Recover Plate button is enabled. For more information on adding and recovering plates, referto the QX ONE Droplet Digital PCR System Instrument Guide.

22 QX ONE Software, Regulatory Edition

Tabs to Functional Windows

Run Status Window
You can view the Run Status window on the QX ONE Droplet Digital PCR System touch screen.
Note: If you are using the software on a computer other than the touch screen, the Run Statuswindow is not available.
Users with the Manage Plate user permission can reorder plates in the software that are still in the
instrument Inbox and cancel plates before or during the run. For information, see the QX ONE Droplet
Digital PCR System Instument Guide.
The Run Status window displays information onn

The runs in process in the top panen

Up to 100 completed runs for the current user in the bottom pane, including plate failures andrecoveries
Note: You set the number of completed runs to show in your user preferences. For information,
see Managing Your Preferences on page 18.

User Guide | 23

Chapter 1 About QX ONE Software

Data Analysis Window
The Data Analysis window displays a list of analysis files created at the end of each run.

From the left panel, you can select files from the following storage areas to open in the Data Analysis
module:n

Recent Datafiles
A list of files that you have opened.

n

My Datafiles or System Datafiles
If the system administrator haso

Not set shared locations in System Settings, all data files are automatically saved to the file path
specified in the user’s preferences.

o

Set shared locations in System Settings, all data files are automatically saved to the path
specified for Datafile Storage Location. Preferred locations override individual user preferences.

Important: For either choice, the specified path must be a local folder on the QX ONE Droplet
Digital PCR System touch screen computer.
Note: For information on files types for your software and edition, see Compatible File Types on
page 30.
n

From the right panel, you can access the Gene Study module. For information, see Gene Study
Module on page 139.

24 QX ONE Software, Regulatory Edition

Tabs to Functional Windows

Chapter 1 Creating or Modifying Templates
The Template Setup window allows you tonCreate new plate, thermal cycling protocol, and report templatesn

Search for existing templatesnModify existing templates

If you are assigned the Create New Template user privilege, you can save new or modified templates forreuse. You can save templates to a personal or system folder (My Templates or System Templates,
respectively), or you can save them to the Shared Templates location.
Note: If your system administrator has specified Preferred Locations in the System Settings window,
the System Templates location overrides the folder specified in your user preferences..

For information on creating or editing templates, seenCreating or Modifying Plate Templates on page 33nCreating or Editing Thermal Cycling Protocol Templates on page 55nCreating or Modifying Report Templates on page 63

User Guide | 25

Chapter 1 Creating or Modifying Templates

System Status Window
The System Status window displays information about your QX ONE Droplet Digital PCR System and QXONE Software.

Depending on your permission level, you cannView or change shared storage folder locations
Important: System administrators can override individual storage locations and set Preferred
Locations for data files and templates. Only administrators can establish or change theselocations.

nView event and maintenance logs, and maintenance reportsnArchive raw data, recover the data from a failed plate run that is stored in a temporary file, and accesstroubleshooting data for review.

Note: The Instrument Calibration tab is enabled only for the Bio-Rad service engineer user account.

26 QX ONE Software, Regulatory Edition

Tabs to Functional Windows

User Management Window
The User Management window displays your active user privileges and user preferences. Your privileges
are assigned to you by a user (usually an administrator) with the Add/Manage users privilege. You cannotchange privileges, but you can change the default settings in user preferences.
Note: Users who are not assigned any of the privileges listed can still execute runs and use theAnalysis module.

User Guide | 27

Chapter 1 Creating or Modifying Templates

Help Window
The Help window provides links to important information about QX ONE Software and Bio-Rad
Laboratories, Inc.

Select the Help tab to display links tonSoftware version informationn

End user license agreement (EULA); see Appendix B, End User License Agreement for information.

n

Bio-Rad technical supportn

Bio-Rad web sitenOpen-source software license information

Note: You can also link PDFs of the instrument and software user guides. For information, see
Linking the User Guides in the Help Window on page 29.

28 QX ONE Software, Regulatory Edition

Linking the User Guides in the Help Window

Linking the User Guides in the Help Window
To facilitate access to user assistance documentation, you can link the instrument guide for the QX ONE
Droplet Digital PCR System and the user guide for your software edition (standard or regulatory).

Important: If you have not received PDFs of the instrument guide and applicable user guide, you candownload them from the Bio-Rad website.

To link the guides
1.

Navigate to the following folder:
C:\ProgramData\Bio-Rad\<Standard or Regulatory>

2.

Within the folder, create another folder and rename it UserGuides.

3.

In QX ONE Software. select the Help tab, and then click the Browse button beside View Software
Manual or View Instrument Manual.

4.

Navigate to the User Guides folder and click Open.
QX ONE Software links the guide. To open the PDF, click the View <manual> button.

5.

Repeat for the remaining guide.

User Guide | 29

Chapter 1 Creating or Modifying TemplatesCompatible File Types
This section describes the file types you can open in QX ONE Software.
The following applies if your organization is using more than one edition of the software:n

You can open files created using Regulatory Edition only in Regulatory Edition.

n

If you open a .qlps or .ddpcrs file in QX ONE Software, Regulatory Edition, the file opens and is saved
as a .ddpcrsone file.

Table 3. Compatible file types

File Type

Extension

DetailsPlate.ddpltPlate template file containing setup details to perform experiments;

template
Plate

this file type opens in the Plate Editor.
.csv

template
ProtocolImportable plate template file containing setup details in CSV format;
this file type opens in the Plate Editor.

.ddthpProtocol template file containing setup details to perform
experiments; this file type opens in the Protocol Editor.

Data.ddpcrsone

Contains the results of an experiment run performed on the QX ONE
Instrument using QX ONE Software, Premium Edition.
Note: If you open a .ddpcrs file in QX ONE Software, the file issaved as a .ddpcrsone file.

Data

.qlps

File type from earlier QuantaSoft™ regulatory versions, for twochannel experiment runs and data analysis; this file type opens in the
Analysis module.

30 QX ONE Software, Regulatory Edition

Compatible File Types

Chapter 1 Creating or Modifying Templates
The Template Setup window allows you tonCreate new plate, thermal cycling protocol, and report templatesn

Search for existing templatesnModify existing templates

If you are assigned the Create New Template user privilege, you can save new or modified templates forreuse. You can save templates to a personal or system folder (My Templates or System Templates,
respectively), or you can save them to the Shared Templates location.
Note: If your system administrator has specified Preferred Locations in the System Settings window,
the System Templates location overrides the folder specified in your user preferences..

For information on creating or editing templates, seenCreating or Modifying Plate Templates on page 33nCreating or Editing Thermal Cycling Protocol Templates on page 55nCreating or Modifying Report Templates on page 63

User Guide | 31

Chapter 1 Creating or Modifying TemplatesThis page intentionally left blank.

32 QX ONE Software, Regulatory Edition

Creating or Modifying Plate Templates
If you are assigned the Create New Templates user privilege, you can save a plate as a template forreuse.
When you choose to add or modify a plate template, the Plate Editor opens, featuring options to set up
your samples in the plate layout and exclude certain wells from droplet generation and droplet reading or
droplet reading only.

LEGEND
Buttons at the top of the window provide quick access to plate editing functions.
The right pane contains the interface for defining your experiment parameters.
The left pane displays the plate grid and configuration information in each well.
The Well Data button toggles to the Well Data table, where you can see your plate setup in atabular format, and then back to the Plate Editor. When the Well Data table appears, the button
name changes to Plate Layout. You cannot edit the Well Data table.

User Guide | 33

Templates that are available to you appear in the default view of the Template Setup tab, as follows:n

Templates created and saved by any user, and designated as shared, appear under Shared
Templates.

nTemplates you created and saved, which are saved to the path you specified in your user
preferences, and appear in your My Templates folder.

Important: If your system administrator has selected the Preferred Locations checkbox in System
Settings, all run files and templates are saved to that location, overriding the location indicated in youruser preferences.

34 QX ONE Software, Regulatory Edition

Creating or Modifying Plate Templates

From the Template Setup or Add Plate window, you can create or modify plate templates. From theTemplate Setup window, you can also modify reports.

\
For detailed information on creating protocol templates, refer to the QX ONE Software User Guide.

User Guide | 35

Opening a New Plate Template
You can open a blank plate template from either the Template Setup window or the Add Plate window.
n

Use Template Setup if you are creating one or more templates to store for future use.

n

Use Add Plate if you are creating a template from a plate you are about to run.

To open a new plate template from the Template Setup window
1.

Tap the Template Setup tab.

2.

Tap Plate Setup.

3.

Tap Edit Plate Layout.
The Template Editor opens, displaying a blank plate layout.

To open a new plate template from the Add Plate window
1.

Tap the Add Plate tab.

2.

Select Plate Setup.
The Plate Configuration window opens to the Plate Information tab by default.

3.

Under Plate Template, tap Create New.
The Template Editor opens, displaying a blank plate layout.

36 QX ONE Software, Regulatory Edition

Opening an Existing Plate Template

Opening an Existing Plate Template
You can open an existing plate template from either the Template Setup window or the Add Plate window.
n

Use Template Setup if you are editing one or more templates to store for future use.

n

Use Add Plate if you are editing a template from a plate you are about to run.

To open an existing plate file from the Template Setup window
1.

Tap the Template Setup tab.

The Plate Setup window appears by default and a list of available plate templates appears.
2.

Select a template and tap Edit Plate Layout.
Tip: You can also double-click the template file to open it.
The Plate Editor opens, displaying the plate template as it is currently configured.

To open an existing plate template from the Add Plate window
1.

Tap the Add Plate tab.

2.

Under Choose existing, select a template from the drop-down list.
The template Plate Editor opens, displaying the template as it is currently configured.

User Guide | 37

Template Plate Editor Window
Use the Template Plate Editor tonCreate new or edit existing plate template files, and save new or modified templates for reusen

Add up to four sample descriptions per welln

Exclude wells from droplet generation and droplet reading, or just droplet reading
Important: When you create an experiment from a template with wells excluded in the platelayout, the instrument pierces the foil for all wells in the physical plate, but generates and/or
reads droplets for active wells only.

To open the template plate editor
1.

Click the Templates tab on the left.

2.

In the Templates window, tap Edit Plate Layout

38 QX ONE Software, Regulatory Edition

Opening an Existing Plate Template

You can also open the template plate editor from the Add Plate window.

LEGENDThe Edit and Exclude buttons are accessible from the top of the screen.
The right pane contains the interface for defining your experiment parameters.
The left pane displays the plate grid and configuration information in each well.
The Well Data button toggles to the Well Data table, where you can see your plate setup in atabular format, and then back to the Plate Editor. When the Well Data table appears, thebutton name changes to Plate Layout.

User Guide | 39

Plate Editor Tools
Use the buttons described in Table 4 when you are creating a template or defining an experiment in the
Plate Template Editor.
Table 4. Plate Editor Buttons

ButtonEdit

Description
Allows you ton Set the experiment type, sample name and type, supermix, and assay
type
n Set the target name, type, and fluorophore for each channeln Add well or plate notes

ExcludeToggle button that allows you to include or exclude wells from droplet
generation and droplet reading, or from droplet reading only

CancelAllows you to cancel and return to the Template Setup window

Save

Opens a dialog box where you can enter a file name and storage location

Clear Selected Wells

Allows you to delete information from selected wells

Undo

Allows you to reverse your last action
Note: This button is enabled only after you have applied at least one
experiment parameter (for example, the selection for Experiment Type).

Well Data or Plate Layout

Toggle button that allows you to access the Well Data table and return toPlate Layout view

Apply

Applies your entries and choices for selected wells

40 QX ONE Software, Regulatory Edition

Opening an Existing Plate Template

Template Setup — Setting Up Your Wells
When you open the Plate Editor from the Template Setup tab, the Edit button is selected by default. You
can assign different experiment parameters to each well or groups of wells.

For each well, or group of wells, you mustnChoose an experiment typen

Select a sample typen

Select a supermixnSelect an assay method

Optionally, you cannEnter up to four sample descriptors for each wellnModify fluorophore assignmentsn

Add notes for each welln

Add notes for the plate

If you are creating a new template, the grid is blank. If you are editing an existing template, previously
saved information appears when you select one or more configured wells.

User Guide | 41

To define well parameters
1.

Select a well or group of wells.

2.

From the Experiment Type dropdown list, select a new or different experiment type. For more
information, see Experiment Types on page 44.

3.

In the Sample Description fields, enter or change up to four words or phrases that describe thesample. For more information, see Sample Descriptions on page 45.

4.

From the Sample Type dropdown list, select a new or different sample type. For more information,see Sample Types on page 45.

5.

From the Supermix dropdown list, select or change the supermix. For more information, seeSupermixes on page 46.

6.

The Assay Type field populates automatically based on your experiment selection. To change it,
select a different assay type from the dropdown list. For information, see the fluorophore options in
Assay Types and Fluorophores on page 47.
Note: Available assay types vary, depending on the selected experiment type.

7.

(Optional) Under Target Info, change the fluorophore assigned to a channel.
The fluorophore information populates automatically for each target, with the maximum number ofrows allowed for the assay method, but you can modify the default fluorophore assignments, if
applicable.
a.

Tap the field dropdown arrow and select an alternative.

b.

To delete a row, tap the minus (-) icon next to the target.

c.

To re-add a row, tap the plus (+) icon.

8.

(Optional) To clear one or more wells at any time, select the wells and tap Clear Selected Wells.

9.

Tap Apply.
Note: You must tap Apply for the software to recognize your new or edited information, andbefore you can save the template. Once applied, you can pause on a well to see thecorresponding information in a tool tip.

42 QX ONE Software, Regulatory Edition

Opening an Existing Plate Template

10. Exclude wells in the plate from droplet generation and droplet reading, or from droplet reading only.
For information, see Template Setup – Excluding Wells.
Important: The QX ONE Droplet Digital PCR System processes all 96 wells in the plate by
default. If you have filled fewer than 96 wells with sample or buffer, you should exclude theremainder.
11. If you are finished with your plate configuration, tap Save in the upper-right corner and then in the
Save dialog box, enter a new name for the template and tap Save again.
Note: You must save the template with a new name. You cannot overwrite existing templates. If
you tap the Save button before the plate layout is complete, the file closes and you must reopen
it from the saved template location.

User Guide | 43

Experiment Types
QX ONE Software offers the experiment types described in Table 5, each enabling different assay options
and analysis tools. You can assign multiple experiment types across the plate, but you can assign only
one experiment type to each well.
Table 5. Experiment types

Type

Description

Direct

DQ uses absolute quantification to determine the concentration (copies/μl) of target

quantification

DNA copies in a sample. DQ measures the number of positive and negative droplets(DQ)

for each target in a well based on their fluorescence amplitude, and uses a Poisson
algorithm to calculate the starting concentration of each target DNA molecule.
Note: The ABS experiment type created in QuantaSoft Software versions 1.4
through 1.7 is automatically mapped into the software as a DQ experiment type.

Copy number

CNV determines concentration (as with DQ) and calculates the copy number of anvariation (CNV)

unknown (CNV) target relative to a known reference or references within the same
well.

Mutation

MUT determines concentration (as with DQ) and calculates the fractional abundance

detection (MUT)

of an unknown mutant present at a low frequency in a wild-type background.

Rare event

RED determines the concentration of a known mutant or a rare target species relative

detection (RED)to a given reference species within a large pool of background DNA.

Drop-off (DOF)

DOF determines absolute quantification of targets for assays designed to detect nonwild type sequences, such as indels and genome edits; DOF is designed to support anassay strategy where one probe counts all alleles and one “drop-off” probe sits on topof a predicted cut site or mutation site.

Gene expression

GEX determines concentration (as with DQ) and calculates relative expression levels(GEX)of an unknown target relative to a known reference or references within the same well.

Residual DNA

RDQ provides a precise method for residual host cell DNA quantification.

quantification

Note: Users can enter a conversion factor to calculate the mass for a well, up to(RDQ)

three decimals. The software displays the result in the Molecular Weight columnof the Data Table.

44 QX ONE Software, Regulatory Edition

Opening an Existing Plate Template

Sample Descriptions
QX ONE Software allows you to enter descriptive words or phrases for your sample in up to four fields per
well. Descriptors can include information such as research type, dilution factors, and so forth.
Tip: Only the information from the first field appears in the Plate Editor layout. However, you can see
entries in the second, third, and fourth fields in the Well Data table, or you can pause on a well to
display a tool tip, as shown in the following graphic.

Sample Types
You can assign multiple sample types within the plate, but you can assign only one sample type to eachwell.
QX ONE Software provides four sample types for selection:nUnknownn

NTC (no template control)n

Pos Ctrl (positive control) where one or more targets are expectedn

Neg Ctrl (negative control), where no response is expected

User Guide | 45

Supermixes
QX ONE Software offers five PCR supermixes. Each supermix is optimized to deliver maximum PCR
efficiency and sensitivity for the amplification and detection of DNA and RNA targets.
Important: Use of unapproved supermixes can harm the instrument and void the warranty.

ddPCR Supermix

Description

Multiplex Supermix

For use in nucleic acid sample preparations to amplify and detect DNA
targets in probe-based experiments involving multiple targets.
Note: Bio-Rad recommends using ddPCR Multiplex Supermix for
Cy5 and Cy5.5 probes assays.

Supermix for Probes (NoFor use in nucleic acid sample preparations, with sensitivity for thedUTP)

amplification and detection of DNA targets using hydrolysis probebased assays.

Supermix for Probes

For use in sample preparation with uracil N-glucosylase (UNG)
decontamination protocols to prevent the reamplification of carryover
PCR products between experiments.

Supermix for Residual
DNA Quantification

For use in residual DNA detection.

One-Step RT Advanced Kit for

For use in absolute quantification of target RNA molecules.

Probes
EvaGreen® Supermix

For use in gene expression.

The One Step RT ddPCR Advance Kit for Probes supermix is time and temperature-sensitive. Therefore,
if you are performing an experiment using this supermix, it is important that the plate does not sit idle in
one of the Inbox slots.

To perform an experiment using this kit
1.

Wait until all plates in the QX ONE Droplet Digital PCR System have processed and then place the
One Step RT plate into the first slot.

2.

Quickly set up and start the run.

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Opening an Existing Plate Template

Assay Types and Fluorophores
QX ONE Software offers the assay methods explained in Table 6.
Table 6. Assay types
Assay type

Description

Single target per

Method assuming up to four probe colors (FAM, HEX/VIC, Cy5 and Cy5.5) and up tochannelfour targets per well, with a single target detected per channel.
This is the default assay type, with the maximum four rows displayed. You can delete
up to three rows.

Amplitude

Method to increase multiplexing up to eight targets per well, with one or two targets

multiplex

detected per channel. The maximum of eight rows are displayed. You can delete up toseven rows.

Probe mix triplex

Triplex mode that allows two groups of three targets each.
Group 1 has one target in FAM, one target in HEX/VIC, and one target in bothchannels. Group 2 has one target in Cy5, one target in Cy5.5, and one target in both.
You can delete a group, but not the individual targets within the group.
Note: The channels within a group are not interchangeable; therefore FAM isalways paired with HEX/VIC and so forth.

Advanced

Method to increase multiplexing rate in any combination of targets detected perclassification

channel. This method requires manual classification of droplets.

Basic drop-off

This mode allows two groups of two targets each. The first group appears by default,
with one target in FAM and HEX/VIC and one target in FAM or HEX/VIC.
This assay type is available only for the DOF experiment type.
You can add one more group of two targets, with one target in Cy5 and Cy5.5, one
target in Cy5 or Cy5.5. You can delete a group, but not the individual targets in thegroup.
Note: The channels within a group are not interchangeable; therefore FAM isalways paired with HEX/VIC and so forth.

User Guide | 47

Table 6. Assay types, continued
Assay type

Description

Double drop-off

This assay type is available only for the DOF experiment type, and allows two groupsof three targets each.
The first group appears by default, and looks for one target in FAM and in HEX/VIC(target type reference) one target in FAM (target type unknown), and one target in
HEX/VIC (target type unknown).
You can add the second group to look for one target in Cy5 and Cy5.5 (target typereference, with two reference copies), one target in Cy5 (target type unknown), andone target in Cy5.5 (target type unknown).
By default, all reference copies are set to 2.
The channels are static for each group. (Channel 1 FAM is always paired with Channel
2 HEX/VIC,Channel 3 Cy5 is always paired with Channel 4 Cy5.5..

The experiment type you select determines the available assay methods.Table 7 displays the sample
type associated with each assay method.
Table 7. Assay types by experiment type
Experiment type
Assay type
DQ

CNV

MUT

ü

ü

Amplitude multiplex

üProbe mix triplex

üSingle target perchannelAdvancedclassification

DOF

GEX

RDQ

ü

ü

ü

ü

ü

ü

ü

ü

ü

ü

ü

ü

ü

ü

RED

ü

ü

Basic drop-offüDouble drop-offü

QX ONE Software automatically populates the fluorophore in each channel for each target but you canchange the default selections.
Use Table 8 to select from the fluorophore options for each assay method listed.

48 QX ONE Software, Regulatory Edition

Opening an Existing Plate Template

Table 8. Fluorophore options
SignalCh1

Assay method

SignalCh3Signal
Ch2

SignalCh4Single target per

FAM

HEX

Cy5

Cy5.5

channel, for1 to 4

None

VIC

None

None

targets

None

Amplitude

FAM HiHEX Hi

Cy5 HiCy5.5 Hi

multiplex, for

FAM LoHEX LoCy 5 LoCy 5.5 Lo

None

VIC HiNone

None

1 to 8 targets

VIC LoNone
Probe mix triplex,

FAM

HEX

Cy5

Cy5.5

for

None

VIC

None

None

6 targets

None

Advanced

FAM

HEX

Cy5

Cy5.5

classification+1-10

1 to 10

1 to 10

1 to 10

None

VIC

None

None

1 to 10
None

User Guide | 49

Template Setup – Excluding Wells
Optionally, you can exclude wells from the Droplet Digital PCR run.
Important: The QX ONE Droplet Digital PCR System processes the entire 96-well plate be default. If
you have added your sample to fewer than 96 wells in the plate, you must exclude the remainder.
The GCR96 cartridges that are inserted into the instrument contain six separate sections, each with tworows of eight wells each. The QX ONE Droplet Digital PCR System automatically generates droplets bysection rather than by well or by individual column. In the QX ONE Software well exclusion design, the firsttwo columns represent the first section, the second two columns the second section, and so forth.
When you select Droplet Generation and Droplet Reading, the software allows you to exclude eachsection from both processes, but if you try to exclude an odd number of columns, QX ONE Software
automatically selects the remaining column.
When you select Droplet Reading, the software allows you to exclude any well or group of wells from thedroplet reading phase.

50 QX ONE Software, Regulatory Edition

Opening an Existing Plate Template

Wells excluded from droplet generation and droplet reading are displayed with a criss-cross pattern.

Wells excluded from droplet reading are identified by a diagonal pattern.

To exclude columns from droplet generation and droplet reading1.

Select one or more column pairs to exclude, and then tap Droplet Generation and Droplet Reading.

Note: If you selected an odd number of columns, the software automatically selects theremaining column to represent a section. You can exclude that column from droplet reading only.
2.

Tap Exclude Selected Wells.

User Guide | 51

To exclude wells from droplet reading1.

Select one or more wells to exclude, either individually or in groups, and then tap Droplet Reading.
Tip: To exclude multiple wells, press the CTRL key, and then select the wells to exclude.

2.

Tap Exclude Selected Wells.

Note: The Exclude Selected Wells button is a toggle button. If you select wells that have been
excluded, the button name changes to Include Selected Wells. If you excluded one or more wells
that should be read, you can select the wells and then tap the button to include them.

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Opening an Existing Plate Template

Creating and Storing Plate Templates from CSV Files
You can create a plate template in a spreadsheet application, save it in .csv format (CSV), and then store
it for use in your droplet reading runs.
Note the following:n

When you create a CSV template, the headings for the columns containing the experiment
information must match the fields and order in the Plate Editor. If they do not match, an error
message appears when you select the template.

nAfter you specify the information in row 1, do not change the field entries until you install a newsoftware version or edition.

n

You can store the CSV file in any location, but the Browse option defaults to your personal templates
storage folder defined in your user preferences (see Managing Your Preferences on page 18), or theshared or preferred location specified by your system administrator (see Shared Settings on page152.

n

CSV templates do not appear in the Templates window. You can select a CSV template file from thePlate Setup dropdown list, or, after you tap Browse, you can navigate to the storage folder and selectthe file.

User Guide | 53

To create and save a CSV template
1.

Open a spreadsheet program and enter the run settings into the template in the format shown below.
Fig. 1: Template CSV Layout Example, Columns A through H

Fig. 2: Template CSV Layout Example, Columns I through R

2.

When you finish populating the fields, navigate to your templates storage location and save the CSV
file to ensure it is available from the Choose Existing drop-down list.

54 QX ONE Software, Regulatory Edition

Creating or Editing Thermal Cycling Protocol Templates
Thermal cycling protocols are specific to the thermal cycling ddPCR phase. When you add or modify aprotocol template from the Template Setup window, the Protocol Editor opens. By default, the editor
opens to a generic 4-step protocol, which includes standard protocol controls to quickly create protocols,
as well as the ability tonQuickly calculate a gradient for a specific temperature range and run time for the plate typen

Edit protocol stepsn

Save protocols for reuse

LEGENDThe lower pane displays the following toolbar commands:
n The trashcan command deletes the selected step.
n The plus sign to the left of the trashcan adds a step before the selected step.
n The plus sign to the right of the trashcan adds a step after the selected step.
The upper pane displays the protocol controls that you can use to customize the protocol.

User Guide | 55

LEGEND , CONTINUED
The main pane displays a graphical representation of the protocol.
The information bar provides quick access to the name of the protocol, view the estimatedprotocol run time, and to save or cancel changes to the protocol.

From the Templates tab you can access the Protocol Templates window, where you can create a new oropen an existing protocol template in the Protocol Editor window. Using the Protocol Editor, you cancreate custom protocols that are specific to the thermal cycling phase of the QX ONE Droplet Digital PCR
System.

For detailed information on creating protocol templates, refer to the QX ONE Software User Guide.

56 QX ONE Software, Regulatory Edition

Opening a New Thermal Cycling Protocol

Opening a New Thermal Cycling Protocol
QX ONE Software provides the following two options to open a new protocol template:n

From the Template Setup windown

From the Add Plate window

To open a new protocol template from the Template Setup window
1.

In the Template Setup window, select the Thermal Cycling Protocol tab.
The Thermal Cycling Protocol window appears, displaying a list of sample and custom protocol
templates.

2.

Tap Add New Protocol.
The Protocol Editor window opens, displaying the default 4-step protocol.

To open a new protocol template from the Add Plate window
1.

In the Plate Setup window, tap Add Plate.

2.

After the plate is loaded, tap Configure Plate.
The Plate Setup window opens, displaying the Required Plate Information window.

3.

In the Thermal Cycling Protocol pane, tap Create New.
The Protocol Editor window appears, displaying the default 4-step protocol.

User Guide | 57

Opening an Existing Thermal Cycling Protocol
QX ONE Software provides sample thermal cycling protocol templates that you can edit and save as new
templates. You can also edit and save a custom protocol as a new protocol template.
To open an existing protocol from the Template Setup window
1.

Select the Thermal Cycling Protocol tab.
The Thermal Cycling Protocol window appears, displaying a list of sample protocol templates.

2.

Locate the protocol of choice.

3.

Select the protocol and tap Edit Protocol at the bottom of the window.
The protocol opens in the Thermal Cycling Protocol Editor window.

4.

(Optional) tap Save to save the protocol with a new name or in a new storage location.

To open an existing protocol from the Add Plate window
1.

In the Plate Setup window, tap Add Plate.

2.

After the plate is loaded, tap Configure Plate.
The Plate Setup window opens, displaying the Required Plate Information window.

3.

In the Thermal Cycling Protocol pane, tap the Choose Existing dropdown arrow, and then select anexisting protocol.

4.

Tap the “i” button to the right of the dropdown arrow.
The Protocol Editor window opens, displaying the selected protocol in a pop-up window.

5.

Tap Edit.

58 QX ONE Software, Regulatory Edition

Thermal Cycling Protocol Editor Window

Thermal Cycling Protocol Editor Window
The Thermal Cycling Protocol Editor includesnStandard protocol controls to quickly create or edit protocolsnAbility to quickly calculate a gradient for a specific temperature range and run time for the plate typen

Ability to edit protocol steps and save protocols for reuse

By default, the window opens to a generic protocol with four steps.

LEGEND
The top section provides quick access to the name of the protocol, estimated protocol run time,and save or cancel functions.
The upper pane displays the protocol controls that you can use to customize the protocol.
The main pane displays a graphical representation of the protocol.
The lower pane displays the following toolbar commands:
n The trashcan command deletes the selected step.
n The plus sign to the left of the trashcan adds a step before the selected
step.
n The plus sign to the right of the trashcan adds a step after the selected
step.

User Guide | 59

Adding Steps to a Protocol Template
To add a step
1.

In the Protocol Configuration window, determine where to insert the new step. Step 1 is selected bydefault. You can add a step before or after an existing step.

2.

On the graph, select a step and do one of the following:3.

n

To insert the new step before the selected step, tap the + icon to the left of the trash can.

n

To insert the new step after the selected step, tap the + icon to the right of the trash can.

Do one of the following:n

To make the step a gradient step, tap the Gradient checkbox.

n

To make the step a goto (repetition) step, tap the Goto checkbox.
Note: If the step type can be converted to Goto, the Goto checkbox is enabled.

4.

Do one of the following:n

To adjust the values in the protocol step options before saving, see Adjusting Step Options on
page 61.

n

To save the protocol with the default step values, tap Save. In the Save dialog box, enter a namefor the protocol and tap Save again.
Note: The protocol closes after you save it. You can reopen the protocol from the Protocol
Templates window.

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Adding Steps to a Protocol Template

Adjusting Step Options
QX ONE Software enables you to change the step options in an existing or added step to customize theprotocol. You can set values for the following step types:nTemperaturen

Gradientn

Goto

Important: You can save the changes to the steps as you go, or save them all at once when you arefinished with your modifications.
To configure a temperature step
1.

From the graph, select the temperature step containing the parameters to be changed.
Temperature steps contain a single temperature line.

2.

Under Step Options, change or enter the following values where applicable:nTemperature (° C)—target temperature (0 to 100° C) for the stepnTime (HH:MM:SS)—hold time (0–64800 sec) for the step
The hold time is the duration after the block has reached the target temperature.

nRamp Rate (° C/sec)—(Optional) the rate (0.1–5° C per second) at which the instrument heats orcools to achieve the target temperature.

n

Increment (° C/cycle)—(Optional) the degrees to increase or decrease the step's temperature
with each successive cycle. The entry must be between -10 and 10° CnExtend (sec/cycle)—(Optional) amount of time (-60–60 sec) to increase or decrease the step's
hold time with each successive cycle.

User Guide | 61

To configure a gradient temperature step
1.

From the graph, select the gradient step containing the parameters to be changed.

2.

Under Step Options, change or enter the following values where applicable:nTemperature (° C)—temperature (30.0–99.0° C) of the last row in the plate.

nTime (HH:MM:SS)—hold time (0–64800 sec.) for the step.
Hold time is the duration after the block has reached the target gradient.
Note: Note: Setting the time to 0 sec instructs the instrument to hold at the current step until
resumed manually from the instrument’s user interface.

nExtend (sec/cycle)—(Optional) amount of time (-60–60 sec) to increase or decrease the step's
hold time with each successive cycle.

nRange—(1°–16° C) range between the lowestr temperature and the highest temperature.
Note: The temperatures for rows A through H automatically populate with updated values
when you change the temperature or temperature range. You can also change thetemperature for each row, and the temperature and range are automatically updated.

To configure a goto (repetition) step
A Goto step loops back to an earlier step and repeats a sequence.
1.

From the graph, select the Goto step containing the parameters to be changed.

2.

Under Step Options, change the following value, if applicable:n

Goto step number—step to which to return to repeat a sequencenNumber of repeats—number of times to repeat the sequence

Deleting Steps from a Protocol Template
Important: You cannot undo this function. Take care when deleting steps.
To delete a step in the protocol
1.

Select the step in the graph.

2.

Tap Delete Step.

3.

Save the protocol.

62 QX ONE Software, Regulatory Edition

Creating or Modifying Report Templates

Creating or Modifying Report Templates
If you are assigned the Create New Templates user privilege, you can create and save a report templateby editing the default report structure or by opening and editing an existing report.

To create or edit a report template1.

Select the Template Setup tab.

2.

Tap Reports and do one of the following:n

To create a new template, tap Add New Template. and tap Edit Report Template.

n

To open an existing template, select the template and tap Edit Report Template.

User Guide | 63

3.

Tap the arrow to the left of each category to expand the list of reportable items.
Important: The Audit Log checkbox is enabled in the Regulatory Edition only.

4.

Select and clear checkboxes to create the report structure.
Note: Use the buttons at the bottom of the screen to expand or collapse all categories at once, orto select or clear all checkboxes at once. You can also drag and drop the section to rearrange
the order of appearance in the report, and drag and drop items in the section to reorder them.

5.

Enter a name for your report in the Report Title field and tap Save.

64 QX ONE Software, Regulatory Edition

Creating or Modifying Report Templates

6.

When the Save dialog box appears, enter a file name.

7.

Select a storage location. Choose from the following:

8.

nSystem Templates (preferred location, if specified by the administrator)n

My Templates (personal storage location specified in user preferences)n

Shared Templates (files saved to this location are available to all users)

Tap Save again to save the report format as a template.

User Guide | 65

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66 QX ONE Software, Regulatory Edition

Chapter 3 Data Analysis Module Overview
As the instrument reads droplets in each well, the QX ONE Droplet Digital PCR System collects raw data.
At the end of each run, QX ONE Software uses the raw data to produce a data file, which contains
analysis information based on the number of positive and negative droplets identified for each target ineach sample. The fraction of positive droplets is fit to a Poisson algorithm to determine the starting
concentration of the target DNA molecule in units of copies/µl input.
This section summarizes the analysis functionality, and also describes the general options governing yourchart displays. More detailed information, including specific information and uses for each analysis
window, as well as information on data calculations, is contained in Chapter 4, Data Analysis
Methodology.
When you select the Data Analysis tab from the touch screen, you can access the Data Analysis module.
When you select the tab from the software installed on a standalone computer, you can also access theGene Study module.

When you open the data file in the Analysis module, you can view the data with the automatic analysis
thresholds and calculations, or you can choose different options to recalculate, replot, or recolor the
results.

User Guide | 67

Chapter 3 Data Analysis Module Overview

The Data Analysis module functionality allows you tonView your droplet data in 1D, 2D, and 3D plotsnAnalyze multiple targets within a well using amplitude multiplexing and probe mixing strategiesnDetect genomic editing events and non-wild type events with a “drop-off assay” analysis optionnVisualize and export data with improved flexibilitynGenerate reports for different analysis typesn

The Gene Study module allows you tonCollect data by adding one or more data filesnView gene expression calculations across multiple experiments

Important: The Gene Study module is accessible from a standalone computer only. The maximum
number of samples you can analyze in a gene study is limited by the size of the RAM and virtual
memory of the standalone computer.

68 QX ONE Software, Regulatory Edition

Opening a Data File for Analysis

Opening a Data File for Analysis
You can open the following file types in the Data Analysis module:
n

.qlps

n

.ddpcrsn.ddpcrsone

To open data files, you must be on a computer on which QX ONE Software is installed and have access tothe storage locations.

To open a data file for analysis
1.

In QX ONE Software, select the Data Analysis tab.

Available data files are displayed under the following headings.
nRecent Datafiles
Displays a list of files you have recently opened.

nSystem Datafiles or My Datafiles
Files are always saved to one of the following locations:no

If preferred locations are enabled, then files for all users are saved there and they appear
under System Datafiles.

o

If preferred locations are not enabled for all users, your files are saved to your personal
folder and appear under My Datafiles.

Shared Datafiles
You can also save your data files to a shared location; these appear under Shared Datafiles.

User Guide | 69

Chapter 3 Data Analysis Module Overview

2.

Tap a file to open it in the Analysis module.
Tabs for each analysis and data window appear on the left of the Analysis window.
Tip: From the touch screen, you can also open the data file from the Run Status window. In thecompleted files section, tap the completed run icon () for the file, and then tap LaunchAnalysis.

Saving a Data File
You can save data files with or without modifications, with the same or different file names, and in thesame or different locations.
Important: When you save a .qlp.qlps.ddpcrs file type in QX ONE Software, the software
automatically saves the file as a .ddpcrsone file. This creates a new file and does not overwrite theoriginal.

To save a data file1.

2.

Do one of the following:n

To save the file under the same file name and in the same location, tap Save.

n

To save the file under a different file name or in a different location, tap Save as, and then do oneof the following:o

If you are saving the file under the same name and in the same location, QX ONE Software
saves the file immediately.

o

If you are saving the file under a different file name or in a different location, navigate to the
new location (if applicable) and enter the new file name. You must be assigned theOverwrite existing file name user privilege to change the file name.

Tap Save.

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Analysis Dashboard
When you open a data file, the Analysis module opens to a Dashboard window containing default analysis
and data panes in a summary view. You can customize the view to change panes, or increase or
decrease the number of panes. For information on the available options in the Dashboard view, see
Dashboard Options on page 72.

Note: If you open a .qlps file, the Plate Editor opens as the default view and you must select the
Dashboard tab to open it.
From the tabs on the left, you can access plate views, amplitude charts, data visualization distribution
charts, droplet counts, and a complete data table. You can also view data specific to the run in the Run
Information window, and run reports on any of the analysis charts from the Reports window.

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Chapter 3 Data Analysis Module Overview

Dashboard Options
From the Dashboard window, depending on the individual dashboard window you have selected, you can
use a range of options as described below:nWhen you select a window, the options that appear in the menu bar correspond to the options you
see if you open the same window from the side menu. For example, if you select the Concentration
chart in the Dashboard, the Concentration menu options appear.

n

You can reset the entire dashboard display by adding or removing rows or columns to display more or
fewer individual windows. For information, see Modifying the Dashboard Display on page 74.

Tap Default View to return to the default display of two rows and two columns.
nFrom any window you canoExpand or collapse the windowoCopy, save, or print the imageoSelect display, scale, axis, and logarithmic optionso

Use the dropdown list to replace the current display with a different displayo

Use the menu bar options for the particular chart type

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Table 9 describes the option sets identified in the following graphic.

Table 9. Dashboard options

Item

Description
Border to identify selected window
Toolbar corresponding to the selected window type in the Dashboard
Dropdown arrow to replace the current display with a new display
Chart options

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Chapter 3 Data Analysis Module Overview

Modifying the Dashboard Display
You can modify the column number and row number to display any or all of the analysis windows
simultaneously.

To change the number of windows in the display
1.

Tap the dropdown arrow for Rows and then select a different number.

2.

Tap the dropdown arrow for Columns, and then select a different number.

You can also use the dropdown menus to change the windows that appear in the Dashboard.

To display a different window in a particular Dashboard slot
1.

Tap the dropdown arrow in the upper-right corner to display the list of available windows, and thenselect a window. The dropdown list closes and the window changes immediately.

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Analysis Display and Output Options
QX ONE Software provides the following option that you can choose or set:n

Universal Options — available from the primary toolbar to save, import, or export data filesnChart Display Preferences — to change how your chart appears in the analysis windownChart Scale Options — to change the scale represented on an axisnChart Menu Options — available in chart views (from theicon) to copy, save, and print chart

images. In Amplitude charts showing data points, you can show or hidenTable Menu Options — available in table views (from theicon) to copy data, export data to

Excel or CSV files, and show or hide columns
You can also expand the chart display to full-screen size and then return to the original display.

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Chapter 3 Data Analysis Module Overview

Universal Options
Universal options appear on the top menu bar, and are available from all windows, although functionality
within the main option might differ according to the analysis window that appears.

Universal options are explained in Table 10, and are available in all Analysis module windows.
Table 10. Universal options
OptionDescription

Save

Saves changes to the analysis file under its current file name or file extension

Save asSaves the file with a different name
If you open a file in the latest application that was created in an earlier application,
the file assumes the file extension of the new application. See Saving a Data File on
page 70 for information.

Import/export

n Imports or exports plate setup data to a CSV filen Imports or exports plate setup data to a plate template filen Exports visible data to a CSV filen Exports amplitude, cluster, and linkage datan Exports and prints the visible charts for the 2D plate layout

Undo

Enabled in the Plate Editor only, after you have made a change

User Settings

Allows you to restore your user preferences to the default settings

To import or export setup or analysis datau

Tap Import/Export.

Table 11 describes the available import/export options.
Note: If there is unsaved data, the software prompts you to save the file before its contents can beimported or exported.

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Table 11. Import/export options

Import/export option

Description

Import plate setup CSV

Import plate layout data from a CSV file to the Plate Editor
Note: This option is only available in the Plate Editor. The new platelayout overwrites the existing layout.

Export plate setup CSVExport a configured plate layout to a CSV file

Import plate setup

Import a plate layout from a plate template (.ddplt) file
Note: This option is only available in the Plate Editor.

Export plate setup

Export the plate layout to a plate template file
Note: This option is only available in the Plate Editor.

Export visible data to CSVExport only the data from the wells you selected in the Well Selector to a CSV
file

Export amplitude data

Export the data to an Excel spreadsheet, and indicates whether or not thetarget was found (1 = yes, 0 = no)Export cluster data

Export the data to an Excel spreadsheet
Note: Includes (for each well) whether or not the target was found (1 =yes, 0 = no), the droplet count, and the mean and standard deviation foreach dye, and the cluster ID.

Export linkage data

Exports to a CSV file the number of copies of a target that are present inoverabundance, versus. the expected value in copies per µL

Export visible charts

Exports all charts that are displayed

Print visible charts

Prints all charts that are displayed

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Chapter 3 Data Analysis Module Overview

Table 12 shows where the options are available.
Table 12. Option availability
Import/export
optionImport plate setup
from CSV
Export plate
setup to CSV
Import plate setup
Export plate
setup

Plate
editor

Plate
view

Plate
2D

Export cluster
data
Export linkage
data

Event
counts

Data
table

ü

ü

ü

ü

ü
ü
ü

data to CSV

data

Data
visualchartsü

Export visible

Export amplitude

Ampl
plots

ü

ü

ü

ü

ü

ü

ü

ü

ü

ü

ü

ü

ü

ü

ü

ü

ü

ü

ü

ü

ü

ü

ü

ü

ü

ü

ü

ü

ü

Export visible
chartsPrint visible
chartsNote: Amplitude plots are available in 1D, 2D and 3D, and Data Visualization charts are
Concentration, Copy Number, Ratio, and Fractional Abundance. In the Dashboard viewcorresponding options are available for each plot or chart displayed.

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Expanding or Reducing the Chart
You can expand or reduce the chart size in the following windows:nDashboardn

1D Amplituden

2D Amplituden

3D AmplitudenConcentrationn

Copy Number

n

Ratio

n

Fractional AbundancenEvent Counts

If you expand an amplitude or data visualization chart to full size, the Well Selector and the Well Data
table are hidden from view until you collapse the chart again.

To expand or reduce the chart size
1.

To increase the chart size to the full window, tap the2.

To reduce the window to its original size, tap theicon in the upper-right corner of the chart.
icon.

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Chapter 3 Data Analysis Module Overview

Selecting Chart Display Preferences
The Chart Display preferences primarily control font sizes and positioning. Table 13 explains eachpreference, as well as the windows in which it is available.
Table 13. Chart display preferences
Preference

Window

Description

Channel or Dye

Plate 2D

Select Channel or Dye to identify your chart accordingly

1D Amplituden If you select Channel, the channel number appears as

2D Amplitude
3D Amplitude

the chart titlen If you select Dye, FAM, HEX, Cy5, or Cy5.5 appears as
the chart title

Axis Data Label

1D Amplitude

Size

2D Amplitude

Select a different font size

3D Amplitude
Concentration
Copy Number
Ratio
Fractional Abundance
Event Counts
Chart Data Label

Concentration

Size

Copy Number

Select a different font size

Ratio
Fractional Abundance
Event Counts
Chart Marker Size

Concentration

Select a different marker size

Copy Number
Ratio
Fractional Abundance
Chart Marker Label

Concentration

Position

Copy Number
Ratio
Fractional Abundance

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Reposition the marker label

Analysis Display and Output Options

1.

Tap the Chart Display button.

To select chart display preferences1.

Tap the Chart Display button.

2.

Select your options.
The display changes immediately to reflect your choices.

3.

Do one of the following:n

To save your choices as your default settings, tap Save as my default. When the successmessage appears, tap OK to close the dialog.

n

To close the menu without saving the settings, tap anywhere in the chart display.

Selecting Chart Scale and Label Options
Chart scale options primarily control chart scale and label displays.

Each preference, as well as the windows in which it is available, is explained in Table 14 on page 82.

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Chapter 3 Data Analysis Module Overview

Table 14. Chart scale options

OptionDescription

Scale

Appears in options for all charts
The Scale option determines the numerical range for your chart data:
Auto — Selected by default; QX ONE Software automatically displays a scale tomatch the data distribution.
Fixed — Selecting Fixed enables the Axis fields, and allows you to manually adjust
the range and increment.

Axis

Appears in options for all charts
When the Axis fields are enabled, you can manually adjust the minimum (Min) andmaximum (Max) points for the data display on each axis, as well as the increments(Incr).
X — Horizontal axis
Y — Vertical axis
Note: You can modify the x axis scale in the 2D Amplitude chart only.

Secondary Y Axis

Appears in options for Concentration only
From the dropdown list, you can select from Channel 2 concentration, ratio, copy
number, or fractional abundance to display the corresponding data in a secondaryrow on the y axis.

Log

Appears in options for all Data Visualization charts (Concentration, Copy Number,
Ratio, and Fractional Abundance)
You can select the Log checkbox to display the y axis in base 10 logarithmic
format.
Note: In the Concentration chart, you can also select Log for the secondary yaxis.

InverseAppears in options for Ratio and Fractional Abundance only
The Inverse option equals the opposing number of the Ratio data point to a total of2, and the opposing number of the Fractional Abundance data point to a total of100.

Data labels

Appears in options for all Data Visualization charts
A data label appears by default for each point in the chart, but you can clear the
checkbox to remove the labels.

Grid Lines

Appears in options for all charts
Gridlines appear by default. You can clear the check box to remove them.

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To select chart scale and label options
1.

Tap theicon in the upper-left corner of the chart to display the corresponding chart options.

2.

To manually define the chart scale, select Fixed.
Note: Auto is the default setting.

3.

In the Min, Max, and Inc fields, enter your lower and upper limits, and the increment, for the y axis.
For 2D Amplitude, you can also change the scale on the x axis.

4.

For Concentration, Copy Number, Ratio, Fractional Abundance, and Event Counts, select an option
for a secondary y axis display. To change to a base 10 logarithmic scale on the y axis, select the Log
checkbox.

5.

For Ratio and Fractional Abundance, to view an inverse calculation, select the Inverse checkbox.

6.

To remove the data labels or grid lines from the display, clear the Data Labels or Grid Linescheckbox.

Using Chart Menu Options

Chart menu options are explained in Table 15.
Table 15. Chart menu options
OptionDescription

Copy

Copies the chart to the clipboard, which you can paste into a separate
document, such as an MS Word file

Save Image as

Saves the chart as an image file

Print

Prints the image

Show Point Values

Shows corresponding point values when you pause on a data point

Tip: Select the Show Point Values checkbox to see the data. Data isavailable for 1D and 2D amplitude only.

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Chapter 3 Data Analysis Module Overview

Chart menu options are available in the following windows:n

1D Amplituden

2D Amplituden

3D AmplitudenConcentrationn

Copy Number

n

Ratio

n

Fractional AbundancenEvent Counts

To use the chart menu options
1.

Select one or more wells in the Well Selector.

Corresponding data appears in the Well Data table and the chart.

2.

3.

In the upper-right corner of the chart, tap the menu icon () and select from the dropdown list.

nSelect Copy to copy the chart to the Clipboard. To paste the data, navigate to the location (suchas a Word file), right-click and select Paste.

n

Select Save Image As and follow the prompts to save the file.

nSelect Print and follow the prompts to print the image.

Tap anywhere in the display to close the dialog box.

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Using Table Menu Options

Table menu options are explained in Table 16.
Table 16. Table options

OptionDescription

Copy

Copies the information from the selected wells to the clipboard

Export to Excel

Exports the data to an .xlsx file

Export to CSVExports the data to a .csv file

Show/HideAdd or remove columns from a table display

Columns

When you select Show/Hide Columns, the following dialog box opens. Select or clear
check boxes to show or hide columns in the data table.

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Chapter 3 Data Analysis Module Overview

Table menu options are available in the following windows:n

1D Amplituden

2D Amplituden

3D AmplitudenConcentrationn

Copy Number

n

Ratio

n

Fractional AbundancenEvent Counts

Tip: You can access the menu icon from the individual windows, or from the windows in the
Dashboard display.

To use the table menu options
If you are using these options in the Data Table, skip to Step 3.
1.

Select wells in the Well Selector.

Corresponding data appears in the Well Data table.

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Analysis Display and Output Options

2.

In the upper-right corner of the table, tap the menu icon (n) and select from the dropdown list.

To copy the data from the selected wells to the Clipboard, select Copy.
Note: To paste the data, navigate to the location (for example, an Excel file) and thenright-click and select Paste.

n

To export your data, select Export to Excel or Export to CSV, and then follow the prompts to savethe file.
Note: Exporting to Excel exports the data in spreadsheet format, while Exporting to CSV
exports the data in comma-delimited format.

n3.

To show or hide the corresponding column in the table, select Show/Hide Columns, and thenselect or clear checkboxes.

Tap anywhere in the display to close the dialog box.

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Chapter 3 Data Analysis Module Overview

Plate View Windows
In the QX ONE Software Analysis module you can open the following windows that contain data on all
wells in the run:n

Plate Editor (analysis view) — Displays the experiment type, sample names, sample types,
supermixes, and assay type for each well in the run (similar to the Template Setup Plate Editor)n

Plate View — Displays concentration results in a text format for each processed welln

2D Plate View — Displays a two-dimensional amplitude chart for each processed well

Plate Editor — Analysis View
You can use the Plate Editor tab in the Analysis module to select wells, and then view or change theexperiment parameters. Similar to other Plate Editor displays, the Analysis Plate Editor displays theexperiment information for each well in the run.
Wells you select in the Analysis Plate Editor determine the data initially displayed in other analysis
windows.

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Plate View
The Plate View tab provides, within the plate layout grid, a text view of the calculated concentration foreach target in each well processed in the run. For information on the concentration results in chart format,
see Concentration on page 123.

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Chapter 3 Data Analysis Module Overview

2D Plate View
To facilitate comparisons of amplitude in the processed wells, the Plate 2D tab provides a
two-dimensional amplitude view in a separate chart for each well in a single display. To see a larger
representation of a well, select the well in the 2D Amplitude view.

To view different color arraysu

Select the channel combination from the dropdown on the left, and then click Update Grid.

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Analysis Windows
In its Analysis module, QX ONE Software provides amplitude displays and data visualization charts in the
Analysis module. For more information on each chart, see Chapter 4, Data Analysis Methodology.

Amplitude Charts
Amplitude charts illustrate the droplets by degree of dye fluorescence. QX ONE Software displays
fluorescence for positive and negative targets identified with FAM, HEX/VIC, Cy5, and Cy5.5 fluorophores
in the following analysis windows:n

1D Amplituden

2D Amplituden

3D Amplitude

Data Visualization Charts
Data visualization charts show calculations based on positive droplet concentrations. QX ONE Softwarecounts the number of positive and negative droplets for each fluorophore in each sample, and then fits thefraction of positive droplets to a Poisson algorithm to determine the starting concentration of the target
DNA molecule in units of copies/µl input in the following analysis windows:nConcentrationn

Copy Number

n

Ratio

n

Fractional Abundance

When you open the analysis data file in QX ONE Software and select one or more wells in the grid, thesoftware plots each calculated Poisson distribution average as a data point in the grid. You can pause on
the data point to see the high, median, and low numbers.
Note: You can display the data using a confidence level of either 68% or 95% (default setting).

Event Counts
Event counts record the number of droplets in which the target was found (positive) and was not found(negative). The Event Counts tab shows a bar chart view of the number of positive, negative, and total
droplets for each of the targets in each of the selected wells.

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Chapter 3 Data Analysis Module Overview

Data Windows
QX ONE Software provides the following table views for your well and run data:nData Table — available from the main tabs on the left, shows your configured experimentparameters, plus all calculations from the run
Note: You can display the data in individual well format, merged format, or both.

nPlate Well Data Table — available in plate setup windows (including the Analysis module Plate
Editor), shows only your configuration information for each individual wellnAnalysis Well Data Table — available in the Analysis module, shows a subset of configuration andrun data for the wells selected in the Well Selector

Data Table Tab
The Data Table window contains columns of data generated for each target in each well, as droplet
clusters are identified through thresholding or manual clustering tools.

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Data Table Column Definitions
Table 17 defines each field in the Data Table.
Table 17. Data Table columns

Column nameContent description

Well

The well location in the plate
Note: The instrument accepts 96-well plates only.

Sample description 1

Words or phrases to describe the sample in each wellSample description 2

Note: Only the sample description 1 data appears

Sample description 3

in plate layout views but you can pause on a well in

Sample description 4

the Plate Editor to display a tool tip containing theentries in all completed Sample description fields.

TargetTarget name from Plate Editor tab

Conc(copies/µL)

Concentration of the target molecules recorded as
copies per microliter

pg/µL
StatusMass concentration of DNA in the well in pg/µl
Contains either Check, OK, Multi, or Manualn If Check, either the automatic analysis for the wellfailed or fewer than 10,000 droplets were counted in
the welln If OK, enough droplets were counted to trigger
automatic analysis
n If Multi, the data was automatically analyzed as part ofa multi-well selectionn If Manual, the droplets were analyzed manually

Status Reason

Identifies the reason for the status (unsuccessful
automatic analysis, not enough droplets counted, or
droplets analyzed manually)

Experiment

Experiment type selected for the well in the Plate Editor

SampleType

Sample type selected for the well in the Plate Editor

TargetType

Target type selected for each dye(FAM, HEX or VIC,
Cy5 and Cy5.5) in the Plate Editor

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Chapter 3 Data Analysis Module Overview

Table 17. Data Table columns, continued

Column nameContent description

Supermix

Supermix selected for the well in Plate Editor

DyeName(s)Dye assigned to channels (1, 2, 3, and 4, respectively)
for the well in the Plate Editor

Copies/20µLWell

Concentration of the target normalized to a volume of 20µL

TotalConfMax

For merged wells the high error bar for the target
concentration of the combined wells at a 95% confidence
interval

TotalConfMin

For merged wells the low error bar for the target
concentration of the combined wells at a 95% confidence
interval

PoissonConfMax

Maximum target concentration normalized for the higherror bar of the droplet Poisson distribution for the 95%
confidence interval

PoissonConfMin

Minimum target concentration normalized for the low
error bar of the droplet Poisson distribution for the 95%
confidence interval

Accepted Droplets

Total number of droplets accepted by quality algorithm

Positives

Number of droplets that contain the target

Negatives

Number of droplets that do not contain the target

Copies/µL linked molecules

Shows number of copies of a target that are present inoverabundance versus the expected value in copies perµL

CNV

Copy number calculated for the target relative to thereference

TotalCNVMax

For merged wells, the high error bar for the copy numberof the combined wells at a 95% confidence interval

TotalCNVMin

For merged wells, the low error bar for the copy numberof the combined wells at a 95% confidence interval

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Table 17. Data Table columns, continued

Column nameContent description

PoissonCNVMax

Maximum copy number normalized for the high error barof the droplet Poisson distribution for the 95%
confidence interval

PoissonCNVMin

Minimum copy number normalized for the low error barof the droplet Poisson distribution for the 95%
confidence interval

ReferenceCopies

Copy number identified for the reference target in the
Plate Editor

UnknownCopies

This field is currently unused in QX ONE Software

Threshold1

Threshold value of first threshold line from left to rightand bottom to top

Threshold2

Threshold value of second threshold line from left to rightand bottom to top

Threshold3

Threshold value of third threshold line from left to rightand bottom to top

ThresholdSigmaAbove

Distance of the threshold from the mean of the negative
cluster, as a multiple of the negative cluster standard
deviation

ThresholdSigmaBelow

Distance of the threshold from the mean of the positive
cluster, as a multiple of the positive cluster standard
deviation

ReferenceUsed

Identifies which target was used as a reference

Ratio

The ratio of the target against the identified reference

TotalRatioMax

For merged wells the high error bar for the ratio of theunknown against the reference of the combined wells at
a 95% confidence interval

TotalRatioMin

For merged wells the low error bar for the ratio of theunknown against the reference of the combined wells at
a 95% confidence interval

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Chapter 3 Data Analysis Module Overview

Table 17. Data Table columns, continued

Column nameContent description

PoissonRatioMax

Maximum ratio of the unknown against the reference
normalized for the high error bar of the droplet Poisson
distribution for the 95% confidence interval

PoissonRatioMin

Minimum ratio of the unknown against the reference
normalized for the low error bar of the droplet Poisson
distribution for the 95% confidence interval

FractionalAbundance

Calculation of fractional abundance of this unknown
target vs. the reference target

TotalFractionalAbundanceMax

For merged wells the high error bar for the fractional
abundance of the combined wells at a 95% confidence
interval

TotalFractionalAbundanceMin

For merged wells the low error bar for the fractional
abundance of the combined wells at a 95% confidence
interval

PoissonFractionalAbundanceMax

Maximum fractional abundance normalized for the higherror bar of the droplet Poisson distribution for the 95%
confidence interval

PoissonFractionalAbundanceMin

Minimum fractional abundance normalized for the low
error bar of the droplet Poisson distribution for the 95%
confidence interval

MeanAmplitudeOfPositives

Mean amplitude value of all droplets that contain thetargetMeanAmplitudeOfNegatives

Mean amplitude value of all droplets that contain notargetMeanAmplitudeTotal

Mean amplitude value of all droplets

ExperimentComments

Data populates this field only from files created in
QuantaSoft™ versions 1.4 – 1.7, if comments wereadded.

MergedWells

96 QX ONE Software, Regulatory Edition

Identifies which wells were merged together

Data Windows

Table 17. Data Table columns, continued

Column nameContent description

TotalConfidenceMax68

For merged wells the high error bar for the target
concentration of the combined wells at a 68% confidence
interval

TotalConfidenceMin68

For merged wells the low error bar for the target
concentration of the combined wells at a 68% confidence
interval

PoissonConfidenceMax68

Maximum target concentration normalized for the higherror bar of the droplet Poisson distribution for the 68%
confidence interval

PoissonConfidenceMin68

Minimum target concentration normalized for the low
error bar of the droplet Poisson distribution for the 68%
confidence interval

TotalCNVMax68

For merged wells the high error bar for the copy numberof the combined wells at a 68% confidence interval

TotalCNVMin68

For merged wells the low error bar for the copy numberof the combined wells at a 68% confidence interval

PoissonCNVMax68

Maximum target concentration normalized for the higherror bar of the droplet Poisson distribution for the 68%
confidence interval

PoissonCNVMin68

Minimum target concentration normalized for the low
error bar of the droplet Poisson distribution for the 68%
confidence interval

TotalRatioMax68

For merged wells the high error bar for the ratio of theunknown against the reference of the combined wells at
a 68% confidence interval

TotalRatioMin68

For merged wells the low error bar for the ratio of theunknown against the reference of the combined wells at
a 68% confidence interval

PoissonRatioMax68

Maximum ratio of the unknown against the reference
normalized for the high error bar of the droplet Poisson
distribution for the 68% confidence interval

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Chapter 3 Data Analysis Module Overview

Table 17. Data Table columns, continued

Column nameContent description

PoissonRatioMin68

Minimum ratio of the unknown against the reference
normalized for the low error bar of the droplet Poisson
distribution for the 68% confidence interval

TotalFractionalAbundanceMax68

For merged wells the high error bar for the fractional
abundance of the combined wells at a 68% confidence
interval

TotalFractionalAbundanceMin68

For merged wells the low error bar for the fractional
abundance of the combined wells at a 68% confidence
interval

PoissonFractionalAbundanceMax68

Maximum fractional abundance normalized for the higherror bar of the droplet Poisson distribution for the 68%
confidence interval

PoissonFractionalAbundanceMin68

Minimum fractional abundance normalized for the low
error bar of the droplet Poisson distribution for the 68%
confidence interval plate list after correcting the issue

TiltCorrected

Indicates if tilt correction was applied to the well
Note: Tilt correction rotates the angles from the
double-negative cluster centroid to each droplet,such that the two single positive cluster centroids
are orthogonal with the double-negative cluster
centroid.

Ch1+Ch2+

Number of droplets that contain both channel 1 andchannel 2 targets

Ch1+Ch2-

Number of droplets that contain just the channel 1 target

Ch1-Ch2+

Number of droplets that contain just the channel 2 target

Ch1-Ch2-

Number of droplets that contain neither channel 1 norchannel 2 targets

Ch3+Ch4+

Number of droplets that contain both channel 3 andchannel 4 targets

Ch3+Ch4-

98 QX ONE Software, Regulatory Edition

Number of droplets that contain just the channel 3 target

Data Windows

Table 17. Data Table columns, continued

Column nameContent description

Ch3-Ch4+

Number of droplets that contain just the channel 4 target

Ch3-Ch4-

Number of droplets that contain neither channel 3 norchannel 4 targets

Plate Well Data Table
From the Plate Editor you can access a window containing the Well Data table, which displays
configuration and run information on all wells containing sample.
Note: You cannot edit this table.
Table 18. Plate Well Data Table columns

Column nameContent description

Well

Well number (A01 through H12)

Sample description 1

Words or phrases to identify each sample, as entered in the Plate Editor

Sample description 2

Note: You can enter up to four sample descriptions for each well.

Sample description 3
Sample description 4
Perform Droplet Generation

Yes, if droplets were generated in the wellNo, if they are not

Perform Droplet Reading

Yes, if wells are readNo, if they are not

Target name

Target name entered in the Plate Editor tab

Conc (copies/µL)
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Table 18. Plate Well Data Table columns, continued

Column nameContent description

Experiment type

Experiment Type selected in the Plate Editor:n Direct Quantification (DQ)n Copy Number Variation (CNV)n Mutation Detection (MUT)n Rare Event Detection (RED)n Drop-off (DOF)
n Gene Expression (GEX)n Residual DNA Quantification (RDQ)
Sample Type

Sample type selected in the Plate Editor:n Unknown
n No template control (NTC)n Positive control (Pos Ctrl)
n Negative control (Neg-Ctrl)

Target Type

Target type selected in the Plate Editor tab

Supermix nameSupermix selected in the Plate Editor:
n ddPCR Multiplex Supermix
n ddPCR Supermix for Probes (no dUTP)
n ddPCR Supermix for Probes
n ddPCR Supermix for Residual DNA Quantificationn One-Step RT-ddPCR Advanced Kit for Probes

DyeName(s)Dye assigned to channels 1, 2, 3, and 4, respectively, for the well in the
Plate Editor

Accepted Droplets

Total number of droplets accepted by quality algorithm

Positives

Number of droplets that contain the target

Negatives

Number of droplets that do not contain the target

Plate Notes

Entries by the user

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Plate Editor Well Data Table
A well data table containing both configuration and run data appears in the analysis windows, next to theWell Selector. This table displays a subset of data columns for only the wells selected in the Plate Editor
or Well Selector. Table 19 defines each column in the consolidated table.
Table 19. Fields in the Analysis Well Data table

Column nameContent description

Well

The well location in the plate the sample is from

Perform Droplet Reading

Yes or No

Experiment Type

Experiment type from the Plate Editor

Sample description 1 – 4The sample name used to identify the well in the Plate Editor tab

SampleType

Sample type from the Plate Editor

Supermix Name

Supermix from the Plate Editor

Assay type

Assay type selected in the Plate Editor

Target Name

Target Name from Plate Editor tab

TargetType

Target type assigned to the target in the Plate Editor

Signal Ch1

Selected fluorophore in Channel 1

Signal Ch2

Selected fluorophore in Channel 2

Signal Ch3

Selected fluorophore in Channel 3

Signal Ch4

Selected fluorophore in Channel 4

Reference Copies

Copy number identified for the reference target in the Plate Editor tab

Well Notes

Entries by the user

Plot?

If selected, target will be used to calculate the copy number for the well

RDQ Conversion Factor

The Residual DNA Quantification conversion factor for the well
Note: RDQ is only available in Residual DNA Quantification
experiments.

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Chapter 3 Data Analysis Module Overview

Viewing Run, Lot, and Audit Information
Use the Run Information window to view the information about the plate and run. You can also view or add
post-run notes regarding the plate. In Regulatory Edition, you can also view the audit logs.
You can also view the Audit Log. For information, see Viewing the Audit Log on page 138.

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As summarized in Chapter 3, Data Analysis Module Overview, the software presents analysis data inseveral different formats. When you open the data file in the Analysis module, you can view the data withthe automatic analysis thresholds and calculations or you can choose different options to recalculate,
replot, or recolor the results.
Data is shown in the following formats:n

Amplitude, which shows each droplet in each well in a scatter plot, in one-dimensional, twodimensional, or three-dimensional viewsnData visualizations, which show estimated target molecule concentration, ratio, and fractional
abundance of one target against another target based on selected error model and confidence
interval
Note: Copy number variation is also calculated if the user ran a CNV experiment.

nEvent counts, which are precise counts of positive and negative droplets for each target in each well

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Chapter 4 Data Analysis Methodology

Amplitude Plot Analysis Options
QX ONE Software provides three amplitude plot displays in which you can view the data with automaticthresholding where applicable, or choose different options to replot or recolor the results.
In the 1D amplitude and 2D amplitude plots, you cannShow well calculations
Note: The 1D amplitude plot shows the data for each selected well in its own section, while the2D amplitude plot shows all selected wells together.

nModify automatic thresholds calculated by QX ONE SoftwarenView data in a heat mapnView histograms (1D amplitude tab only)nRe-apply automatic analysisnApply tilt correction to automatic analysis

In the 3D amplitude plot, you cannRotate the plot to gain a better view of the droplets in 3D orientationnView thresholds previously set in 1D or 2D amplitude displaynView 2D data behind 3D droplets in clusters or heat maps

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Table 20 explains the analysis options that are available in both 1D amplitude and 2D amplitude plots.
Table 20. 1D and 2D amplitude tool options

Icon

Mode

Description
Data appears with the thresholds or clusters

View mode

assigned by QX ONE Software.
When this icon is selected, the view is locked andyou cannot modify the thresholds.

Heat map

Shows the distribution of droplets, with higher
density of droplets shown as hot.

Automatic analysis

Disables tilt correction and automaticallyreanalyzes the data.
Enables tilt correction and automaticallyreanalyzes the data.

Automatic analysis withtilt correction

Note: Tilt correction rotates the angles fromthe double-negative cluster centroid to each
droplet, such that the two single positive
cluster centroids are orthogonal with the
double-negative cluster centroid.

Reset

Resets the data to the original automaticallyanalyzed data.
Choose whether to display axis with channel

Chart display options

number or fluorophore, and change the size ofaxis markers, data labels, and axis labels.

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Chapter 4 Data Analysis Methodology

Table 21 explains the analysis options available only for 1D amplitude plots.
Table 21. 1D amplitude tool options

Icon

Mode

Automatic thresholding

Description
Individual Wells: Clusters are generated for each selected well
separately.
Combined Wells: Droplets from all selected wells are combined, andclusters are generated on that combined set.
Positive Control Wells: Thresholds are generated for each channel
based on any positive controls currently selected, and applies thosethresholds to all currently selected wells.
Note: If you change the thresholds manually, you can tap Resetto return to the automatic thresholds.

Threshold, single

For a single well or multiple wells, the user can manually input

wellthresholding values in the 1D amplitude plot.
You can tap anywhere in the plot to apply threshold lines to the data.

Threshold, multiple
wells

To adjust the threshold valuesn Drag the lines horizontally in the plot arean Enter a new threshold value in the boxes at the end of eachthreshold linen Tap the up and down arrows to incrementally adjust the numbers

Threshold, single

For a single well or multiple wells, QX ONE Software automaticallywell SD

calculates and populates thresholding values with standard

Threshold, multiple

deviations (SD).

wells SD

Important: SD thresholds are not recommended for RMD, sincethese clusters are not typically located at right angles to eachother.
Note: The SD thresholds are set at a limit that is less than themean number of positive targets, and more than the mean
number of negative targets. Starting from where the user taps inthe plot, SD locates the threshold between the positive andnegative clusters such that the classification error is minimized.

Show histogram

Hides or displays histogram plots for 1D amplitude

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Table 22 explains the analysis options available only for 2D amplitude plots.
Table 22. 2D amplitude tool options

Icon

Mode

Automatic thresholding

Description
Individual Wells: Clusters are generated for each selected well
separately.
Combined Wells: Droplets from all selected wells are combined, andclusters are generated on that combined set.
Positive Control Wells: The software generates thresholds for eachchannel based on any positive controls currently selected, and applies
those thresholds to all currently selected wells.
Note: If you change the thresholds manually, you can tap Resetto return to the automatic thresholds.

Threshold line

Use the Threshold line icons to establish thresholds across a pair ofchannels, dividing droplets into distinct clusters separated by different
colors.
You can tap anywhere in the plot to apply threshold lines to the data.
To adjust the threshold values, you cann Drag the lines horizontally or vertically in the plot arean Enter a new threshold value in the boxes at the end of eachthreshold linen Tap the up and down arrows to incrementally adjust the numbers

Threshold line SD

QX ONE Software automatically calculates and populates thresholdline values with standard deviations (SD).
Important: SD thresholds are not recommended for RMD, sincethese clusters are not typically located at right angles to eachother.
Note: The SD thresholds are set at a limit that is less than themean number of positive targets, and more than the mean
number of negative targets. Starting from where the user taps inthe plot, SD locates the threshold between the positive andnegative clusters such that the classification error is minimized.

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Chapter 4 Data Analysis Methodology

Icon

Mode

Description

Threshold cluster

Use the Threshold Cluster icons to establish thresholds across a pairsquareof channels, dividing droplets into distinct clusters separated byThreshold cluster

different colors.

circleTo classify your droplets, you canThreshold clustern Tap any of the three Threshold Cluster Mode buttons (square,
circle, or free form)

freeformn Draw the chosen shape around the cluster of droplets
Note: When using the cluster mode tools to draw a shape arounda cluster of droplets, a dialog box with color selections by target
appears in the window to help you select the correct target
combination.

Target names are shown along the two axes based on the signal
values assigned during plate setup. Tap the colored box that best
represents the location of the cluster of droplets selected toautomatically classify the droplets as positive or negative for eachgiven target.
Note: The Cluster dialog box display varies according to the plexmode.

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Table 23 explains the analysis options available only for 3D amplitude plots.
Table 23. 3D amplitude tool options

Icon

Mode

Description

dropdown menu containing

Choose to show 3D droplets, 2D

options for data visualization

amplitude data behind the 3D clusters,
thresholds, and 2D color options.

Axis titles

Choose whether to display axis withchannel number or fluorophore name.

Reset

Reset the display to the default
orientation.

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Chapter 4 Data Analysis Methodology

1D Amplitude
Use the 1D Amplitude analysis window to view droplet amplitudes for each channel, in selection order, foreach well selected in the Well Selector.

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Amplitude Plot Analysis Options

The 1D Amplitude plots illustratenn

All accepted droplets, with a visual representation of the positive and negative droplets in each welloPositive droplets appear in color, while negative droplets appear in gray.

o

The Well Selector displays an A in each well where QX ONE Software automatically thresholded
the droplet data. An M appears in any well in which you manually changed the threshold.

Amplitude in a histogram plot

Note: The Histogram icon is enabled by default so a Histogram plot appears automatically foreach fluorophore (channel).
n

Amplitude in a Heat Map view

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Chapter 4 Data Analysis Methodology

When you select the 1D Amplitude tab, the toolbar options that allow you to change thresholds and showor hide the heat map and histogram appear above the plots.

Note: For detailed information on each toolbar option, see Amplitude Plot Analysis Options on page
104.
Default color assignments for 1D amplitude plots are
Positive FAM

=

Blue

Positive HEX

=

Green

Positive Cy5=

Red

Positive Cy5.5=

VioletNegative=

Gray

The images in this section display data from a direct quantification experiment involving four targets:
Negative droplets appear in grayscale in each plot.

For experiments where enough droplets are recognized, QX ONE Software automatically configures anamplitude threshold, which distinguishes between positive and negative droplets.
Note: Well data must meet certain quality metrics (minimum of 10,000 droplets) before the software
will automatically calculate a threshold above which droplets are considered positive. You can use
the manual thresholding tools to ensure correct quantification of positive and negative droplets.

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By default, the Histogram icon (.

) is enabled so the Histogram plot automatically appears with theamplitude plot.

To manually adjust the thresholds
1.

Select one or more wells.

2.

Tap any of the threshold icons () to set manual thresholds:nRegular threshold for a single well; when multiple wells are selected, you can set a different
threshold for each welln

Regular threshold for multiple wells, where you can set one threshold for all selected wellsnStandard deviation threshold for a single well; when multiple wells are selected, you can set a
different threshold for each wellnStandard deviation for multiple wells, where you can set one threshold for all selected wells

3.

Tap on the location in the plot where you want to draw the new threshold.

4.

When the line appears, use the up or down arrows, or tap and drag the threshold, to adjust thelocation.

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Chapter 4 Data Analysis Methodology

QX ONE Software displays the manual threshold and changes the color of the droplets that are now
above or below the threshold.
Note: You can also manually enter a threshold number.
When you manually modify a threshold, the well selector displays an M for manual instead of the A for
Automatic.

5.

(Optional) To reset to the automatic threshold, tap Auto.

6.

(Optional) To adjust the automatic threshold to consider tilt correction, tap Auto with Tilt.

7.

(Optional) To reset everything to the original display, tap Reset.

To view a heat map representation of the plot datau

Tap the heat map () icon.

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2D Amplitude
For the wells you select in the analysis view of the Plate Editor, the 2D Amplitude window illustrates
fluorescence for four targets. The images in this section display data from an experiment involving fourtargets. Each plot shows individual cluster representations for first and second target positive droplets,
droplets containing both targets, and droplets containing neither target.

When you select the 2D Amplitude tab, the toolbar options allow you to change thresholds, manuallycluster wells, and show or hide the heat map above the plots.

For detailed information on each toolbar option, see Amplitude Plot Analysis Options on page 104.

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Chapter 4 Data Analysis Methodology

Default color assignments for 2D amplitude plots are:
Positive FAM

=

Blue

Positive HEX/VIC

=

Green

Positive FAM and HEX/VIC=

Orange (may appear brown on some monitors)

Positive Cy5=

Red

Positive Cy5.5=

VioletPostive Cy5 and Cy5.5=

Light Blue

Double negative

=

Gray

Note: Well data must meet certain quality metrics (minimum of 10,000 droplets) before QX ONE
Software will automatically calculate a threshold above which droplets are considered positive. You
can use the manual thresholding tools to ensure correct quantification of positive and negative
droplets.

To use the 2D amplitude tools
1.

Open your data file in the Analysis module and select the 2D Amplitude tab.

2.

Choose a threshold line or cluster option.
Note: Use the cluster option when the data is not orthogonal and the threshold lines do notadequately divide the populations of droplets.
n

To adjust threshold lines, select a threshold line type () and then tap in the plot to apply

crosshair lines. Use any of the following methods:oDrag the lines horizontally or vertically in the plot area.

oEnter a new threshold value in the boxes at the end of each threshold line.

o

Tap the up and down arrows to incrementally adjust the numbers.

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3D Amplituden

To adjust thresholds using the cluster option, select a cluster type () and draw thechosen shape around the cluster of droplets to classify.

3.

(Optional) To reset to the automatic threshold, tap Auto.

4.

(Optional) To adjust the automatic threshold to consider tilt correction, tap Auto with Tilt.

5.

(Optional) To reset everything to the original display,tap Reset.

3D Amplitude
When you select the 3D Amplitude tab, the toolbar options that allow you to select the well and choose
which channels to display appear above the chart.
For the well selected from the toolbar, the 3D amplitude plot illustrates three targets in a single chart.

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Chapter 4 Data Analysis Methodology

To use the different 3D amplitude viewing tools
1.

Select Well.

2.

Select the dye for the x, y, and z axis.

3.

Tap Show and the following menu appears:

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3D Amplitude

4.

Select one or more of the Show 2D options to display 2D amplitude data behind the 3D droplets.
Show 3D Droplets is selected by default. You can also shown

2D data on different axis combinationsn

Display thresholds on the x, y, and z axis
Note: You must set the thresholds on the 1D or 2D amplitude plots to display them in 3D. .

n

2D colors in clusters or a heat map

5.

Tap Chart Display to show the labels by color or by channel.

6.

Tap Reset Display to return to the default display.

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Chapter 4 Data Analysis Methodology

Data Visualization Chart Analysis Options
QX ONE Software also provides chart displays where you can view the data from the original calculations,or choose different options to recalculate the results.
In charts reflecting Data Visualization distributions, you cannShow individual or merged well datan

Change the error model and confidence level

Note: For event counts, you can also alter the view mode to see positive, negative, or all droplet
counts.
Table 24 explains the available options.
Table 24. Chart tool options for Data Visualization distribution charts
Icon

Mode

Description

Show bothShows the calculated total or
percentage as a merged calculation for
all selected wells and as an individual
calculation for each selected wellShow merged

Shows the calculated total or
percentage as a combined number for
all selected wells with the same
parameters
Note: Well setup must match foreach well before the software canmerge them.

Show individualShows the calculated total or
percentage as an individual calculationfor each selected well

X-axis well

120 QX ONE Software, Regulatory Edition

Shows the well number on the x axis

Data Visualization Chart Analysis Options

Icon

Mode

Description

X-axis sample

Shows a partial sample name on the xaxis
Note: You can select Well andSample simultaneously.

Error model andSelect an error model and confidence

confidence interval

level
Note: The default choices are
Poisson and 95%, to yield thehighest confidence in the results.

Important: The software uses thefollowing multipliers in its
calculations:n For 95% confidence interval, thesigma multiplier is 1.96
n For 68% confidence interval, thesigma multiplier is 1.0

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Chapter 4 Data Analysis Methodology

Table 25 identifies the additional options available in the Event Count window. These options areavailable for event counts only.
Table 25. Additional options for event counts
Icon

Mode

Description

View mode

Displays in a bar chart the number of positive droplets for each target inpositiveeach well

View mode

Displays in a bar chart the number of negative droplets for each target innegativeeach well

View mode

Displays in a bar chart the number of total droplets in each welltotal

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Data Visualization Chart Analysis Options

Concentration
Concentration measures the number of copies of the target molecules in each µl of sample based on thedefault settings. Use the toolbar to merge or separate the calculated results for each well, and torecalculate your results based on a different error model and confidence interval.
For information on toolbar options, see Data Visualization Chart Analysis Options on page 120.

The following image displays concentration data, across a sequence of wells.

QX ONE Software plots the averaged concentration for each target as a data point. Pause on a data point
to see the high, median, and low numbers.

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Chapter 4 Data Analysis Methodology

Concentration Calculation
The software uses the formula and variables shown below.

Where:-ln

Negative natural logarithm

Vdroplet

Volume of droplet

Nneg

Number of negative dropletsN

Total number of droplets

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Data Visualization Chart Analysis Options

Confidence Interval Calculation
Note: If you select a 68% confidence interval, the multiplier is changed from 1.96 to 1.00.

Where:

Vdroplet

Volume of droplet

Nneg

Number of negative dropletsN

Total number of dropletsp

p-value (probability of finding the target in the sample)

¶∂

Delta

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Chapter 4 Data Analysis Methodology

Copy Number
Important: To see copy number variation data, you must run a copy number variation (CNV)
ddPCR experiment.
Copy number measures the count of the target gene, relative to the reference gene, to calculate thenumber gene targets per genome. Use the toolbar to merge or separate the calculated results for eachwell, and to recalculate your results based on a different error model and confidence interval. For
information on toolbar options, see Data Visualization Chart Analysis Options on page 120.

The following image displays copy number data, across a sequence of wells.

QX ONE Software plots each copy number variation as a data point. Pause on a data point to see thehigh, median, and low numbers.

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Data Visualization Chart Analysis Options

Copy Number Calculation
The software uses the formula and variables shown below.

Where:A

Target concentration

B

Reference concentration

NB

Number of reference copies in genome (usually 2)

Confidence Interval Calculations
Note: If you specify the lesser confidence interval when you define your error model, the softwareuses 68% in its calculations instead of 95%.

.

Where:A

Target concentration

B

Reference concentration

NB

Number of reference copies in genome (usually 2)

CIA

Poisson confidence interval of target (95% max - 95% min)CIB

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Chapter 4 Data Analysis Methodology

Notes:
Copy Number analysis involves determining the number of copies of a given target DNA sequence ascompared to an invariant reference DNA sequence. Use the CNV analysis to determine structuralvariations, such as ploidy changes (addition or deletion of chromosomes), deletions (missing partial
chromosomes) or duplications (repeated target sequences) in the genome.
The ability to discriminate between consecutive CN states has been a technical challenge in copy number
assessment. Fundamentally, as CN state increases, the percentage difference in target genomic material
between states decreases. For example, for a given target locus, a CN of 3 is 50% more abundant inconcentration per genome than a CN of 2, while a CN of 5 is only 25% more abundant per genome than a
CN of 4, and so on.
Due to the massive partitioning of the reaction across up to 20,000 droplets per well, and the absolutenature and precision of the ddPCR concentration measurement, ddPCR CNV analysis enables thequantitative discrimination required to resolve consecutive copy number states beyond CN 3. For
example, a 20% difference in target concentration between a CN 5 versus CN 6 sample can be
reproducibly resolved using ddPCR. This discrimination is both a function of concentration differences
between consecutive CN states, as well as the amount of DNA assayed per well.

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Data Visualization Chart Analysis Options

Ratio
Ratio determines the quotient of two target molecules in each µl of sample based on the default settings.
T1:T2
Use the toolbar to merge or separate the calculated results for each well, and to recalculate your results
based on a different error model and confidence interval. For information on toolbar options, see Data
Visualization Chart Analysis Options on page 120.

The following image displays ratio data, across a sequence of wells.

QX ONE Software plots each ratio as a data point. To see the confidence level numbers, pause on thedata point.

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Chapter 4 Data Analysis Methodology

Ratio Calculation
The software uses the formula and variables shown below.

Where:A

Target concentration

B

Reference concentration

Note: If no reference is selected, the target with the highest dye concentration in the highest channel
will automatically be considered as the reference. If multiple references are selected, the geometricmean is used.

Confidence Interval Calculations
Note: f you specify the lesser confidence interval when you define your error model, the softwareuses 68% in its calculations instead of 95%.

Where:A

Channel 1 target concentration

B

Channel 2 target concentration

CIA

Confidence interval of Channel 1 target (95% max - 95% min)

CIB

Confidence interval of Channel 2 target (95% max - 95% min)

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Fractional Abundance
Fractional Abundance determines the relative abundance of your targets in each µl of sample based on
the default settings. Use the toolbar to merge or separate the calculated results for each well, and torecalculate your results based on a different error model and confidence interval. For information on
toolbar options, see Data Visualization Chart Analysis Options on page 120.

The following image displays fractional abundance data across a sequence of wells.

QX ONE Software plots the fractional abundance as a data point. To see the high and low confidence
interval numbers, pause on the data point.

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Chapter 4 Data Analysis Methodology

Fractional Abundance Calculation
The software uses the formula and variables shown below.

Where:A

Target concentration

B

Reference concentration

Note: If no reference is selected, the target with the highest dye concentration in the highest channel
will automatically be considered as the reference. If multiple references are selected, the geometricmean is used.

Confidence Interval Calculations
Note: If you specify the lesser confidence interval when you define your error model, the softwareuses 68% in its calculations instead of 95%.

Where:A

Channel 1 target concentration

B

Channel 2 target concentration

CIA

Confidence interval of Channel 1 target (95% max - 95% min)

CIB

Confidence interval of Channel 2 target (95% max - 95% min)

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Event Counts
The Event Counts tab shows a bar chart view of the number of positive and negative droplets for each of
the targets in each of the selected wells.

Use the toolbar to change the display by combining or separating data for each well. You can also show
data by well name or by sample name, and you can show any combination of positive, negative, or
combined droplet counts.

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Chapter 5 Analysis Module Reporting
Use the Reporting function to create a variety of reports in the QX ONE Software Analysis module.
You can create reports on the fly from any analysis file or, if you need to generate specific reports on aregular basis, you can set up report templates for reuse.
This section contains information on how to create analysis reports.

Creating Analysis Reports
You can generate PDF reports on selected data in each analysis file.
For the checkboxes you select in the report configuration, the software displays corresponding charts anddata for the selected wells. In each report header, the software automatically displays the user name ofthe person creating the report, date and time stamps, and the name of the analysis file used to generate
the report.

User Guide | 135

Chapter 5 Analysis Module ReportingTo create an analysis report
1.

Open an analysis data file and tap Reports.
Note: If you make changes to the file, you must save the changes before you can set up and run
a report.

2.

Do one of the following:nWork in the default report layout, which appears automatically.

n

Select a report template from the Template dropdown list.

3.

Under Name, tap the arrow to the left of each item to expand the list of available columns.

4.

Keep the original configuration, or select and clear check boxes to create a new report format.

5.

(Optional) Use the options located in the lower portion of the window to expand or collapse the tree,
and to select or clear all checkboxes.

6.

In the Report Title field, enter a name for your report. You can use up to 30 characters.

7.

(Optional) If the report should be private, select the Password Encrypt Report? checkbox and thenenter a password in the Password field.
The password must contain between 6 and 20 characters, with at least 1 letter and 1 number.

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Creating Analysis Reports

8.

(Optional) To save the structure as a new report template, tap Save and then enter a name, andselect a storage location.

Choose from:n

My Templates, to keep the template for your personal usenShared Templates, to make the template available to all users

n

Browse, to navigate to a different folder

Note: You can save a template if you are assigned the Create New Templates user privilege.
9.

To generate the PDF report, tap Generate Report.

10. When the File Download prompt appears, select Open to open the file immediately or Save to savethe file to your C drive.
11. To view a saved file, navigate to the file location and open it in a PDF viewer.

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Chapter 5 Analysis Module Reporting

Viewing the Audit Log
You can view the audit log entries for the data file using the Audit Log viewer found in the Run Information
tab in the Analysis module.

To view the Audit Logu

In the Analysis module, select the Run Information tab, and then select the Audit Log tab to display
the audit log entries.

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Chapter 6 Gene Study Module
You access the Gene Study module from the Data Analysis window.
Note: The Gene Study module is not available from the touch screen computer.

To create a gene study, you add one or more data files and then compare gene expression data from one
or more ddPCR experiments.
Note: The maximum number of samples you can analyze in a gene study is limited by the size of thecomputer’s RAM and virtual memory.

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Chapter 6 Gene Study Module

To open the Gene Study moduleu

Click Gene Study.

The Gene Study dialog box includes two tabs:nStudy Setup — manages the run files in the gene study
Important: Adding or removing data files in a gene study does not change the data in theoriginal file.

nStudy Analysis — displays the gene expression data for the combined runs.

From these tabs you can add files to set up a gene study, analyze your data in charts and plots, and run
reports on the gene study data.

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Gene Study Options

Gene Study Options
The Gene Study module provides the menus and corresponding options described below.
nFile — Open, save, and close the gene study, and add filesnSettings — Restore the default layoutnTools — Create a report

To open a gene study file1.

Tap File Ú Open.

2.

In the Open dialog box, navigate to and select the data file, and then tap Open.

To save a gene study file1.

Tap File and select Save or Save As.

2.

If prompted, enter a file name and tap Save.

3.

If prompted to overwrite an existing file, tap Yes or No.

To close a gene study fileu

Tap File Ú Close.
Note: If you have not saved the file, an advisory prompt appears.

To add data files
1.

Tap File Ú Add Data Files.

2.

In the Open dialog box, navigate to the data files to be added to the study.

3.

Select one or more files and then tap Open to add the files to the Gene Study window.

To restore default settingsu

Tap Settings Ú Restore default settings.
Note: At least one modified data file must be open.

To generate a reportu

See Gene Study Reports on page 146.

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Chapter 6 Gene Study Module

Setting up the Gene Study
Use the Study Setup window ton

Add data files to include in the studynRemove data files from the studynSelect data files for viewing and analysisnShow the plate view for a data file142 QX ONE Software, Regulatory EditionSetting up the Gene Study

Table 26 defines each column in Study Setup.
Table 26. Gene Study Setup Fields
Column Title

Description

File Name

Name of the data file

Date Created

Date the file was imported into the Gene Study module

Plate View

Opens a map of the plate with the data for each dye in each well included in theGene Study

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Chapter 6 Gene Study Module

Adding Data Files
To add data files
1.

On the Study Setup tab, tap Add Data Files.

2.

In the Open dialog box, navigate to the data files to be added to the study.

3.

Select one or more files and then tap Open.
The files are added to the Study Setup window.

Removing Data Files
To remove data filesuSelect the checkbox next to the file and then tap Remove.
The file is removed from the Study Setup window.
Note: The Remove button is disabled until you select a file.

Selecting Data Files for Analysis
To select data files for analysis
1.

Select the checkboxes next to the files to be included in the analysis.

2.

Select the Study Analysis tab to see the analysis results.

144 QX ONE Software, Regulatory EditionSetting up the Gene Study

Showing the Plate View
To show the plate view
1.

In the Plate View column, tap the button for the plate view to be displayed.

The Plate view appears.

2.

Click Close to close the view.

User Guide | 145

Chapter 6 Gene Study Module

Gene Study Reports
Use the Gene Study Report dialog box to arrange the gene study data into a report. Table 34 lists all theoptions available for a gene study report.
Table 27. Gene Study Report Categories
Category

OptionDescription

Report Information

Date, user name, data file name, data file path, and the

Headerselected well group
Gene Study File List

List of all the data files in the gene study

Notes

Notes about the data report

Study Analysis Bar Chart
Analysis Settings

List of the selected parameters

Chart

Gene expression bar chart showing the data

Target Names

List of targets in the gene study

Sample Names

List of samples in the gene study

Data

Spreadsheet that shows the data

Target Stability

Target stability data

Inter-run Calibration

Inter-run calibration data

Study Analysis Clustergram, Scatter Plot, Volcano Plot, and Heat Map
Analysis Settings

Settings for each chart type

Chart

Gene expression chart showing the data

Data

Spreadsheet listing the data in each target

146 QX ONE Software, Regulatory Edition

Gene Study Reports

Creating a Gene Study Report
Before creating a report, make any needed adjustments to the gene study data and charts.

To create a gene study report
1.

Select Tools Ú Reports in the Gene Study menu to open the Report dialog box.

2.

Select the options you want to include in the report.
The report opens with default options selected. Select or clear the checkboxes to change wholecategories or individual options within a category.
For information on the available options to display, see Gene Study Reports on page 146.

3.

Change the order of categories and items in a report.
Drag the options to the required position. Note that Items can be reordered only within the categories
to which they belong.

4.

Click Update Report to update the Report Preview window with any changes.

5.

Print or save the report:a.

Click the Print Report button on the toolbar to print the current report.

b.

Select File Ú Save to save the report in PDF, MHT or MHTML file format and select a location tosave the file.
Note: MHT and MHTML are Microsoft formats.

c.
6.

Select File Ú Save As to save the report with a new name or in a new location.

(Optional) To save the current report settings as a template, select Template Ú Save or Save As.

User Guide | 147

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148 QX ONE Software, Regulatory Edition

Chapter 7 System Utilities
From the System tab, you can manage tasks related to the instrument, software, data, and storage.
nSystem Settings — View shared and assigned storage locations, and select an alternate graphics
rendering method.

n

Event Log — View logged data about all software activities.

n

Maintenance Log — Record maintenance activities.

n

Maintenance Reports — PDF maintenance records from Bio-Rad maintenance.

nSystem Data — Archive raw data to clear up space in your primary storage areas, access data fortroubleshooting, and use the data recovery function.

User Guide | 149

Chapter 7 System Utilities

System Settings Tab
When you select the System tab, the System Settings window opens by default.

n

Use the Datafile and Template Storage tab to view storage locations:oPreferred Locations — if the checkbox selected, storage for all users is limited to a single folder
for data files and a single folder for templates.

oShared Settings — if the Preferred Locations checkbox remains cleared, then shared locations
can be defined, but remain optional. Users can choose to save their files to their personal folders
specified in User Preferences or to the shared location.

Note: The system administrator specifies shared and preferred storage locations, and can also
define a backup location for data file storage if the priority folder is full.
n

Use the Instrument tab to select the instrument model that is connected to QX ONE Software.

n

Use the Software Settings tab to change the graphics rendering method.

150 QX ONE Software, Regulatory Edition

System Settings Tab

Preferred Location
If the administrator sets Preferred Location file paths, then data files and templates for all users areautomatically saved to the specified folders. Preferred Locations override selections in individual user
preferences.
Important: Only users assigned the System Settings user privilege can set preferred locations.

To specify preferred locations
1.

Select the Use for all users checkbox.

2.

(Optional) Keep the default paths or modify them to alternative secure storage locations.

3.

Tap Save.

4.

When the confirmation message appears, tap OK.

User Guide | 151

Chapter 7 System Utilities

Shared Settings
Under Shared Settings, you can choose to save your files to locations that are available to all users.

The software allows the software administrator to set a primary storage folder and a backup storage
folder. Bio-Rad recommends routinely checking available storage space and if necessary, using theArchive function.
Important: Only users assigned the System Settings user privilege (typically your software
administrator) can change storage locations in the System Settings window.

To specify shared folder locations
1.

In each of the storage path fields, enter a file path or tap Browse to search for the folder.

2.

Tap Save.

3.

When the confirmation message appears, tap OK.

152 QX ONE Software, Regulatory Edition

System Settings Tab

Software Settings
The Software Settings tab provides an option to modify the chart rendering process to improve the chart
appearance.
Important: The poor rendering is associated with Intel graphics cards only. For more information,
contact Bio-Rad Technical Support. Do not change the setting unless instructed to do so by Bio-Rad
Technical Support.

Complete the following steps if directed to do so by Bio-Rad Technical Support.

To change the graphics rendering method1.

Under Chart Settings, tap Alternate.

2.

Click Save.

3.

Close QX ONE Software.

4.

To enable the change, restart the touch screen computer, and then reopen the software.

User Guide | 153

Chapter 7 System Utilities

System Log Files
From the System tab, you can access log files for system and software events (event log), andmaintenance activities (maintenance log).

Event Log
Users who are assigned the Maintenance user privilege can view the system event log, which contains
timestamped information on all software activities.

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System Log Files

To view the Event Log
1.

Select the System Utilities tab.

2.

Select Event Log.

3.

Tap Open Log to choose either the debug log or the history log.

4.

Review the log entries.

5.

(Optional) To automatically generate and display a PDF document, tap PDF Report.
Tip: To restrict access to the file, you can assign a password. Select the PDF Password checkbox and then enter a password in the field. The file is saved in the Log Reports folder and can be
opened only by entering the password.

User Guide | 155

Chapter 7 System Utilities

Maintenance Log
All users can view the maintenance log, which contains a list of maintenance records. The MaintenanceLog tab is available from the instrument touch screen only. Users who are assigned the Maintenance user
privilege can set up maintenance records, specify associated activities, and generate PDF reports.
Tip: This guide also contains a form on which you can document completion of maintenanceactivities. For information, see .

To add or view an activity
1.

Select the System Utilities tab.

2.

Select Maintenance Log. Note that activities that are already scheduled appear in the grid, sorted bynewest activity first.

3.

Do one of the following:n

Scroll to view a particular activity.
Important: You can view or add static activities; you cannot modify them.

n

Add an activity; tap the + icon next to New Activity.

A line item appears in the bottom section, with all fields except Activity completed automatically.
4.

In the Activity field in the bottom section, type the activity into the Activity field, and tap Save.

5.

Repeat to add another activity.

6.

(Optional) Tap PDF Report to create a report of the added activities.

156 QX ONE Software, Regulatory Edition

Maintenance Reports

Maintenance Reports
Reports generated from the Bio-Rad service engineer when performing maintenance on your instrument
are stored on the Maintenance Reports tab. The Maintenance Reports tab is available from the instrument
touch screen only.

To open a reportu

Tap the list item.

Following is an example of a Color Calibration report:

User Guide | 157

Chapter 7 System Utilities

System Data
In the System Data window, you cannArchive raw datan

Access troubleshooting datan

Recover data

Archiving Raw Data
When the storage space on the QX ONE instrument touch screen QX ONE Droplet Digital PCR Systemapproaches the limit displayed under Disk Space, you can free up space by moving raw data to an archive
storage folder in a different location, such as a network or USB drive.
Important: You must be assigned the System Data user privilege to archive data.

To archive raw data
1.

Select the System Utilities tab.

2.

Select System Data.
The Archive Raw Data tab is selected by default.

3.

Under Archive Progress, tap the calendar icons and select a From date and a To date to define a date
span.

4.

Tap Query to search for files dated within the date span.

5.

Tap Browse to search for and select the target storage location.

6.

Tap Archive.

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System Data

Troubleshooting Data
If issues occur with the QX ONE Software, you can access data to assist in troubleshooting the problem.
Important: You must be assigned the System Data user privilege to access troubleshooting data.

To access data for troubleshooting purposes
1.

Select the System Settings tab.

2.

Select System Data > Troubleshooting.

3.

Tap the From and To calendar icons to select a date range.

4.

Select any of the following checkboxes:nSystem Logsn

Raw Plate Datan

Version DatanCalibration Data

5.

Tap Browse and navigate to the data folder in the target storage location.

6.

Tap Collect Data.

User Guide | 159

Chapter 7 System Utilities

Recovering Data
If a run fails on the QX ONE Droplet Digital PCR System, you can use the Data Recovery tab in System
Data to recover data that has been saved to a temporary file.
Important: You must be assigned the System Data user privilege to access the Data Recovery tab.

To create a..ddpcrsone file from the data in the temporary file1.

Tap the System Utilities tab.

2.

Tap System Data, and then tap Data Recovery.

Temporary files that are available for conversion are listed in the top pane, which is scrollable.
3.

Tap a file, and then tap Browse

160 QX ONE Software, Regulatory Edition

System Calibration

4.

Navigate to the target storage location on your computer or network, and then click Recover.
The following advisory message appears.

5.

Click Yes.
A dialog box appears with a progress bar, and automatically closes when the conversion is complete.

The .ddpcrsone file is stored in the folder you specified.

System Calibration
You cannot execute runs on the QX ONE Droplet Digital PCR System unless the instrument is properly
calibrated. Only a Bio-Rad service engineer can calibrate your instrument and use the Instrument
Calibration options in QX ONE Software. The option is available from the touch screen only, and isdisabled for all other users.
If your instrument requires service, contact Bio-Rad Technical Support.

User Guide | 161

Chapter 7 System Utilities

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162 QX ONE Software, Regulatory Edition

Chapter 8 Managing Users
Use the User Management window to add, change, or remove QX ONE Software users, and set orchange user privileges and preferences.

The Bio-Rad service engineer sets up an initial user with all privileges, including Add/Manage users, on
the touch screen. Your software administrator can use this account to set up additional users withappropriate privileges.
You can set up local or domain accounts. If the instrument touch screen computerconnected to the QX
ONE Droplet Digital PCR SystemnCannot access your company’s network, your IT department or system adminisrator can set up usernames as local Windows accounts.

n

Can access your company’s network, QX ONE Software can recognize the Active Directory usernames.

You can create user accounts with any user name convention and password character structure, as longas it complies with your organizational requirements. The software is designed to validate each user
against the name it finds in either the Active Directory or local domain, so users must log in with the proper
domain and user name.

User Guide | 163

Chapter 8 Managing Users

Adding Users
Before you can add users to QX ONE Software, you must be logged in with the initial superuser account,or another account that is assigned the Add/Manage Users privilege.
Important: Before you begin, ensure that all users either have local Windows user accounts on thelocal computer or user accounts in Active Directory (network domain).

To add a user
1.

Open QX ONE Software and sign into the application.

2.

Tap the User Management tab on the left

3.

In the User Name field on the right, enter the user name as follows:n

If the computer is connected to a network and you are using Active Directory accounts, enter<domain name>/<user name>. For example, global/johnsmith.
Important: Active Directory users must log in every time with the domain name preceding
the user name.

n

If the computer is not connected to a network, enter <local computer name>/<user name>. For
example, analysiscomputer/johnsmith.
Important: To locate the computer name, right-click on the yellow folder in the lower menu
bar and select File Explorer. When the dialog box appears, the computer name is located at
the top of the left panel. Local users are not required to reenter the computer name after the
initial login.

164 QX ONE Software, Regulatory Edition

Adding Users

4.

Tap Check Name.
One of the following occurs:

5.

n

If the software recognizes the network domain or computer name and the user name, thesoftware displays a validation message.

n

If the user is not recognized, an error message appears. Do one of the following:o

For network users, ensure there is a working connection to the network and then verify yourentry is accurate.

o

For local users, verify the entry is accurate. If the user is not set up locally or on the network,
contact your system administrator for assistance.

When the This user is valid message appears, tap OK, and then tap Add at the bottom of the pane.
An Are you sure you want to add this user? message appears.

6.

Tap Yes.
A confirmation message appears.

7.

Tap OK.

8.

Continue until all users are added.

User Guide | 165

Chapter 8 Managing Users

Adding or Removing User Privileges
Only a user assigned the Add/Manage users privilege can add or remove user privileges. By default,
individual users without privileges can set up and execute a run, open files, view and analyze results.
For descriptions of all user privileges, see Viewing User Privileges on page 16.

Table 28 contains a sample set of privileges assigned by user roles common to a laboratory. User roles
can be defined and used in any scenario, but might be required in regulatory environments.
Table 28. Example User Roles

Superuser

Lab
Manager

Add/manage usersüü

Create new templates

ü

ü

ü

View data files created by

ü

ü

ü

ü

ü

ü

ü

ü

Privilege

Group
Lead

Technician/Student Internüother users
Override existing ddPCR file
name
System settingsü

MaintenanceüArchive data filesü

166 QX ONE Software, Regulatory Edition

üAdding or Removing User Privileges

Table 28. Example User Roles, continued

Privilege

Superuser

Lab
Manager

Group
Lead

Technician/Student Intern

Access the module recovery

ü

ü

ü

ü

ü

ü

ü

tool
Manage Plates

You can assign the Create new templates privilege to non-administrative roles if anyone can create andsave a template. If template formats are restricted, then you should assign this permission toadministrative or supervisory roles only.
Note: Bio-Rad recommends defining at least one additional superuser as a backup, as well as otheradministrative and standard roles based on your needs. Guest users can open shared templates,
execute runs, and perform analysis but are not typically assigned other privileges.

To add or modify privileges
1.

Tap the Add/Manage Users tab to open the User Management window.

2.

Select a user from the Current Users list. The name appears in the User Name field. A network orcomputer name and backslash might appear before the user name.

3.

Select or clear the checkbox for each privilege in accordance with the user’s role in using thesoftware. You can assign user privileges in any combination.

4.

When the confirmation message appears, tap Yes to save the changes, and then tap OK to close the
pop-up.

User Guide | 167

Chapter 8 Managing Users

Removing Users
Only a user assigned the Add/Manage Users privilege can remove users. Removing a user from thesoftware does not remove the user from other systems, such as Windows, or databases.

To remove a user
1.

Tap the Add/Manage Users tab to open the User Management window.

2.

In the Current Users pane, select the user and tap Remove. QX ONE Software displays aconfirmation message.

3.

Tap Yes to remove the user, and then tap OK to close the dialog box.

Modifying User Preferences
Users assigned the Add/Manage Users privilege can change the preferences for other users. Individual
users can also change their own preferences.

To modify a user's preferences1.

Tap the Add/Manage Users tab, and then enter the user name.

2.

Change any of the following preferences for the user:nEnter a different file path for user data files and templates, and choose to keep them private.
Important: You can also specify Preferred Locations in System Settings, to override all filepaths specified in individual preferences. For information, see System Settings Tab on page
150.

nEnter a different default system timeout period.

nEnter a different number of completed plates to show in the Status section, up to a maximum of
100.

3.

Tap Save. QX ONE Software displays a confirmation message.

4.

Tap Yes to save the changes, and then tap OK to close the dialog box.

168 QX ONE Software, Regulatory Edition

Appendix A Experiment Examples
This section contains setup and analysis information for the following sample experiments:n

Direct Quantification, using the Amplitude Multiplex assay typen

Copy Number Variation, using the Probe Mix Triplex assay typenDrop-Off, using the Basic Drop-off Assay type for genome edit detectionnDirect Quantification, using the Advanced Classification Method assay type

User Guide | 169

Appendix A Experiment Examples

Direct Quantification Multiplexed
You can increase the number of targets to be evaluated by using the Amplitude Multiplex option.
Optionally, to expedite plate setup you can copy and paste data from one well to another, and you can tap
Undo to undo an applied well setting. You cannot change the supermix that was used during datacollection.

Setting Up Experiment Parameters
To set experiment parameters
1.

Open the analysis data file and select the Plate Editor tab.

2.

Select one or more wells to be assigned well analysis attributes.

3.

Change the experiment type to Direct Quantification (DQ), and then tap Apply.

4.

(Optional) Change the sample description identifiers.

5.

(Optional) Change the sample type. The default type is Unknown.
Tip: For automatic thresholding of positive control wells, you must select Pos Ctrl.

6.

Under Assay Type, select Amplitude Multiplex and tap Apply. Eight target rows appear.

7.

Change the target name and type (unknown or reference) for each target in the well.
The software defaults to FAM Lo, FAM Hi, HEX Lo, HEX Hi, Cy5 Lo, Cy5 Hi, Cy5.5 Lo, and Cy5.5 Hi,
respectively, for each target.

170 QX ONE Software, Regulatory EditionDirect Quantification Multiplexed

8.

(Optional) Change the high and low target identifiers.
Amplitude Multiplex allows for up to two assays in each channel, indicated by Lo and Hi labels. In thismultiplex mode, each target may have a signal for only one channel. You can

9.

nSelect a different dye, if the channel supports two dyes (for example change HEX to VIC or
VIC to HEX)n

Select a different channel for the target (for example, change FAM to HEX)n

Change Hi to Lo or Lo to Hi

Tap Apply.

User Guide | 171

Appendix A Experiment Examples

Viewing and Adjusting Results
Automatic thresholding is not available for Amplitude Multiplex experiments.
For clusters that are easily identifiable across the chart, use Threshold Lines mode to set manualthresholds. If the data is not orthogonal and the threshold lines do not adequately divide the populations ofdroplets, use Threshold Clusters mode to set manual thresholds.

To set thresholds in the 2D Amplitude window
1.

Select the 2D Amplitude tab.

2.

On the toolbar, tap the Threshold Line Mode button.

3.

Tap anywhere on the plot to apply “best fit” threshold lines to the data.

4.

To adjust the thresholds, drag the lines horizontally or vertically in the plot area.
Tip: You can also enter a specific threshold value in the boxes at the end of each threshold line.

172 QX ONE Software, Regulatory EditionDirect Quantification Multiplexed

To apply and adjust manual clusters
1.

Tap any of the three cluster mode buttons (square, circle, or freehand).

2.

Press the left mouse button and draw the chosen shape around the cluster of droplets to be classified
first.
Tip: When you release the mouse button, a pop-up tool appears to help you select the correct
target combination. Target names are shown along the two axis based on the signal values
assigned during plate setup.

3.

To classify droplets as positive or negative for each given target, select the colored box that best
represents the location of the cluster of droplets.

User Guide | 173

Appendix A Experiment Examples

Copy Number Variation Triplexed
A CNV experiment with probe mix triplexing uses two targets and a reference in two groups of three toproduce analysis data. Optionally, to expedite plate setup you can copy and paste data from one well toanother, and you can tap Undo to undo an applied well setting.

Setting Up Experiment Parameters
To set experiment parameters
1.

Open the analysis data file and select the Plate Editor tab.

2.

Select one or more wells to be assigned well analysis attributes.

3.

Set the experiment type to Copy Number Variation (CNV), and tap Apply.

4.

(Optional) Change the sample description identifiers.

5.

(Optional) Change the sample type. The default type is Unknown.
Tip: For automatic thresholding of positive control wells, you must select Pos Ctrl.
Note: You cannot change the supermix that was used during data collection.

6.

Tap Apply.

7.

From the Assay Type dropdown list, select Probe Mix Triplex and tap Apply.
QX ONE Software displays three targets.
Important: You cannot add or delete triplex targets.

8.

Enter target names and select the target type (unknown or reference) for each target in the well.
This creates two groups of there targets each.

174 QX ONE Software, Regulatory Edition

Copy Number Variation Triplexed

9.

Assign the targets as follows:

Group 1
TargetTarget Type

Signal
Ch1

Signal Ch2

Ref
CopiesPlot?

Target 1

Unknown

FAM

None

N/A

N/A

Target 2

Unknown

None

HEX/VIC

N/A

N/A

Target 3

Reference

FAM

HEX/VIC

2

Yes

TargetTarget Type

SignalCh3SignalCh4

Ref
CopiesPlot?

Target 4

Unknown

Cy5

None

N/A

N/A

Target 5

Unknown

None

Cy5.5

N/A

N/A

Target 6

Reference

Cy5

Cy5.5

2

Yes

Group 2

10. Tap Apply.

User Guide | 175

Appendix A Experiment Examples

Viewing and Adjusting Results
Note: Linear thresholding is not available for Probe Mix Triplex experiments.

To see the results
1.

After all wells are configured, select the 2D Amplitude tab to evaluate the data and manually set
clusters.

2.

Tap any of the three cluster mode buttons (square, circle, or freehand).

3.

Press the left mouse button and draw the chosen shape around the cluster of droplets to be classified
first.
Tip: When you release the mouse button, a pop-up tool appears to help you select the correct
target combination. Target names are shown along the two axis based on the signal values
assigned during plate setup.

4.

To classify droplets as positive or negative for each given target, select the colored box that best
represents the location of the cluster of droplets.

176 QX ONE Software, Regulatory Edition

Drop-Off Assays for Genome Edit Detection

Drop-Off Assays for Genome Edit Detection
Optionally, to expedite plate setup you can copy and paste data from one well to another, and you can tap
Undo to undo an applied well setting.
This type allows two groups of two targets per well; Group 1, shown by default, with 1 target in FAM and
HEX (reference), and 1 target in FAM or HEX (unknown). You can add a second group with 1 target in
Cy5 and Cy5.5 (reference) and 1 target in Cy5 or Cy5.5 (unknown).
Note: The channels within each group are not interchangeable therefore FAM is always paired with
HEX/VIC and Cy5 with Cy5.5.

User Guide | 177

Appendix A Experiment Examples

Setting Up Experiment Parameters
To set experiment parameters
1.

Open the analysis data file and select the Plate Editor tab.

2.

Select one or more wells to be assigned well analysis attributes.

3.

Set the experiment type to Drop-Off (DOF) and tap Apply.

4.

(Optional) Change the sample description identifiers.

5.

(Optional) Change the sample type. The default type is Unknown.
Tip: For automatic thresholding of positive control wells, you must select Pos Ctrl.
Note: You cannot change the supermix that was used during data collection.

6.

Tap Apply.
QX ONE Software automatically assigns the assay method as Basic Drop-Off.

7.

Enter the target names, and then designate one target as Reference and the other as Unknown.
The Reference target enables ratio calculation of results.

8.

Tap Apply.

178 QX ONE Software, Regulatory Edition

Drop-Off Assays for Genome Edit Detection

Viewing and Adjusting Results
Thresholding is not available for Drop Off experiments.

To apply and adjust clusters
1.

After all wells are configured, select the 2D Amplitude tab to evaluate the data and manually set
clusters.

2.

Tap any of the three cluster mode buttons (square, circle, or freehand).

3.

Press the left mouse button and draw the chosen shape around the cluster of droplets to be classified
first.
Tip: When you release the mouse button, a pop-up tool appears to help you select the available
cluster assignments. Target names are shown along the two axis based on the signal values
assigned during plate setup.

4.

To classify droplets as positive or negative for each given target, select the colored box that best
represents the location of the cluster of droplets.

User Guide | 179

Appendix A Experiment Examples

Direct Quantification with the Advanced Classification Method
The Advanced Classification Method analysis mode allows the user complete control of droplet
identification, and does not force or preassign any clusters. This allows a higher degree of multiplexing
through radial dye mixing. Optionally, to expedite plate setup you can copy and paste data from one wellto another, and you can tap Undo to undo an applied well setting.
Important: This mode is intended for experienced users of Droplet Digital PCR.

Setting Up Experiment Parameters
To use the advanced classification method
1.

Open the analysis file and select the Plate Editor tab.

2.

Select one or more wells to be assigned well analysis attributes.

3.

Set the experiment type to Direct Quantification (DQ) or RED and tap Apply.

4.

(Optional) Change the sample description identifiers.

5.

(Optional) Change the sample type. The default type is Unknown.
Tip: For automatic thresholding of positive control wells, you must select Pos Ctrl.
Note: You cannot change the supermix that was used during data collection.

6.

Tap Apply.

7.

Under Assay Type, select Advanced Classification Method and tap Apply.

8.

Use the + icon to add up to ten targets.

180 QX ONE Software, Regulatory EditionDirect Quantification with the Advanced Classification Method

9.

Enter the target name and type (unknown or reference) for each target in the well.

10. (Optional) If you select Reference, you can change the number of reference copies. The default
is 2.
n

If no reference is selected, the target with the highest dye concentration in the highest channel isused as the reference. For example, if there are targets in all four channels, the target in Channel
4 is used; however if there are targets only in the first three channels, then Channel 3 is thereference target.

n

If more than one reference is selected in the well, checkboxes appear to the right for using thereference in the plot.

Note: Each Reference target enables ratio calculations in the results.
11. Assign the first target an expected signal in a channel.
12. Assign your last target a signal in a channel that was not selected for your first target.
Each target in between will be assigned a signal depending on mixture ratio of the target.
13. Tap Apply to confirm all well settings.

User Guide | 181

Appendix A Experiment Examples

Viewing and Adjusting Results
To view and adjust results
1.

Tap the 2D Amplitude tab.
All droplets are unclassified by default and are colored red. Only the Threshold Cluster mode isavailable for setting thresholds in Advanced Classification experiments in the 2D plot.

2.

Use one of the Threshold Cluster mode tools (box, circle or freehand) to assign droplet clusters totargets.
When you select a droplet cluster, a cluster assignment box appears.

3.

Select a radio button to identify the composition of the cluster.
For example, if the cluster is composed of positive droplets for Target-2, select the positive radio
button for Target-2 and the negative radio buttons for the remainder.
Important: For calculations to occur, you must have at least one positive cluster for each target,
as well as the assigned negative cluster.
Note: The software sets aside unassigned for calculation purposes. If you are unsure ofdroplet/cluster composition, leave it unassigned (red).
You can view the concentration results in the data table and in the Concentration tab.
If one of your samples was identified as a Reference under AssayType, you can view a ratio ofconcentration for unknown versus reference in the Ratio tab.

182 QX ONE Software, Regulatory Edition

Appendix B End User License Agreement
For research use only.
The Bio-Rad Droplet Digital PCR system is composed of a droplet generator, thermal cycler, droplet
reader, and related components, in a single instrument ( of the Droplet Generator and Droplet Reader andrelated components (collectively “Hardware”) and Bio-Rad’s proprietary QX Software and related files(collectively “Software”). The Hardware and Software are collectively referred to herein as the “System”.
BIO-RAD LABORATORIES, INC. (“BIO-RAD”) LICENSES THE SOFTWARE UNDER THE TERMS AND
CONDITIONS OF THIS AGREEMENT AND SOFTWARE DOCUMENTATION AS SET FORTH HEREIN(THIS “AGREEMENT”). BY YOUR PURCHASE OR EVALUATION OF THE SYSTEM OR USE OF THE
SOFTWARE, YOU (“YOU” or “END USER”) UNDERSTAND AND AGREE TO BE BOUND BY THISAGREEMENT. IF YOU ARE ENTERING INTO THIS AGREEMENT ON BEHALF OF A LEGAL ENTITY(A “COMPANY”), YOU REPRESENT THAT YOU HAVE THE AUTHORITY TO BIND, AND HEREBY
BIND, SUCH COMPANY TO THIS AGREEMENT. IN THE FOREGOING CASE, THE TERMS “END
USER,” “YOU” OR “YOUR” SHALL REFER TO YOUR COMPANY AND ALL OF ITS INDIVIDUAL
AUTHORIZED USERS OF THE SYSTEM.
1. Grant of License. Subject to the terms and conditions in this Agreement, Bio-Rad hereby grants to End
User a limited, non-exclusive, non-transferable, license to use the Software and any accompanying user
manuals (“Documentation”) (i) only on workstations that are either owned or operated by End User in theoperation of its business; (ii) only in conjunction with the System, or in the case of a Bio-Rad authorized
trial, only for use with sample data that may be provided by Bio-Rad for the limited purposes of evaluating
the Software; and (iii) only by You and Your Company’s employees to whom you provide access to the
System (“Authorized Users”). You agree that each Authorized User shall be bound by this Agreement.
2. Copies. End Users that have purchased the System may make a maximum of five (5) copies of the
Software for analyzing data at up to five (5) End User workstations and/or for backup or archival purposes.
In the case of a Bio-Rad authorized trial, End Users may make no more than one copy of the Software.
For any copies authorized by this Section, End User shall reproduce all copyright and other proprietary
notices in such copy. The original, and any copy of the Software, in whole or in part, shall at all times be
the sole and exclusive property of Bio-Rad. End User shall keep a record of each copy made, where suchcopy is located and in whose custody it is in.
3. Restrictions. The Software may not be incorporated in, or used in conjunction with, any other product,system or hardware other than the Hardware (except on authorized End User work stations as provided in
Section 1(i) above). End User shall not (a) modify, reverse engineer, disassemble, decompile orotherwise attempt to derive source code from the Software; (b) delete any copyright notices and/or other

User Guide | 183

Appendix B End User License Agreement

legends of ownership from the Software or Documentation or any copies made in accordance with thisAgreement; (c) create derivative works of the Software or Documentation; (d) assign, sublicense, rent,
loan or otherwise provide to any third party in any way the Software or Documentation, whether or notincorporated or merged into other programs or materials; or (e) allow the Software or Documentation to be
used directly by third parties, other than for End User’s internal use, or provide a copy of the Software or
any portion thereof or the Documentation to any third parties without Bio-Rad’s prior written consent.
4. Ownership. As between Bio-Rad and End User, Bio-Rad owns all rights, title, interest and ownership in
the Software and Documentation and all proprietary rights in the System and any subsequent releases,
versions or modifications to the System. Bio-Rad’s rights include, but are not limited to, all copies of the
Software and Documentation and any patent rights (including but not limited to patent applications anddisclosures), copyrights, trademark rights, trade secret rights, and any other intellectual property rightrecognized in any country or jurisdiction in the world as well as the content and related services and any
suggestions, ideas, enhancement requests, feedback, recommendations or other information provided by
End User or any other party relating to the System or any component thereof. Under the terms of thisAgreement, End User receives only a limited right to use the Software and Documentation. The Software
is protected both by United States laws and international treaty provisions. The System may include third
party products. Please read our accompanying documentation help files for further information. By using
the System, End User represents and warrants that it has complied and will continue to comply with theterms of use of any third party products that are covered by the System.
5. End User Responsibilities. End User is responsible for maintaining the security of End User’s login
information including any user names and passwords for End User’s use of the System. Your computer
systems must be selected, maintained, and controlled solely by You.
6. Term and Termination. This Agreement will become effective upon the first date on which End User
accesses the Software and will continue perpetually unless terminated by either party in accordance withthis Section 6 or, in the case of trials, as may be otherwise provided by Bio-Rad in applicable terms. BioRad may terminate this Agreement immediately if End User fails to comply with any provision of thisAgreement. Upon termination of this Agreement, all of End User’s rights and licenses to use the Software
shall cease immediately without any further action by Bio-Rad. End User shall cease all use of the
Software, and shall (i) return to Bio-Rad all existing copies of the Software, or (ii) furnish Bio-Rad evidence
satisfactory to Bio-Rad that the original and all copies of the Software have been destroyed. The following
provisions shall survive termination of this Agreement: Section 4, and Sections 6 – 11.
7. Confidentiality. “Confidential Information” means Bio-Rad’s non-public information relating to theSoftware, the Documentation, the System or any component thereof that may be disclosed to, or
accessed by, End User through use of the System, Software and/or Documentation. End User agrees not
to disclose or use any Confidential Information except as permitted in this Agreement. Access to the
Confidential Information shall be restricted to the End User’s employees and agents, who, in each case,need to have access to carry out a permitted use and are bound to maintain the confidentiality of such
Confidential Information. The Confidential Information, and all copies of part or all thereof, shall be and

184 QX ONE Software, Regulatory Editionremain the exclusive property of Bio-Rad, and End User shall acquire only such rights as are expressly
set forth in this Agreement and only for as long as such rights are in effect. Upon Bio-Rad’s request or
upon termination of this Agreement, End User shall return to Bio-Rad or destroy all copies of Confidential
Information in End User’s possession.
8. Indemnification. End User agrees to indemnify and hold Bio-Rad and its officers, agents, employees,
partnering agencies and/or associations harmless from any claim or demand or fines or fees, including
reasonable attorneys' fees, due to or arising out of End User’s breach of this Agreement or violation of any
applicable laws.
9. Warranty Disclaimers. Bio-Rad makes no warranties or representations concerning the Software orsystem or any results to be achieved through use of the software or system. Bio-Rad disclaims all
warranties, express or implied, including the warranties of merchantability and fitness for a particularpurpose, and all implied warranties arising from a course of dealing, usage, or trade practice. Bio-Rad
does not warrant that the System will be uninterrupted or error-free.
10. LIMITATION OF LIABILITY. IN NO EVENT SHALL BIO-RAD BE LIABLE FOR ANY LOSTREVENUE, PROFIT, OR DATA, OR FOR SPECIAL, INDIRECT, CONSEQUENTIAL, INCIDENTAL, OR
PUNITIVE DAMAGES HOWEVER CAUSED AND REGARDLESS OF THE THEORY OF LIABILITY,
ARISING OUT OF THE USE OF OR INABILITY TO USE THE SOFTWARE OR SYSTEM, INCLUDINGLOSSES RESULTING FROM LOSS, MISAPPROPRIATION OR UNAUTHORIZED ACCESS TO OR
MODIFICATION OF ANY END USER DATA, OR FROM MISTAKES, OMISSIONS, OR DELAYS IN ANY
TRANSMISSION OF INFORMATION, OR FROM INTERRUPTIONS IN ANY TELECOMMUNICATIONSCONNECTIONS TO THE SOFTWARE OR SYSTEM, VIRUSES OR FAILURES OF PERFORMANCE,OR FROM THE IMPACT OF THE SOFTWARE OR SYSTEM ON ANY SYSTEM, EVEN IF BIO-RAD HAS
BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGES. IN NO EVENT SHALL BIO-RAD’STOTAL LIABILITY TO YOU, WHETHER IN CONTRACT, TORT (INCLUDING NEGLIGENCE), OR
OTHERWISE, EXCEED THE AMOUNT OF ANY LICENSE FEE PAID BY YOU TO BIO-RAD FOR THE
SOFTWARE OR SYSTEM. The foregoing limitations shall apply even if the remedy fails of its essential
purpose. Because some states or jurisdictions do not allow limitation or exclusion of consequential
damages, the above limitation may not apply to End User.
11. General.
1. Compliance with Laws. You agree to comply fully with all relevant export laws, regulations, treaties, andorders (“Export Laws”) to assure that neither the System nor any components thereof are (i) exported,
directly or indirectly, in violation of Export Laws; or (ii) are intended to be used or are used for any
purposes prohibited by the Export Laws.
2. Assignment. End User may not assign this Agreement in whole or in part without the prior writtenconsent of Bio-Rad, except that End Users who have purchased the System may assign this Agreement(along with a transfer of the System) without such consent upon written notice to Bio-Rad in connection
with a merger, acquisition, corporate reorganization or sale of all or substantially all of its assets, unlessUser Guide | 185

Appendix B End User License Agreement

such transaction would result in an assignment to an entity reasonably deemed to be a direct competitorof Bio-Rad. This Agreement will inure to the benefit of, and will be binding upon, the parties and theirsuccessors and permitted assigns.
3. Notices. Notices shall be given by personally delivering or mailing same by first class U.S. mail, if to
Bio-Rad to: Bio-Rad Laboratories, 5731 West Las Positas Blvd., Pleasanton, CA 94588 or other address
as Bio-Rad may specify in writing; and if to End User to End User at the address provided by End User
during the respective purchase order, sign-up or registration process. Notice shall be deemed to havebeen given three (3) days after deposit in first class U.S. Mail, or if personally delivered, upon delivery.
4. Governing Law and Venue. This Agreement will be construed in accordance with and governed by thelaws of the State of California, USA, without giving effect to any choice of law rule that would cause theapplication of the laws of any other jurisdiction. The exclusive venue for actions relating to this Agreement
is the federal and/or state courts in Alameda County, California.
5. Audit Rights. Bio-Rad or a Bio-Rad agent may examine End User’s System, desktop computers,
laptops, servers, books and records for the purposes of verifying End User’s compliance with the terms ofthis Agreement, upon reasonable notice and during normal business hours at End User’s place ofbusiness. End User shall have the right to request that the agent acting on behalf of Bio-Rad sign anappropriate non-disclosure agreement with respect to any End User confidential information that may beviewed during an audit.
6. Relationship of the Parties. Nothing contained in this Agreement creates a joint venture, partnership,
agency or similar endeavor between the parties. Each party is acting solely as an independent contractor
and neither party has any power or authority to direct or indirectly bind or act on behalf of the other.
7. Agreement Construction. This Agreement (including accompanying purchase orders, if any) sets forth
the entire agreement between the parties and supersedes all prior agreements, understandings anddiscussions regarding the subject matter hereof. No purported amendment or modification of any
provision of this Agreement will be binding unless set forth in a written document agreed upon by theparties. No waiver by either party of any default of the other party will be held to be a waiver of any other orsubsequent default. No waiver shall be effective unless it is in writing and is signed by the party against
which it is asserted. If a provision contained or referred to in this Agreement is determined to be legallyinvalid or unenforceable, that provision will be ineffective to the extent of the invalidity or unenforceability
without affecting the remaining provisions of the Agreement, which will continue to be valid andenforceable to the fullest extent permitted by law.

186 QX ONE Software, Regulatory Edition

Appendix B Ordering Information
This appendix contains descriptions and catalog numbers for new or replacement items.

QX ONE Droplet Digital PCR System
Table 29 contains information on the QX ONE Droplet Digital PCR System components.
Table 29. QX ONE Droplet Digital PCR System

Product

Description

Catalognumber

QX ONE Droplet Digital
PCR System

Instrument used for droplet generation, PCR, droplet

12006536

reading, and data analysis
This purchase includes the QX ONE Accessory Kit(formerly catalog number 12011478), which contains thefollowing:n PX1 GCR Sealer Support Blockn QX ONE Droplet Digital PCR System ddPCR System
Waste bottles
n Keyboardn Power cord, compatible within North American Warranty card

QX ONE Software

instrument control, assay setup, and data collection andStandard
Edition, N/A

analysis

Regulatory

Connects to the QX ONE Droplet Digital PCR System for

Edition,12012078
Plate Sealer

PX1 PCR Plate Sealer

1814000

User Guide | 187

Appendix B Ordering Information

ddPCR Materials and Consumables
Table 30 contains information on additional items for performing ddPCR.
Table 30. Additional ddPCR materials and consumables

Product

Description

Catalog
Number

Reagents for Probe Detection
PCR Supermix

n ddPCR Supermix for Probes (No dUTP)

1863023
1863024
1863025

n ddPCR Supermix for Probes

1863026
18630101863027
1863028

n ddPCR Multiplex Supermix

12005909
12005910
12005911

n ddPCR Supermix for Residual DNA Quantification

1864037
1864038
1864039
1864040n One-Step RT-ddPCR Advanced Kit for Probes

18640211864022

DTT (dithiothreitol)

2 ml DTT

12012171

ControlddPCR Buffer Control for Probes

1863052

QX ONE Droplet

QX ONE Droplet Generation Oil for Probes, 15 plates

12006058

QX ONE Droplet Reader Oil, 10 plates

12006057

Digital PCR System
Droplet Generation Oil
QX ONE Droplet
Digital PCR System
Droplet Reader Oil

Consumables

188 QX ONE Software, Regulatory Edition

Table 30. Additional ddPCR materials and consumables, continued

Product

Description

Catalog
Number

Cartridge

n GCR96 Cartridge, pk of 1

12006858

n GCR96 Cartridges, pk of 10

12006859

Foil plate seal

GCR96 Foil Heat Seal

12006843

Waste bottle

QX ONE Droplet Digital PCR System ddPCR System Waste

12006060

bottleMaterials from Other Suppliers
Pipets (Rainin)

Pipet tips (Rainin)n 8-channel multichannel pipet, 2–20 µl

L8-20 XLSn Single channel pipet, 2-20 µl

L-20 XLSn Single channel pipet, 100–1,000 µl

L-1000 XLS

n 20 µl filter tips

GP-LTS-A10µL-/F-

n 1,000 µl filter tips

960/10GP-LTS-A1000µL-/F768/8

Reagent troughMatrix Reagent Reservoirs (Thermo Fisher Scientific)

8093

Vortexer

Any

N/A

Centrifuge

Centrifuge with plate rotor capable of at least 1,150 rcf

N/A

50 ml BD Luer-Lok Tip

Syringe for removing liquid from the overflow reservoir

309653

QX ONE Droplet Digital PCR System IQ/OQ/PQ service

IQ 12012331

Syringe
Services
Bio-Rad service

OQ 12012333
PQ 12012332

User Guide | 189

Appendix B Ordering Information

190 QX ONE Software, Regulatory Edition

10000148670 Ver D US/EG

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