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User Manual - Agilent

Agilent has prepared this manual as a technical reference for the ProSize data analysis software for use with the Fragment Analyzer, ZAG DNA Analyzer and.

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ProSize data analysis software user manual D0002111

ProSize Data Analysis Software
User Manual

Notices

Document Information
Document No: D0002111 Rev. A EDITION 02/2020
Copyright
© Agilent Technologies, Inc. 2020
No part of this manual may be reproduced in any form or by any means (including electronic storage and retrieval or translation into a foreign language) without prior agreement and written consent from Agilent Technologies, Inc. as governed by United States and international copyright laws.
Agilent Technologies, Inc. 5301 Stevens Creek Blvd. Santa Clara, CA 95051
Trademark acknowledgements
ProSize software is a registered trademark of Agilent Technologies, Inc.
Fragment Analyzer, ZAG DNA Analyzer and Femto Pulse Systems are a trademark of Agilent Technologies, Inc.
Windows is a registered trademark of Microsoft Corporation.
Revisions and Updates
This manual is subject to change without notice. This manual may be revised and updated periodically as components and/or maintenance procedures are modified. These updates will be provided to all manual holders on record. Contact Agilent if you have questions regarding availability of updates. Please take care to keep your contact information current with Agilent so that revisions and updates can be provided in a timely manner.

Warranty
The material contained in this document is provided "as is," and is subject to being changed, without notice, in future editions. Further, to the maximum extent permitted by applicable law, Agilent disclaims all warranties, either express or implied, with regard to this manual and any information contained herein, including but not limited to the implied warranties of merchantability and fitness for a particular purpose. Agilent shall not be liable for errors or for incidental or consequential damages in connection with the furnishing, use, or performance of this document or of any information contained herein. Should Agilent and the user have a separate written agreement with warranty terms covering the material in this document that conflict with these terms, the warranty terms in the separate agreement shall control.
Technology Licenses
The hardware and/or software described in this document are furnished under a license and may be used or copied only in accordance with the terms of such license.
Restricted Rights Legend
U.S. Government Restricted Rights. Software and technical data rights granted to the federal government include only those rights customarily provided to end user customers. Agilent provides this customary commercial license in Software and technical data pursuant to FAR 12.211 (Technical Data) and 12.212 (Computer Software) and, for the Department of Defense, DFARS 252.227-7015 (Technical Data Commercial Items) and DFARS 227.7202-3 (Rights in Commercial Computer Software or Computer Software Documentation).

Safety Notices
CAUTION
A CAUTION notice denotes a hazard. It calls attention to an operating procedure, practice, or the like that, if not correctly performed or adhered to, could result in damage to the product or loss of important data. Do not proceed beyond a CAUTION notice until the indicated conditions are fully understood and met.
WARNING
A WARNING notice denotes a hazard. It calls attention to an operating procedure, practice, or the like that, if not correctly performed or adhered to, could result in personal injury or death. Do not proceed beyond a WARNING notice until the indicated conditions are fully understood and met.

In This Guide
Agilent has prepared this manual as a technical reference for the ProSize data analysis software for use with the Fragment Analyzer, ZAG DNA Analyzer and Femto Pulse parallel electrophoresis systems.
This document includes software system requirements, installation procedures, software operation, troubleshooting guide, and technical support information.
This document is intended for use by technical personnel that are proficient with analytical instrumentation operation. A certain level of training and expertise is assumed and fundamentals are not addressed herein. Information is presented in a section-by-section format using screen captures and written descriptions. If questions remain after reviewing a given topic, please contact your corresponding Agilent Support or Service Representative.
1 ProSize System Overview
This chapter gives an overview about the ProSize data analysis software.
2 ProSize System Requirements and Installation
This chapter provides information on the requirements and installation instructions for the ProSize software.
3 ProSize Main Screen
This chapter provides an overview of the ProSize software main screen with the functions and menus available to the user. The file menu for opening and closing data files is also covered in this chapter.
4 ProSize Configuration
This chapter provides an overview of the Global Configuration dialog of the ProSize software. It also provides information on setting individual configuration parameters in opened data files.
5 ProSize Size Calibration Screen
This chapter describes the size calibration screen (also referred to as calibration curve screen). The calibration curve screen is used to select the conditions for calibrating the size of unknown sample peaks/smears.

ProSize Data Analysis Software User Manual

6 ProSize Overlay Samples
This chapter gives an overview of the possible ways to compare samples in the ProSize software.
7 Exporting Data from ProSize
This chapter provides an overview of the options available for exporting processed data from the ProSize software. The following sections describe how to open the Export Data window; provide an outline of various data export options; describe how to perform data export; and give examples of exported data.
8 Generating Reports from ProSize
This chapter provides an overview of the options available for generating reports from the ProSize software. The following sections describe how to open the Generate Report window; provide an outline of various report generation options; describe how to perform the report generation; and give examples of reported data.
9 ProSize View Capillary Positions
This chapter briefly covers the tools and functions of the View Capillary Positions window.
10 ProSize Troubleshooting Guide
This chapter gives an overview of troubleshooting the ProSize software.
11 Batch Processing Using ProSize
This chapter explains how to perform a batch process and provides an outline of the various exporting options.
12 ProSize Enhanced Security Features
This chapter explains how to perform a batch process and provides an outline of the various exporting options.

ProSize Data Analysis Software User Manual

Content

1 ProSize System Overview 7 About the System 8
2 ProSize System Requirements and Installation 10 System Requirements 11 ProSize Software Installation Instructions 13 Starting the ProSize Program 16
3 ProSize Main Screen 18 Main Screen (No Data File Open) 19
4 ProSize Configuration 75 About Configuration 76 Overview Global Configuration Dialog 80 Set Individual Parameters 108
5 ProSize Size Calibration Screen 127 About Size Calibration 128
6 ProSize Overlay Samples 144 Overlay Options 145
7 Exporting Data from ProSize 163 Export Data Utility 164
8 Generating Reports from ProSize 177 Generate a Report 178 Examples of Generated Reports 182
9 ProSize View Capillary Positions 187 About View Capillary Positions 188

ProSize Data Analysis Software User Manual

Content
10 ProSize Troubleshooting Guide 198 ProSize Software Error Messages 199
11 Batch Processing Using ProSize 201 Batch Processing 202
12 ProSize Enhanced Security Features 207 Activate Enhanced Security Feature 208

ProSize Data Analysis Software User Manual

ProSize System Overview

About the System 8
ProSize Data Analysis Software 8

This chapter gives an overview about the ProSize data analysis software.

ProSize Data Analysis Software User Manual

ProSize System Overview

About the System

ProSize Data Analysis Software
Agilent's ProSize software is designed for analyzing the raw data from the parallel capillary electrophoresis instruments and reporting the size (in base pairs for dsDNA or nucleotides for RNA) and concentration (in ng/mL or nmol/L) of separated DNA or RNA fragments. The ProSize software performs the following general functions:
· Reads the raw data files generated from the instrument operational software.
· Provides calibration of DNA size (in bp) or RNA size (in nt) from standard mixtures and conversely measures DNA/RNA size of unknown fragments in a sample.
· Provides quantitative measurements of DNA/RNA concentrations present in a sample either from relative comparisons to an internal/external standard peak or by comparison to a calibration DNA/RNA ladder.
· Exports size and concentration data as well as raw electropherogram trace data (time vs. fluorescence intensity) in a generic comma separated values (.csv) file format; and digital electropherogram trace or gel view images in .jpg, .bmp, or .png image formats.
· Generates PDF format sample reports containing user specified information including measured DNA/RNA size, DNA/RNA concentration, digital gel images, experimental information, and/or other various calculated sample properties.
· Provides option for enhanced security features and digital signature generation.
This manual serves as a guide to the ProSize software and will assist the user in taking advantage of the many benefits of the parallel capillary electrophoresis instruments.

ProSize Data Analysis Software User Manual

ProSize System Requirements and Installation

System Requirements 11
General Software Requirements 11 Operating System 11 PC Recommendation 12
ProSize Software Installation Instructions 13
Starting the ProSize Program 16
Exit the ProSize software 17

This chapter provides information on the requirements and installation instructions for the ProSize software.

ProSize Data Analysis Software User Manual

ProSize System Requirements and Installation

System Requirements

General Software Requirements
Report Review
A PDF viewer is required to read Adobe PDF formatted reports. Install the most recent version of Adobe Reader. The software is available free of charge for download at www.adobe.com.
Data File Export
· The ProSize software exports data in a generic comma-separated values (.csv) file format, which can be read by most spreadsheet and database management programs. Install a spreadsheet program (for example, Excel, OpenOffice) to open and read exported .csv files.
· Electropherogram and digital gel images are exported in a .jpg, .bmp, or .png image format. A suitable program for viewing these file formats should be installed.

Operating System

The ProSize software must be installed on a computer running a Windows operating system to function properly.

Table 1 Supported operating systems Operating System Windows

Details Windows 7 or Windows 10, 32-bit or 64-bit

ProSize Data Analysis Software User Manual

ProSize System Requirements and Installation

PC Recommendation

The table provides the recommended hardware configuration for the ProSize software.

Table 2 Recommended hardware configuration

Item

Details

Processor speed (CPU)

Intel Core 2, or faster

Physical memory (RAM)

4 GB

Hard disk

500 GB (for accommodation of raw data files)

Graphic resolution

1280 x 800 minimum screen resolution 1280 x 1024 recommended

ProSize Data Analysis Software User Manual

ProSize System Requirements and Installation

ProSize Software Installation Instructions

To Install the Software
1 Download the appropriate installer from the Agilent website. 2 Navigate to the folder ProSize data analysis software. 3 Run the installer (640,980 KB) and follow the instructions and prompts
provided by the installation wizard:

Figure 1

Default directory locations for the ProSize data analysis software and supporting National Instrument software

a Provide the installation directory. The default directory is C:\ProSize data analysis software\.
A shortcut of the ProSize software is created in the Start menu.
Supported software from National Instruments will also be installed under C:\Program Files\National Instruments\. b Complete the following installation steps.

ProSize Data Analysis Software User Manual

ProSize System Requirements and Installation

ProSize Software Installation Instructions

c Restart the computer to complete the installation process.
Upon successful installation of the ProSize software, several sub-folders will have been created within the C:\ProSize data analysis software\ directory (see Figure 2):
· Configurations: This folder contains ProSize global configuration setting files, optimized for different application kits for the parallel capillary electrophoresis instruments.
· Ladders: This folder contains ProSize sizing ladder information files (.txt format), for use with different application kits for the parallel capillary electrophoresis instruments.
· Support: This folder contains supporting files for ProSize software operation.
· Training Test Runs: This folder contains data files, for use with a set of tutorial videos which are loaded upon installation of the ProSize software.
· Video: This folder contains ProSize tutorial videos, which can be accessed via the Help menu as described in the following section.

Figure 2 Sub-folders created within the C:\ProSize data analysis software\ directory

ProSize Data Analysis Software User Manual

2
NOTE

ProSize System Requirements and Installation ProSize Software Installation Instructions
4 Update the Configurations, Ladders and Video folders within the C:\ProSize data analysis software\ directory to the latest versions. You can obtain the most recent version of software and files from Agilent support. To update the folder files: a Navigate to C:\ProSize data analysis software\. b Delete the contained folders Configurations, Ladders, and Video. c Paste the folders Configurations, Ladders, and Video supplied from Agilent into the same directory C:\ProSize data analysis software\.
After installation of the ProSize 3.0 software, the system will install System.Data.SQLite. Please follow all screen instructions on this installation.
To properly read PDF reports:
1 Install the latest version of Adobe Reader on your computer. This software is available free of charge for download at www.adobe.com.

ProSize Data Analysis Software User Manual

ProSize System Requirements and Installation

Starting the ProSize Program

1 In the Windows Start menu, navigate to ProSize data analysis software. Alternatively, create a shortcut from your desktop to launch the program.
A splash screen appears showing the software version (Figure 3).

Figure 3

Splash screen displaying version information upon starting the ProSize software (Note: This example shows software version 4.0. This manual refers to version 1.0, and higher)

When the software is open, a screen with a white background screen is displayed. At the upper part of the screen you see two active main menu options (see Figure 4):
· File > Open File: Use this drop-down menu to open data files. Alternatively, from the toolbar, select to open your data.
· Help > About: Shows information about the software version and Agilent contact information.
· Help > Tutorial Videos: Offers a set of tutorial videos on how to use the software. The other commands of this menu are discussed in detail in Chapter 3, "ProSize Main Screen".

ProSize Data Analysis Software User Manual

ProSize System Requirements and Installation

Exit the ProSize software

Figure 4 Main screen of the ProSize software
Exit the ProSize software
To close the ProSize software, select Exit from the File menu, or press .

ProSize Data Analysis Software User Manual

ProSize Main Screen

Main Screen (No Data File Open) 19
Opening a Data File in ProSize Software 23 Configuration Dialog 25 Main Screen (Data File Open) 27 Plate Map 31 Digital Gel Image Toolbar 33 Electropherogram Trace Toolbar Menu 37 Electropherogram Trace Context Menu 41 Electropherogram Trace Context Menu RNA Mode 45 Peak Table DNA Mode 47 Peak Table NGS Mode 49 Peak Table gDNA Mode 51 Peak Table Total RNA Mode 52 Peak Table mRNA Mode 54 Peak Table Small RNA Mode 55 Peak Table Mutation Analysis Mode 56 Flag Analysis Table 57 Smear Analysis Table 59 Advanced Flag Analysis Table 62 Set Individual Parameters 63

This chapter provides an overview of the ProSize software main screen with the functions and menus available to the user. The file menu for opening and closing data files is also covered in this chapter.

ProSize Data Analysis Software User Manual

ProSize Main Screen

Main Screen (No Data File Open)

The functions and menu items available from the main screen of the ProSize software prior to opening a *.raw data file are summarized in Table 3.

Figure 5 Main screen of ProSize software (no data file open).

Table 3 Main Menu screen menu items (active with no data file open)

Menu Command File Open File
Close File

Description
Opens a Select folder dialog for locating and opening a raw data file (*.raw extension) generated by the parallel capillary electrophoresis instruments for data analysis (Figure 7 ). Note that if the configuration name (ProSize) and the method name (FA Software) do not exactly match, the first time a data file is opened in the ProSize software, the Global Configuration dialog is displayed. The screen allows you to select the appropriate predefined data configuration file (*.ini extension), or to customize a configuration file be selecting the data analysis mode (DNA, RNA, NGS, Mutation Analysis, gDNA, Restriction Digest) and analysis settings (Figure 9).
Closes the ProSize software. When closing, the data file, all settings and operations are automatically saved.

ProSize Data Analysis Software User Manual

ProSize Main Screen

Main Screen (No Data File Open)

Table 3 Main Menu screen menu items (active with no data file open)

Menu Command Administration Configuration
Event Report Option > Option dialog settings Set Default Data Path
Language Capillary Array Format Display Option DNA Quality Number (DQN) Molarity Calculation %(Conc.) based on Image Resolution Edit Configuration Email

Description
Opens a pop-up menu with configuration settings. The user can enable/disable the following settings: · Login required · Enhanced security (for more details, refer to Chapter 12, "ProSize
Enhanced Security Features") · Auto Logoff Time · Minimum Password Length · Password Expiration · Allowed Login Attempts · Previous Unique Passwords
Opens the Event Log reporting the User ID, Computer Name, Event Time, Operation, File Path (when relevant), and a Description of the event.
Allows you to set the default file path for accessing and saving data files. In the Select folder dialog, navigate to the desired directory and select the folder to be set to open data files (Figure 7). The default directory for opening *.raw data files is C:\Agilent\Data\.
Allows you to select the language of the program (English, German, etc.).
Allows you to arrange the data from left to right in the Digital Gel Image display by Row (A1, A2, A3, etc.) or by Column (A1, B1, C1, etc.).
Allows you to select the view of the data by selecting Scale to Sample or the Autoscale function.
Allows you to enable or disable DQN.
Allows you to Use Peak Maxima or Use Avg. Size for all molarity calculations.
Allows you to select the preferred concentration units, ng/µL ... nmole/L, or pg/µL ... pmole/L.
Define the image resolution as 1X, 2X, 3X, or 4X used for exporting electropherograms or gel images.
Opens the Global Configuration dialog (Figure 9), where you can set parameters for data analysis mode, peak and marker integration, quantification, ladder size, etc.
Allows you to set up an email account to send results to.

ProSize Data Analysis Software User Manual

ProSize Main Screen

Main Screen (No Data File Open)

Table 3 Main Menu screen menu items (active with no data file open)

Menu Command Project Load Project Create Project Batch Processing Batch Data Process View Error Log Help User Manuals Tutorial Videos
About

Description
Opens a Select Folder dialog for locating and opening a Project data file (*.proj extension) generated for data analysis. Allows you to open the Project screen and create a project by selecting different *.raw data files.
Allows you to process a set of *.raw files within a selected folder, and to define an output folder within which processed files are sent. Allows you to view the error log associated with a particular batch data process.
Access all ProSize User manuals. Allows you to open and view a selection of tutorial videos to aid in the use of the ProSize software. Alternatively, the tutorial videos can be opened directly from the C:\ProSize data analysis software\Video directory. Displays software information, version information, copyright information and Agilent technical support contact information.

The Options pop-up dialog is shown in Figure 6. The properties of the dialog are described in Table 3 above.

ProSize Data Analysis Software User Manual

ProSize Main Screen

Main Screen (No Data File Open)

Figure 6 Option dialog

Figure 7 Set Default Data Path selection screen

ProSize Data Analysis Software User Manual

ProSize Main Screen

Opening a Data File in ProSize Software

To Open a Data File

1 From the menu, select File > Open File (alternatively, select toolbar).

from the

The Select Folder dialog opens (Figure 7). 2 Navigate to your raw data file (*.raw extension) for data analysis. 3 Select the folder with the raw data.

In the ProSize software, the Open screen is displayed, showing the selected file directory (Figure 8).

If you need to navigate to a higher-level directory, select the folder right of the file path and a standard file browser will open.
4 In the Open screen, click a data file.

to the

A file previewer window will be displayed on the right side of the screen, containing a digital gel image of the data, a Sample Information list, any typed Note, and a summary of the experimental Method used to collect the data file (Figure 8). If the data file has not been previously processed in ProSize, the heading of the digital gel image will be labeled Non-processed Data. If the data file has been previously processed, no heading will be displayed on the digital gel image.
5 If the correct data file is verified, select Open to load the data file into ProSize. Select Cancel to close the Open screen without loading a file.

ProSize Data Analysis Software User Manual

ProSize Main Screen

Opening a Data File in ProSize Software

Figure 8 Open file data selection and previewer screen

ProSize Data Analysis Software User Manual

ProSize Main Screen

Configuration Dialog

NOTE

Upon selecting Open, the data file will be processed if the matching predefined data analysis configuration file is available (for example, DNF-473-33 - SS NGS Fragment 1-6000bp.ini). The first 10 characters of the configuration file name must match the first 10 characters of the separation method used to automatically process the data.
A minimum of 10 characters must be used to name both the method (instrument) and configuration (ProSize).
Should there be no matching configuration file, the Global Configuration dialog will be loaded when opening a previously unprocessed data file. Should the wrong configuration file be chosen, the user can navigate to the Option window, and select Edit Configuration to load the correct configuration.
Upon opening, there are ten primary tabs in the Global Configuration dialog (Figure 9). The settings are discussed in detail in Chapter 4, "ProSize Configuration".

Figure 9 Global Configuration dialog (Advanced Settings tab is selected)

ProSize Data Analysis Software User Manual

3
NOTE NOTE

ProSize Main Screen Configuration Dialog
You may create a customized configuration by selecting the proper data analysis mode (DNA, RNA, etc.) and analysis settings.
In most cases, predefined, optimized global configuration files are available to load for each Agilent Reagent Kit. The configuration files are labeled according to the particular kit number, for example, DNF-910-33 - DNA 35-1500bp.ini.
After the configuration settings have been loaded, select Apply to open the data file in the main screen of the ProSize software.

ProSize Data Analysis Software User Manual

ProSize Main Screen

Main Screen (Data File Open)

Once a data file is opened in ProSize, several additional main screen functions are enabled as summarized in Table 4. The opened data is displayed in the main screen as shown in Figure 10 (12-capillary data) and Figure 11 (96-capillary data).

Table 4 Additional Main screen menu items (active only with data file open)

Menu Option

Description

File

Close File

Closes the currently opened data file and displays the main screen. All settings up to the point of closing the data file will be saved and reloaded when reopening the data file.

Generate Report or

Opens a menu for configuring and generating an Adobe .pdf report, containing user specified information (electropherograms, gel images, peak information, etc.). For more information, see Chapter 8, "Generating Reports from ProSize".

Export Data or

Opens a menu for configuring and exporting user specified data to a user specified folder. Types of data include gel images, electropherograms, peak tables, size calibration data, etc. For more information, see Chapter 7, "Exporting Data from ProSize".

Analysis

Show Size Calibration or

Opens the Calibration Curve screen, which contains input data associated with the size calibration ladder. For more information, see Chapter 5, "ProSize Size Calibration Screen".

Overlay Samples or

Opens the Overlay Samples screen, where you can overlay gel images or electropherograms from individual lanes or different data files. For more information, see Chapter 6, "ProSize Overlay Samples".

View Capillary Positions

Opens the View Capillary Positions screen, which allows you to view and modify the alignment of the detector signal with the capillary positions. For more information, see Chapter 9, "ProSize View Capillary Positions".

Quick Display

Displays the Quick Display window, which shows non-normalized electropherograms for all 12 capillaries (or windows of 12 capillaries each for a 96-capillary array format) enabling quick review of the data.

Display Separation Parameters Displays the Separation Parameters window, which allows the user to view the current, voltage, and pressure that occurred during the separation.

ProSize Data Analysis Software User Manual

ProSize Main Screen

Main Screen (Data File Open)

Figure 10 Main screen of ProSize software (12-capillary data file open)

1 2
3 4
6

11 12

14 13
Figure 11 Main screen of ProSize software (96-capillary data file open)

ProSize Data Analysis Software User Manual

ProSize Main Screen

Main Screen (Data File Open)

The primary accessible functions from the main screen are shown in Figure 11, and are summarized in the legend table:

1 Main screen toolbar

2 Digital Gel image

Digital Gel Image of the data set (see also Figure 14, Table 6). The sample wells are normally displayed in increasing order for each row. Starting on the left side starting with well A1 (A1, A2, A3-A12; followed by B1-B12, etc.). Change the view to ascending order of the well columns from left to right (A1, B1, C1, etc...). From the Option menu, select Capillary Array Format Column. To toggle back to a row-by-row format, select Capillary Array Format Row.

3 Plate map

Shows the currently selected well highlighted in green (A1 is the default location on opening the file). Click on the appropriate well by using the keyboard arrows, or double-clicking the appropriate lanes of the digital gel image. You can select any well of the plate for individual viewing. The functions available within the Plate Map menu are summarized in Table 5.

4 Info

Click to access experimental information regarding the method used to collect the data file.

5 Sample ID navigation Enables Sample ID navigation. The Sample ID list as entered into the instrument software will be displayed, enabling you to navigate the samples by Sample ID and to edit sample names if desired. Close the Sample ID navigation view by right-clicking in the list, and select Close.

6 Contrast

Use the slider bar to change the intensity contrast of the gel image.

7 Re-scale

Re-scale the electropherogram image and gel image by sliding the bar between the two images.

8 Electropherogram An Electropherogram trace of the currently selected well. The well position and sample name (if

trace

imported) is displayed in the upper left corner of the trace. Right-click the screen to process or

annotate the data (Table 7 and Table 8).

9 Scaling y-axis

Units for the y-axis can be changed by right clicking the axis title and selecting RFU or nmole/L.

10 Scaling x-axis

Units for the x-axis can be changed by right-clicking the axis title and selecting time scale or size scale.

11 Set individual parameters

Select to reveal the configuration window, for which most settings can be individually adjusted for each sample as desired (see Figure 13). A brief summary of the primary configuration functions are presented in this section. For more detailed information, refer to Chapter 4, "ProSize Configuration".

12 Peak table

Displays information about the selected and integrated sample peaks and markers (see also Figure 18). Depending on the configurations: · A Flag Analysis tab will be shown if the flag configuration function contains any flag conditions
(Figure 34). · A Smear Analysis tab will be shown if the smear analysis configuration contains any entries
(Figure 33). · An Advanced Flag tab will be displayed if any advanced flag functions are used (Figure 36).

13 Expand Electropherogram trace

Select to expand the Electropherogram trace across the bottom panel which provides you with more zoom options (see Figure 12).

14 Filename

Upon opening a data file, the currently selected Filename/directory is displayed in the lower left toolbar.

ProSize Data Analysis Software User Manual

ProSize Main Screen

Main Screen (Data File Open)

Figure 12 Expanded Electropherogram Trace

Figure 13 Main screen with data file open and open configuration dialog (Set Individual Parameters dialog)

ProSize Data Analysis Software User Manual

ProSize Main Screen

Plate Map

The functions available within for the Plate Map are summarized in Table 5.

Table 5 Plate Map menu functions

Menu Option Plate Map

Description
Used to select a sample well for adjusting marker/peak integration, assigning flag or smear analysis conditions, viewing Peak Table information and Electropherogram Trace, or adjusting lane contrast in the Digital Gel Image. Possibilities to select a well: · Click the well. · Change the well selection via the keyboard arrows. · Double-click the lane in the digital gel image. · Right-click the sample wells to overlay them in the Electropherogram
section of the screen. · Select the Sample ID in the Sample ID navigation pane.

To display a well/sample name, place the mouse cursor over the well.

ProSize Data Analysis Software User Manual

ProSize Main Screen

Plate Map

Table 5 Plate Map menu functions

Menu Option Experimental Information

Description
Shows the instrument system software user interface, containing the experimental information, Capillary Array information, instrument system information, and Notes fields for the collected data file. To close the window, click .

Sample ID navigation

The Sample ID list will be displayed. In the list, you can navigate the samples by the Sample ID previously entered into the instrument system software. Double-click on a sample name allows you to edit the Sample ID information. Right-click the sample ID allows you to Import a .txt file. Select Close to exit the Sample ID list and return to the plate map view.

ProSize Data Analysis Software User Manual

ProSize Main Screen

Digital Gel Image Toolbar

The toolbar options above the digital gel image are summarized in Table 6. The gel image is annotated on both the left and right side with the sample sizing ladder in bp or nt. The currently selected lane is bracketed by vertical cursors (Figure 14).
Right-click on any sample lane will overlay the samples in the electropherogram view. Double-click on a sample lane to view a single sample well in the electropherogram view.
Additional information is available under Help > Tutorial Videos > ProSize Gel Image Functions.

Figure 14 Main screen zoomed to the digital gel image and its toolbar menu

Table 6 Digital Gel Image toolbar

Toolbar Option Copy Full Gel Image
Copy Selected Gel Lane Image

Description
Copies an image of the full Digital Gel Image to the clipboard, for pasting in another program such as Microsoft PowerPoint. If zoomed into the gel image, it will copy the visible portion of the image. The left and right side of the gel image is annotated with the sizing ladder in the copied gel image.
Copies an image of the currently selected gel lane to the clipboard, for pasting in another program such as Microsoft PowerPoint. If zoomed into the gel image, it will copy the visible portion of the gel lane image. The left side of the gel lane image is annotated with the sizing ladder in the copied image.

ProSize Data Analysis Software User Manual

ProSize Main Screen

Digital Gel Image Toolbar

Table 6 Digital Gel Image toolbar Toolbar Option Show Cursor
Hide Cursor Create Annotation

Description
Displays a horizontal line cursor across the digital gel image, annotated by size (bp or nt) corresponding to the current cursor position. The cursor can be dragged to any position along the gel image by holding the mouse over the cursor and pressing and holding the left mouse button. When the cursor is active and displayed, a similar vertical cursor annotated by size is displayed in the Electropherogram Trace, whose movement corresponds to that in the gel image. Up to four cursors can be displayed at one time.
To deactivate the cursor, press the button a second time.
To create customized annotations in the Digital Gel Image display. When selected, the Add Annotation window opens. You can type desired annotation into the field, and select OK. The annotation will be displayed in the Digital Gel Image window.

ProSize Data Analysis Software User Manual

ProSize Main Screen

Digital Gel Image Toolbar

Table 6 Digital Gel Image toolbar Toolbar Option Edit Annotations

Description
Opens the Annotation Editor window. Allows you to: · Edit the annotation text by typing in the text field. · Change the color of the annotation by left clicking on the
annotation color. · Delete one annotation by selecting Delete next to each
annotation, or delete all annotations by selecting Delete All.

Zoom AutoFit Auto Intensity Normalize Intensity to Lower Marker
Normalize Intensity to Upper Marker
ProSize Data Analysis Software User Manual

Select Apply, to confirm your settings.
When selected, zooming to an area by clicking and dragging is enabled.
Autoscales the Digital Gel Image x-/y-axis view for all 12 or 96 lanes. The autoscaled image will display the gel from the selected lower marker to upper marker, or if only a lower marker only is used from the lower marker to the end of the separation window.
Autoscales the intensity of the Digital Gel Image view for all 12 or 96 lanes. The viewed intensity across the image will reflect the actual observed fluorescence signals from each lane.
Normalizes the intensity of the Digital Gel Image view for the lower marker to the same value for all 12 or 96 lanes, adjusting the relative intensities of all bands in each lane accordingly. The viewed intensity across the image will as a result reflect the fluorescence signals from each lane after normalization to the lower marker, as is done when calculating the concentration of samples using the lower marker.
Normalizes the intensity of the Digital Gel Image view for the upper marker to the same value for all 12 or 96 lanes, adjusting the relative intensities of all bands in each lane accordingly. The viewed intensity across the image will as a result reflect the fluorescence signals from each lane after normalization to the upper marker, as is done when calculating the concentration of samples using the upper marker.
35

ProSize Main Screen

Digital Gel Image Toolbar

Table 6 Digital Gel Image toolbar Toolbar Option Enhanced Contrast
Gel Image Color
1:1 Gel Image Hide/Show Marker Peaks Hide/Show non-integrated peaks Gaussian Fit

Description
Increases the contrast of an individual lane while holding other lanes constant. When selected, the Lane Contrast Control window opens. Drag the scroll bar to adjust the contrast for the currently selected lane in the gel image. Once adjusted, click OK to accept changes, or select Cancel to discard your changes. Note: The enhanced contrast setting works on the currently selected lane only (marked with vertical cursors in gel image). The Auto Intensity, Normalize Intensity to Lower Marker, and Normalize Intensity to Upper Marker functions will undo any previous enhanced contrast operations.
Changes the color scheme for the Digital Gel View, the gel image in the Overlay Samples screen and any exported or printed report. Options for the gel image color: · White on Black (white bands on black background) · Black on White (black bands on white background) · Green on Black (green bands on black background) · Red on Black (red bands on black background) · Pseudo Color (green to red bands on blue background)
Displays the gel image in an adjustable window for changing the aspect ratio of the gel image (useful for producing figures for papers). The adjusted image can be copied to the clipboard.
Toggle to hide or show the marker peaks in the digital gel image screen. This is useful when comparing to a slab gel result which does not use lower/upper normalization markers.
Toggle to hide or show non-integrated peaks in the digital gel image screen. This is useful for eliminating noise peaks from the gel image.
Toggle to fit all peaks to a narrow Gaussian peak.

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ProSize Main Screen

Electropherogram Trace Toolbar Menu

The options available in the Electropherogram Trace toolbar are summarized in Table 7.
Additional information is available under Help > Tutorial Videos > ProSize Electropherogram Functions.

NOTE

Figure 15 Main screen zoomed to the Electropherogram Trace and its toolbar menu. Note the y-axis scale is set to the sample peaks and not the marker peak heights.
In version 4.0 of the ProSize software, the Electropherogram Trace will automatically autoscale to the largest observed sample peak, regardless of the lower/upper marker peak heights. To view the entire y-axis scale including marker peaks, in the Option window, select Autoscale as the Display Option (see Figure 6).

ProSize Data Analysis Software User Manual

ProSize Main Screen

Electropherogram Trace Toolbar Menu

Table 7 Electropherogram Trace toolbar functions

Toolbar Option Zoom
Pan Autoscale
Copy Units

Description
Enables zooming in the x- and y-axis of the Electropherogram Trace. To zoom-in: Place the mouse over the trace, click and drag it outward to expand a box area to define the zoom region. Release the mouse button to apply. The zoomed image can be copied to the clipboard. The zoom region will be preserved when viewing other samples in the Plate Map. To undo the zoom, use the Autoscale function.
Enables panning of the image. Move the image around with the mouse cursor.
To autoscale the Electropherogram Trace x-/y-axis display. The autoscaled image will display the full trace from the start to the end of the separation when using the Time Scale and Size Scale display modes; when using the Uniform Size Scale the autoscaled display will start at 0 bp. Note: the y-axis scale will automatically zoom to the highest detected sample peak, regardless of the marker peak heights. To view the entire y-axis scale including markers, go to Option window, and as Display Option, select Autoscale.
Copies an image of the current view of the Electropherogram Trace to the clipboard, for pasting in another program such as Microsoft PowerPoint. Any zoom, annotation, baseline and/or peak start/end point displayed will be copied in the image. The well ID and sample name will be copied in the top left of the trace, and the x-/y-axis will correspond to the currently selected view in the copied image.
Displays a menu for changing the peak annotation of the Electropherogram Trace. Only integrated peaks are annotated. Units for the peak annotation: · None · Peak ID (labels in order as 1, 2, etc.) · Migration Time (min:sec) raw migration time · Peak Height (in RFUs) · Corrected Peak Area (Peak Area/Migration Time) · Size (in bp or nt) · Average Size (in bp or nt) · Concentration (in ng/µL) · nmole/L

ProSize Data Analysis Software User Manual

ProSize Main Screen

Electropherogram Trace Toolbar Menu

Table 7 Electropherogram Trace toolbar functions

Toolbar Option Create Annotation

Description
To create customized annotation in the Electropherogram Trace display. In the Add Annotation dialog, the user can type desired annotation into the field. Click OK and the annotation will be displayed in the Electropherogram Trace window.

Edit Annotations

To move the annotation to the desired position on the trace: Click the annotation and drag it to the desired position. An arrow will appear at the opposite end of the annotation upon dragging. Multiple annotations can be created by repeatedly selecting Create Annotation. Any created annotations will be copied to the clipboard with the Copy function.
Opens the Annotation Editor window. Allows the user to: · Edit the annotation text by typing in the text field. · Change the color of the annotation by left clicking on the annotation
color. · Delete one annotation by selecting Delete next to each annotation,
or delete all annotations by selecting Delete All.

Show/Hide Baseline

Select Apply to confirm your settings.
Toggle to display or hide the baseline drawn for the peak integration, shown as an orange line. Displaying the baseline helps if adjustments need to be made to the baseline to better define the actual peak area via the Peak Width (sec), Valley to Valley, or Manual Baseline Setpoints tools. Note: It is highly recommended to enable the display of the baseline, to ensure the correct baseline is being drawn to the data, especially with DNA/RNA smears.

ProSize Data Analysis Software User Manual

ProSize Main Screen

Electropherogram Trace Toolbar Menu

Table 7 Electropherogram Trace toolbar functions

Toolbar Option

Description

Show Peak Start/End Edge

Toggle to diplay or hide the start and end points used for peak integration, shown as vertical orange lines. Displaying the peak integration start/end points helps if adjustments need to be made to the peak integration to better define the actual peak area. The user can change the peak start/end points by adjusting the Peak Width (sec), or by using the right click menu tools in the Electropherogram Trace such as Split Peak or Move Peak Start/End Points (see Table 8). The user can change the baseline drawn (and subsequently the peak start/end points) via the Peak Width (sec), Valley to Valley, or Manual Baseline Setpoints tools. Note: It is highly recommended to enable the display of the baseline, to ensure the correct integration is being drawn to each peak, especially with DNA/RNA smears.

Expand Electropherogram Trace Expands the Electropherogram Trace across the bottom panel covering the Peak Table. Provides more user control over zoom.

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ProSize Main Screen

Electropherogram Trace Context Menu

The context menu of the trace, the y-axis, and the x-axis contains functions for adjusting marker peak assignment, peak integration and scaling the x-axis of the Electropherogram Trace (Figure 16). Functions are also provided for copying the trace and exporting the data, and are summarized in Table 8.
Additional information is available under Help > Tutorial Videos > ProSize Electropherogram Functions.

Figure 16 Main screen zoomed to the Electropherogram Trace and context menus. Note that the y-axis scale is set to the sample peaks and not the marker peak heights.

Table 8 Electropherogram Trace context menu commands

Menu commands Accept Change
Cancel
Undo
Set As Lower Marker/ Set As Upper Marker

Description
Accepts any manual modification to the performed peak integration (Add Peak, Split Peak, Merge Peaks, Delete Peak, Move Peak Start/End Points). After the manual modification has been made, select Accept Change to apply the changes to the trace.
Cancels any manual modification to the peak integration (Add Peak, Split Peak, Merge Peaks, Delete Peak, Move Peak Start/End Points). Select Cancel to discard the changes applied to the trace.
To undo any manual modification applied to the peak integration (Add Peak, Split Peak, Merge Peaks, Delete Peak, Move Peak Start/End Points). Select Undo to revert back one step.
To automatically set any integrated peak to the lower or upper marker, and correspondingly re-normalize the trace to the new marker. Right-click the peak to be reassigned as the marker. Select Set As Upper (or Lower) Marker. The peak will be set as the marker and renormalized.

ProSize Data Analysis Software User Manual

ProSize Main Screen

Electropherogram Trace Context Menu

Table 8 Electropherogram Trace context menu commands

Menu commands Add Peak
Split Peak Merge Peaks Delete Peak Move Peak Start/End Points

Description
To manually add a peak which has not been auto-integrated by the Peak Analysis settings and to define the start and end point for integration. Zoom-in to the region where the peak is to be added. Right-click this region and select Add Peak. Two red vertical cursors will appear; the left cursor defines the new peak start point and the right cursor the new peak end point. Drag each cursor to the desired position, then right-click, and select Accept Change to add the peak for integration. Note: The Add Peak function will not add a peak that is located outside the lower/upper marker window (or before lower marker if using only lower marker).
To manually split a peak which is currently integrated into two peaks and to define where the split occurs. Zoom-in to the region where the peak is to be split. Right-click this region, and select Split Peak. A red vertical cursor will appear. Drag the cursor to the desired location, then right-click, and select Accept Change to split the peak into two peaks for integration.
To merge any number of peaks and integrate as a single peak. Zoom-in to the region where the peak is to be split. Right-click this region, and select Merge Peaks. Two red vertical cursors will appear; the left cursor defines the left most peak to merge and right cursor defines the right most peak to merge. Drag the cursor within that peak's start/end point region to merge, then right click, and select Accept Change to merge the peaks into a single peak for integration.
To manually delete a peak which has been integrated. Zoom-in to the region where the peak is to be deleted. Right-click this region, and select Delete Peak. A red vertical cursor will appear. Drag the cursor to the desired peak location, then right-click, and select Accept Change to delete the peak from the integration.
To change the currently positioned start/end integration points of a peak. Zoom-in to the region where the peak is located. Place the cursor between the start and end points of the integration. Right-click and select Move Peak Start/End Points. Two red vertical cursors will appear at the current start/end points. Drag the cursors to the desired locations, then right-click and select Accept Change to apply the new start/end point positions to reintegrate the peak. Note: This function will not change the baseline; only the start and end points are affected. To change the baseline, the user must adjust the via the Peak Width (sec), Valley to Valley, or Manual Baseline Setpoints tools.

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ProSize Main Screen

Electropherogram Trace Context Menu

Table 8 Electropherogram Trace context menu commands

Menu commands Copy Image to Clipboard
Export Data to Clipboard

Description
Copies an image of the current view of the Electropherogram Trace to the clipboard, for pasting in another program, such as Microsoft PowerPoint. Any zoom, annotation, baseline and/or peak start/end point displayed will be copied in the image. The well ID and sample name will be copied in the top left of the trace, and the x-/y-axis will correspond to the currently selected view in the copied image.
Copies the electropherogram x-data (time; in seconds) and y-data (RFUs) in a .csv format that can be pasted directly into common spreadsheet programs such as Microsoft Excel. The x-data is copied into column 1, and the y-data into column 2 of the file upon pasting. To export, right-click and select Export Data to Clipboard. Open a spreadsheet, select the desired cell and paste the copied data. Note: In the Time Scale and Size Scale display modes, the exported data will be: · x = time, and · y = RFU.

Export Data to Excel
Time Scale Size (bp) Scale

This function requires Microsoft Excel to be installed on the computer to function properly. Selection will automatically open Microsoft Excel and copy the electropherogram x (time; in seconds) and y (RFUs) in a .csv format. The x-data is copied into column 1, and the y-data into column 2 of the file upon pasting. To export, right-click and select Export Data to Excel. The file can then be processed and saved as desired. Note: In the Time Scale and Size Scale display modes, the exported data will be: · x = time, and · y = RFU.
Changes the x-axis to units of migration time in hours, minutes and seconds (HH:MM:SS). To change, right-click and select Time Scale. The x-axis scale will change to time and the trace with autoscale.
Changes the x-axis to units of size (bp in DNA, NGS, gDNA, Mutation Analysis or Restriction Digest modes; nt in RNA modes). The display will use underlying units of time and convert the time to size from the current Size Calibration settings. To change, right-click and select Size Scale. The x-axis scale will change to a size scale and the trace with autoscale.

ProSize Data Analysis Software User Manual

ProSize Main Screen

Electropherogram Trace Context Menu

Table 8 Electropherogram Trace context menu commands

Menu commands
Set Display Starting Time (min)

Description
To set-up the displayed start time at the x-axis. Right-click the x-axis and select Set Display Starting Time (min). In the dialog, enter a start time (in minutes), and select Apply to confirm your entry.

Set nmol/L

To set-up the scaling for the y-axis. Allows you to change the scale from RFU to nmol/L. To change, right-click the y-axis, and select Set nmol/L. To change the y-axis scale back to RFU, right-click the y-axis, and select RFU.

ProSize Data Analysis Software User Manual

ProSize Main Screen

Electropherogram Trace Context Menu RNA Mode

NOTE

In RNA (Eukaryotic), RNA (Prokaryotic), or RNA (Plant) analysis modes, additional commands are shown in the Electropherogram Trace context menu (see Figure 17 and Table 9).
The currently selected 16S/18S ribosomal RNA peak is highlighted pink, and the currently selected 23S/25S/28S ribosomal RNA peak is highlighted blue in the Electropherogram Trace when in these RNA analysis modes.

Figure 17 Zoomed-in Electropherogram Trace screen showing context menu in RNA (Eukaryotic) mode. Note that the ribosomal RNA peaks are shaded pink and blue, respectively.

Table 9 Electropherogram Trace context menu commands (only available in RNA mode)

Menu commands RNA (Eukaryotic) Mode Set As 18S

Description
To manually assign a peak as the 18S ribosomal RNA peak for calculating the sample's RNA Quality Number (RQN). Place the mouse over the peak to be reassigned as the 18S peak. Right-click and select Set As 18S. The peak will be set as the 18S peak and the RQN recalculated.

ProSize Data Analysis Software User Manual

ProSize Main Screen

Electropherogram Trace Context Menu RNA Mode

Table 9 Electropherogram Trace context menu commands (only available in RNA mode)

Menu commands Set As 28S
RNA (Prokaryotic) Mode Set As 16S
Set As 23S
RNA (Plant) Mode Set As 18S
Set As 25S

Description
To manually assign a peak as the 28S ribosomal RNA peak for calculating the sample's RNA Quality Number (RQN). Place the mouse over the peak to be reassigned as the 28S peak. Right-click and select Set As 28S. The peak will be set as the 28S peak and the RQN recalculated.
To manually assign a peak as the 16S ribosomal RNA peak for calculating the sample's RNA Quality Number (RQN). Place the mouse over the peak to be reassigned as the 16S peak. Right-click and select Set As 16S. The peak will be set as the 16S peak and the RQN recalculated.
To manually assign a peak as the 23S ribosomal RNA peak for calculating the sample's RNA Quality Number (RQN). Place the mouse over the peak to be reassigned as the 23S peak. Right-click and left click Set As 23S. The peak will be set as the 23S peak and the RQN recalculated.
To manually assign a peak as the 18S ribosomal RNA peak for calculating the sample's RNA Quality Number (RQN). Place the mouse over the peak to be reassigned as the 18S peak. Right-click and select Set As 18S. The peak will be set as the 18S peak and the RQN recalculated.
To manually assign a peak as the 25S ribosomal RNA peak for calculating the sample's RNA Quality Number (RQN). Place the mouse over the peak to be reassigned as the 25S peak. Right-click and select Set As 25S. The peak will be set as the 25S peak and the RQN recalculated.

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ProSize Main Screen

Peak Table DNA Mode

NOTE

The Peak Table displays information about the selected and integrated sample peaks and markers (Figure 18). The data in this table can be directly exported via its context menu. The fields of the Peak Table are summarized in Table 10.
When the Show Marker Information on Peak Table setting is selected (Global Configuration dialog > Advanced Settings tab), entries for the lower and/or upper marker will also be visible in the table.

Figure 18 Peak Table form on main screen (DNA mode)

Table 10 Peak Table functions (only available in DNA mode)

Item Size Conc. (ng/µL)
% (Conc.)

Description
Reports the calculated size in bp (DNA, NGS, Mutation Analysis, gDNA, Restriction Digest modes) or nt (RNA modes) for each integrated peak.
Reports the calculated concentration in units of nanogram per microliter (ng/µL) for each integrated peak. The concentration is calculated from comparison to the lower or upper marker, or to the calibration sizing ladder referenced to the lower marker depending upon the current settings in the Quantification tab of the Global configuration dialog. Any factoring from dilution of the input sample is also calculated via the Quantification settings.
Reports the calculated % of total concentration (not including markers) for each integrated peak in the sample.

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ProSize Main Screen

Peak Table DNA Mode

Table 10 Peak Table functions (only available in DNA mode)

Item nmole/L
TIC TIM Total Conc.:
DQN Export Data to Clipboard

Description
Reports the calculated concentration in units of nanomoles per liter (nmole/L) for each integrated peak. The concentration is calculated from comparison to the lower or upper marker, or to the calibration sizing ladder referenced to the lower marker depending upon the current settings in the Quantification tab of the Global configuration dialog. Any factoring from dilution of the input sample is also calculated via the Quantification settings. Note: This calculation is only approximate for smear peaks, as the molecular weight of the DNA/RNA fragments constituting the smear is a mixture of many different length species.
Reports Total Integrated Concentration (TIC), in ng/µL of all integrated peaks for the sample (not including markers). Note: The TIC does not factor in the concentration for peaks or smears that are not integrated in the sample result.
Reports Total Integrated Molarity (TIM), in nmole/L of all integrated peaks for the sample (not including markers). Note: The TIM does not factor in the molarity for peaks or smears that are not integrated in the sample result.
Reports the calculated total concentration in ng/µL of all peaks for the sample (not including markers), regardless of whether the peaks are integrated or not. Note: The Total Conc. takes into account all of the total peak area above the baseline after the lower marker when using the lower marker only for normalization, or the total peak area above the baseline and between the lower and upper markers when using both markers for normalization. The Total Conc. does not include peak area below the lower marker or after the upper marker.
Reports the DNA Quality Number, which is a percent/10 of DNA above a user-defined threshold (defined in Global Configuration dialog > Advanced Settings tab).
Right-click in the table and select Export Data to Clipboard. The peak table information is exported to the clipboard for pasting directly into common spreadsheet programs such as Microsoft Excel. The full table is exported.

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ProSize Main Screen

Peak Table NGS Mode

In NGS analysis mode, additional information about selected peaks and/or smears is displayed in the Peak Table (Figure 19 and Table 11).
This additional information is useful when examining DNA smear characteristics for NGS applications.

Figure 19 Peak Table form on main screen (NGS mode).

Table 11 Additional Peak Table functions (only available in NGS mode)

Item From (bp) To (bp) Avg. Size

Description
Reports the size (in bp) of the starting integration point of a DNA peak/smear (i.e., minimum size value).
Reports the size (in bp) of the ending integration point of a DNA peak/smear (i.e., maximum size value).
Reports the mean size (in bp) of the integrated DNA peak/smear, accounting for the distribution of the concentration of the peak/smear. Therefore, the Avg. Size will reflect the size corresponding to 50% of the total peak/smear concentration being below and above this value.

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ProSize Main Screen

Peak Table NGS Mode

Table 11 Additional Peak Table functions (only available in NGS mode)

Item CV%
DQN

Description
Reports the distribution of concentration from the mean for a peak/smear, in terms of the coefficient of variation (CV), where CV is the ratio of the standard deviation () to the mean (): CV(%) = / The value is reported as a percentage; smaller values indicate a tighter size distribution of the integrated peak/smear concentration.
Reports the DNA Quality Number, which is a percent/10 of DNA above a user-defined threshold (defined in Global Configuration dialog > Advanced Settings tab).

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ProSize Main Screen

Peak Table gDNA Mode

In the gDNA analysis mode, additional information about the selected peaks and/or smears is displayed in the Peak Table (Figure 20 and Table 12).
This additional information is useful when examining genomic DNA.

Figure 20 Peak Table form on main screen when in gDNA mode

Table 12 Additional Peak Table functions (only in gDNA mode)

Item GQN
Threshold

Description
Reports the sample's calculated Genomic Quality Number (GQN). This value is based upon the concentration of sample above and below the User defined Size Threshold (bp) and is based on a scale of 1 (poorest) to 10 (highest). GQN is reflected as percent/10 above the user-defined threshold (defined in Global Configuration dialog > Advanced Settings tab).
The user defined size (bp) used in the calculation of GQN.

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ProSize Main Screen

Peak Table Total RNA Mode

In the RNA (Eukaryotic), RNA (Prokaryotic), or RNA (Plant) analysis modes, additional information is displayed to the right of the Peak Table (Figure 21 and Table 13).
This additional information is useful for examining RNA sample characteristics and for calculating a sample's RQN (RNA Quality Number).

Figure 21 Peak Table form on main screen when in RNA modes

Table 13 Additional Peak Table functions (only available in RNA mode)

Item RNA Concentration (ng/µL)

Description
Reports the calculated total concentration in ng/µL of all peaks for the sample (not including the markers), regardless of whether the peaks are integrated or not. Note: The RNA concentration takes into account all of the total peak area above the baseline after the lower marker when using the lower marker only for normalization. The RNA concentration does not include peak area below the lower marker.

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ProSize Main Screen

Peak Table Total RNA Mode

Table 13 Additional Peak Table functions (only available in RNA mode)

Item 28S/18S (Eukaryotic), or 23S/16S (Prokaryotic), or 25S/18S (Plant)
RNA Quality Number

Description
Reports the calculated concentration ratio of the selected ribosomal RNA peaks. This ratio is an indication of the quality of the RNA sample and is used in calculating the sample's RNA Quality Number (RQN).
Reports the sample's calculated RNA Quality Number (RQN). This value is based upon several factors including sample concentration, ribosomal RNA peak ratios and sample purity, and is based on a scale of 1 (poorest) to 10 (highest). Note: If the ribosomal peaks are not detected or fall below a threshold concentration level, NaN will be displayed and no RQN value will be reported.

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ProSize Main Screen

Peak Table mRNA Mode

In the mRNA analysis mode, additional information is displayed to the right of the Peak Table (Figure 22 and Table 14).
This additional information is useful for examining RNA sample characteristics and for calculating a sample's % rRNA contamination.

Figure 22 Peak Table form on main screen when in mRNA mode

Table 14 Additional Peak Table functions (only available in mRNA mode)

Item % rRNA Contamination

Description
Reports the calculated ribosomal RNA contamination in the sample (not including the markers), regardless of whether the peaks are integrated or not.

ProSize Data Analysis Software User Manual

ProSize Main Screen

Peak Table Small RNA Mode

In the Small RNA analysis mode, additional information is displayed to the right of the Peak Table (Figure 23 and Table 15).
This additional information is useful for examining Small RNA sample characteristics and for calculating a sample's % micro RNA.

Figure 23 Peak Table on main screen when in small RNA mode

Table 15 Additional Peak Table functions (only available in small RNA mode)

Item %microRNA
smallRNA (pg/µL) microRNA (pg/µL)

Description
Reports the sample's calculated %microRNA within the small RNA defined region. This value is based upon several factors including sample concentration, Maximum miRNA (nt), and Maximum Small RNA (nt). Default setpoint is 4 nt but can be changed by the user.
Reports the concentration from the Lower Marker to the Maximum Small RNA (nt) setpoint. Defualt setpoint is 40 nt but can be changed by the user.
Reports the concentration from the Lower Marker to the Maximum miRNA (nt) setpoint. Default setpoint is 300 nt but can be changed by the user.

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ProSize Main Screen

Peak Table Mutation Analysis Mode

In the Mutation Analysis mode, if the combined size of a pair of integrated DNA peaks adds up to the size of the largest integrated DNA peak (assumed to be the intact gene), within the % Error defined in the Advanced Settings tab of the Global Configuration dialog, those peaks are highlighted in both the Electropherogram Trace and Peak Table (Figure 24).

NOTE

Figure 24 Electropherogram Trace and Peak Table highlighted for positive result in Mutation Analysis mode (combined size within preset % Error of 15%)
In order for peaks indicating a positive result in the Mutation Analysis mode to be highlighted in the Electropherogram Trace and Peak Table, the Valley to Valley Baseline? setting must be enabled in both the Peak Analysis and Marker Analysis tabs of the Global Configuration dialog.

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ProSize Main Screen

Flag Analysis Table

NOTE

If the configuration contains any Flag settings, a Flag Analysis tab will also be visible as a separate tab with the Peak Table (Figure 25 and Table 16). The flagged results are displayed in rows, with each row corresponding to a sample well of the plate. The columns are titled by the flag tag which is the field used to name the particular flag condition. The results are output in a binary format with 0 = FALSE and 1 = TRUE.
The information in the Flag Analysis tab can be exported to the clipboard for pasting into common spreadsheet programs by right clicking the mouse and left clicking Export Data to Clipboard. Further information on the flag functions can be found in Chapter 4, "ProSize Configuration" or under Help > Tutorial Videos > Flag Analysis.

Figure 25 Flag Analysis tab on main screen

Table 16 Flag Analysis tab

Item Well/Sample Name
Columns

Description
The first column labels the sample well; the second Sample ID column lists the entered sample name.
Each column is titled by the respective tag entry and flag conditions for each entry.

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ProSize Main Screen

Flag Analysis Table

Table 16 Flag Analysis tab Item Column/Row Data
Export Data to Clipboard

Description
Data is reported in binary format, following the specified criteria for each flag condition: 0 = FALSE (conditions not met) 1 = TRUE (conditions met)
From the context menu of the table, select Export Data to Clipboard. The peak table information is exported to the clipboard for pasting directly into common spreadsheet programs such as Microsoft Excel. The full table is exported.

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ProSize Main Screen

Smear Analysis Table

NOTE

If the configuration of the Smear Analysis tab contains any filter settings, a Smear Analysis tab will also be visible as a separate tab with the Peak Table (Figure 26 and Table 17).
The calculated results table shows for each sample row the following columns (for details, see Table 17):
· the sample ID and well position
· the customized smear size Range
· the calculated concentration in ng/µL for the defined size range
· the % Total, which is the percent of total sample concentration in the smear range
· the nmole/L concentration of the defined smear range
· the Avg. Size
· %CV of the defined smear range
Smear Analysis ranges are color coded and a vertical dotted line will show on the electropherogram view showing the range and reporting the corresponding average size for the smears (see Figure 27).
If a sample well has not been selected for the Smear Analysis, its row will be left blank. If multiple size ranges are selected for a sample, multiple rows will be displayed. In Figure 26, all wells have two smear ranges selected: 190 bp to 500 bp, and 500 bp to 900 bp.
The information in the Smear Analysis tab can be exported to the clipboard for pasting into common spreadsheet programs. Select Export Data to Clipboard from the context menu. Further information on the flag functions are discussed in Chapter 3, "ProSize Main Screen" or in the Help > Tutorial Videos > Smear Analysis.

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ProSize Main Screen

Smear Analysis Table

Figure 26 Smear Analysis tab on main screen

Figure 27 Electropherogram view when Smear Analysis tab selected on main screen

Table 17 Smear Analysis tab

Item ID Range

Description
Lists the Sample Well: Sample Name.
Lists the smear range, in size (bp). The smear range corresponds to the Start Size and End Size entered in the Smear Analysis tab (Global Configuration dialog). Note: If the Apply function has not been made to the respective well, it will be blank in the table.

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ProSize Main Screen

Smear Analysis Table

Table 17 Smear Analysis tab

Item ng/µL % Total nmole/L Average Size
% CV
Export Data to Clipboard

Description
Reports the concentration of the defined smear range in ng/µL.
Reports the % of the total concentration of the sample that resides in the defined smear range.
Reports in alternate concentration units of nmole/L for the defined smear range.
Reports the mean size (in bp) of the defined smear range, accounting for the distribution of the concentration of the smear. Therefore, the Avg. Size will reflect the size corresponding to 50% of the total smear concentration being below and above this value.
Reports the distribution of concentration from the mean for the defined smear, in terms of the coefficient of variation (CV), where CV is the ratio of the standard deviation () to the mean (): CV(%) = / The value is reported as a percentage; smaller values indicate a tighter size distribution of the smear concentration.
From the context menu of the table, select Export Data to Clipboard. The peak table information is exported to the clipboard for pasting directly into common spreadsheet programs such as Microsoft Excel. The full table is exported.

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ProSize Main Screen

Advanced Flag Analysis Table

The Advanced Flag tab of the Global Configuration dialog offers flag settings. An Advanced Flag Analysis tab will be displayed as a separate tab with the Peak Table (Figure 28). The flagged results are displayed, with each page representing a single well.
The Advanced Flag Analysis tab reports the following results columns:
· size concentration
· % Concentration
· nmole/L
· RFU
To see results for all wells simultaneously, export the data to a .csv file using the Export button in the main menu.
Further information on the flag functions can be found in Chapter 4, "ProSize Configuration" or under Help > Tutorial Videos > Advanced Flag Analysis.

Figure 28 Advanced Flag Analysis tab on main screen

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ProSize Main Screen

Set Individual Parameters

When a data file is initially loaded into the ProSize software, the settings for selection of lower/upper markers, sample peak integration, size calibration, quantification, and any flag/smear analysis are globally set for all samples in the Global Configuration dialog (for more information, see Chapter 4, "ProSize Configuration").
After the data is loaded, any of the common configuration parameters can be individually edited for each sample independently (local setting), or changes can be made and applied to all samples (global setting), or to a customized subset of samples.
To view and adjust the configuration settings for the currently viewed lane/sample, select Set Individual Parameters on the right side of the Peak Table. This will reveal the configuration dialog, for which most settings can be individually adjusted for each sample as desired (Figure 29). To toggle back to the Peak Table, select Show Results.

Figure 29 Main screen with data file open and configuration dialog open (clicked)

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Set Individual Parameters

The individual settings within each tab are described in detail in Chapter 4, "ProSize Configuration". The following sections describe the settings that apply to individual configuration tabs.
Configuration Peak Analysis
The Peak Analysis tab contains settings to define the sample peak integration, data analysis filter, analysis region, and manual baseline set points (Figure 30). These functions are described in detail in section "Global Configuration - Peak Analysis" on page 84.
New settings in the Peak Analysis tab are applied to the current selected sample well only, or to all samples of the sample plate (see Table 18).

Figure 30 Peak Analysis tab in Set Individual Parameters dialog

ProSize Data Analysis Software User Manual

ProSize Main Screen

Set Individual Parameters

Table 18 Additional Peak Analysis settings

Setting Apply to All
Apply to Selected

Description
All current settings of the Peak Analysis tab will be applied to all samples of the loaded data file. This is therefore a global function. Note: Any previous individually tuned Peak Analysis tab settings for different samples of the data file will be overwritten when using this function.
All current settings of the Peak Analysis tab will be applied to the specified wells of the sample plate. Upon selecting this function, the Selected Samples plate map menu will be displayed to select the wells of the sample plate to apply the current settings to.

Click the desired wells, rows or columns, and click OK to apply; click Cancel to abort the operation. Note: Any previous individual setting of the Peak Analysis tab for the selected samples of the data file will be overwritten when using this function.

ProSize Data Analysis Software User Manual

ProSize Main Screen

Set Individual Parameters

Configuration Marker Analysis
The Marker Analysis tab contains settings to define the marker peak selection and marker peak integration (Figure 31). These settings are described in detail in "Global Configuration - Marker Analysis" on page 89.
New settings in the Marker Analysis tab can be applied to the current selected sample well only, or to all samples of the sample plate (see Table 19).

Figure 31 Marker Analysis tab Set Individual Parameters dialog

ProSize Data Analysis Software User Manual

ProSize Main Screen

Set Individual Parameters

Table 19 Additional Marker Analysis settings

Setting Apply to All
Apply to Selected

Description
All current settings of the Marker Analysis tab will be applied to all samples of the loaded data file. This is therefore a global function. Note: Any previous individually tuned Marker Analysis tab settings for different samples of the data file will be overwritten when using this function.
All current settings of the Marker Analysis tab will be applied to the specified wells of the sample plate. Upon selecting this function, the Selected Samples plate map menu will be displayed to select the wells of the sample plate to apply the current settings to.

Select the desired wells, rows or columns, and click OK to apply; click Cancel to abort the operation. Note: Any previous individual setting of the Marker Analysis tab for the selected samples of the data file will be overwritten when using this function.

ProSize Data Analysis Software User Manual

ProSize Main Screen

Set Individual Parameters

Configuration Quantification
The Quantification tab contains settings to assign a marker or ladder for quantification; its working concentration and any dilution factor applied to the sample prior to injection (Figure 32). These functions are described in detail in "Global Configuration - Quantification" on page 96.
New settings in the Quantification tab are applied to the current selected sample well only, or to all samples of the sample plate (see Table 20).

Figure 32 Quantification tab in Set Individual Parameters dialog

ProSize Data Analysis Software User Manual

ProSize Main Screen

Set Individual Parameters

Table 20 Additional Quantification settings

Setting Apply to All
Apply to Selected

Description
All current settings of the Quantification tab will be applied to all samples of the loaded data file. This is therefore a global function. Note: Any previous individually tuned Quantification tab settings for different samples of the data file will be overwritten when using this function.
All current settings of the Quantification tab will be applied to the specified wells of the sample plate. Upon selecting this function, the Selected Samples plate map menu will be displayed to select the wells of the sample plate to apply the current settings to.

Select the desired wells, rows or columns, and click OK to apply; click Cancel to abort the operation. Note: Any previous individual setting of the Quantification tab for the selected samples of the data file will be overwritten when using this function.

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ProSize Main Screen Set Individual Parameters
Configuration Smear Analysis
The Smear Analysis tab contains settings to enable the user to specify a size range(s) for which to integrate and calculate the total concentration in ng/mL (Figure 33). These functions are described in detail in "Global Configuration Smear Analysis" on page 99
New settings in the Smear Analysis tab are applied to the current selected sample well only, or to all samples of the sample plate (see Table 21).
If Display Smear Range is enabled, two color coded vertical dotted cursors are displayed in the Electropherogram Trace screen. These cursors define the entered values for the Start Size (bp) and the End Size (bp) for the currently selected smear range. The colors can be changed by left clicking on the color next to the smear range. The start and end points of the smear range can be adjusted by clicking on a cursor peak, and dragging the respective cursor left or right; the smear analysis region will be automatically updated to the new cursor location.

Figure 33 Smear Analysis tab in Set Individual Parameters dialog with Display Smear Range enabled

ProSize Data Analysis Software User Manual

ProSize Main Screen

Set Individual Parameters

Table 21 Additional Smear Analysis settings

Setting Apply to All
Apply to Selected

Description
All current settings of the Smear Analysis tab will be applied to all samples of the loaded data file. This is therefore a global function. Note: Any previous individually tuned Smear Analysis tab settings for different samples of the data file will be overwritten when using this function.
All current settings of the Smear Analysis tab will be applied to the specified wells of the sample plate. Upon selecting this function, the Selected Samples plate map menu will be displayed to select the wells of the sample plate to apply the current settings to.

Select the desired wells, rows or columns, and click OK to apply; click Cancel to abort the operation. Note: Any previous individual settings of the Smear Analysis tab for the selected samples of the data file will be overwritten when using this function.

ProSize Data Analysis Software User Manual

ProSize Main Screen

Set Individual Parameters

Configuration Flag Analysis
The Flag Analysis tab contains settings to enable the user to specify specific criteria (size or concentration range) to be met within the data, and generate a binary output (0 = false, 1 = true) of the results (Figure 34). These settings are described in detail in "Global Configuration - Flag" on page 101.
New settings in the Flag Analysis tab are applied to all samples of the sample plate. Some of these settings are described in Table 22.

Figure 34 Flag Analysis tab in Set Individual Parameters dialog

Table 22 Additional Flag Analysis settings

Setting Save Flag Parameters
Load Flag Parameters Remove All Flag Parameters

Description
The current Flag Analysis settings will be saved as a file (*.Flag extension) that can be loaded into different data files. A file browser menu will open to navigate and save the desired filename and location; click OK to save the file, or Cancel to discard your changes.
The previously saved Flag Analysis settings will be loaded from a file (*.Flag extension). A file browser menu will open to navigate and load the desired filename and location; click OK to load the file, or Cancel to discard your changes.
All settings will be cleared from the tab.

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ProSize Main Screen Set Individual Parameters
Configuration Advanced Settings
The Advanced Settings tab contains settings to assign the data analysis mode for processing data (Figure 35). All settings of this tab are described in detail in "Global Configuration Advanced Flag" on page 102.
The Advanced Settings parameters are applied to all samples in the data file. Individual samples cannot use different analysis modes. It is assumed all data is processed with a single analysis mode in a single experiment or data file.

Figure 35 Advanced Settings tab in Set Individual Parameters dialog

ProSize Data Analysis Software User Manual

ProSize Main Screen

Set Individual Parameters

Configuration Advanced Flag Analysis
The Advanced Flag Analysis tab contains settings to specify specific criteria (size, concentration range, various logic criteria) to be met within the data, and generate a table with results (Figure 36). These settings are described in detail in "Global Configuration Advanced Flag" on page 102.

Figure 36 Advanced Flag tab in Set Individual Parameters dialog

ProSize Data Analysis Software User Manual

ProSize Configuration

About Configuration 76
Access Configuration Information in the ProSize Software 77
Overview Global Configuration Dialog 80
Global Configuration - Advanced Settings 81 Global Configuration - Peak Analysis 84 Global Configuration - Marker Analysis 89 Global Configuration - Peak Analysis for Reference Cap 92 Global Configuration - Size Calibration 93 Global Configuration - Quantification 96 Global Configuration - Smear Analysis 99 Global Configuration - Flag 101 Global Configuration Advanced Flag 102 Global Configuration Inclusion Region 106
Set Individual Parameters 108
Set Individual Parameters - Advanced Settings 110 Set Individual Parameters - Peak Analysis 110 Set Individual Parameters Marker Analysis 112 Set Individual Parameters Quantification 113 Set Individual Parameters Smear Analysis 115 Set Individual Parameters Flag Analysis 117 Set Individual Parameters DNA Quality Number 118 Set Individual Parameters Advanced Flag Analysis 119 Set Individual Parameters Inclusion Region 124

This chapter provides an overview of the Global Configuration dialog of the ProSize software. It also provides information on setting individual configuration parameters in opened data files.

ProSize Data Analysis Software User Manual

ProSize Configuration

About Configuration

NOTE

The Global Configuration dialog (or Set Individual Parameters dialog for opened data files) is used to load, edit, or save the data analysis mode and settings for processing the data file, such as size calibration, marker/sample peak integration, quantification, smear analysis, and/or flag analysis criteria.
By using established method configurations for the different types of sample analysis on the instrument, the user can streamline data analysis with the ProSize software. This requires, that the first 10 characters of the ProSize configuration file name matches the first 10 characters of the parallel capillary electrophoresis system.
In most cases, predefined, optimized global configuration files are available to load when processing data collected from different Agilent Reagent Kits. The configuration files are labeled according to the particular kit number, e.g., DNF-910-33 35-1500bp.ini. These configuration files are available from Agilent upon request if needed. The configuration files preloaded upon software installation are located in the C:\ProSize data analysis software\Configurations folder.

ProSize Data Analysis Software User Manual

ProSize Configuration

Access Configuration Information in the ProSize Software

NOTE

There are two ways to access configuration information in the ProSize software:

Global Configuration Dialog

This option is used to define how an unprocessed data file is first opened in the ProSize software. The user is automatically prompted with the Global Configuration dialog when opening an unprocessed data file if the corresponding separation method used does not match the naming configuration (Figure 37).

Alternatively, once a data file is opened, the user can modify at any time the

configuration by selecting Option, and clicking Edit Configurations

In most cases, predefined, optimized global configuration files are available to load when processing data collected from different Agilent Reagent Kits. The configuration files are labeled according to the particular kit number, for example, DNF-910-33 35-1500bp.ini. These configuration files are available from Agilent upon request if needed. The configuration files preloaded upon software installation are located in the C:\ProSize data analysis software\Configurations folder.

ProSize Data Analysis Software User Manual

ProSize Configuration

Access Configuration Information in the ProSize Software

Figure 37 Global Configuration dialog
Set Individual Parameters Dialog
This dialog is used to modify or change configuration settings after a data file is opened. The Set Individual Parameters dialog can be accessed in an open data file by selecting on the right side of the main menu (Figure 38). Note that this icon toggles between the Set Individual Parameters and Show Results ( ) form, which displays the Peak Table and Smear Analysis or Flag Analysis tables.
For some modes (DNA, NGS), the Set Individual Parameters dialog also has a DQN tab, which allows users to set a threshold for a DNA Quality Number. The percent of DNA above the threshold, divided by 10, gives the DQN (which is an indication of quality, based on a scale of 1 to 10).

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ProSize Configuration Access Configuration Information in the ProSize Software
Using Set Individual Parameters dialog, the currently selected tab is applied independently from the other configuration tabs. In addition, if the user select [Enter] after changing a setting, the change is only applied to the currently selected sample; therefore this is a "local" setting. To apply the change made in the tab to all samples, click Apply to All; to apply the change only to selected samples, click Apply to Selected.
The settings within each tab of the Global Configuration and Set Individual Parameters dialog perform the same functions. Each tab of the configuration is discussed in further detail in the following sections of this chapter.

Figure 38 Set Individual Parameters icon for configuration settings after a data file is opened

ProSize Data Analysis Software User Manual

ProSize Configuration

Overview Global Configuration Dialog

There are ten tabs in the Global Configuration dialog to define the appropriate data analysis mode and settings for processing the data, as described in the following sections.
At the bottom of the Global Configuration dialog, the user is able to Save, Load, or Apply the configuration settings (see Table 23).

Table 23 Global Configuration dialog buttons

Button

Description
Saves the global configuration as a user defined file (*.ini format). The settings from all tabs are saved, regardless of which tab is open when saving the file.
Loads a predefined configuration file (*.ini format). Note than upon installation, predefined configuration files are installed for most Agilent Reagent Kits in the in the C:\ProSize data analysis software\Configurations folder.
Applies the current global configuration settings to the data file. If the data file is unprocessed, pressing the Apply button will open the data file with the currently loaded settings. If the data file is already open, the new settings will be applied to the data, overwriting any previously defined individual configuration settings.
Cancels all changes and closes the global Configuration window.

ProSize Data Analysis Software User Manual

ProSize Configuration

Global Configuration - Advanced Settings

The settings of the Advanced Settings tab allow the user to assign the data analysis mode for processing data (Figure 39). The settings are described in detail in Table 24.

Figure 39 Advanced Settings tab in Global Configuration dialog

Table 24 Advanced Settings configuration tab

Settings Mode DNA

Description
For analyzing standard dsDNA fragment (for example, PCR product) samples where discrete, Gaussian peaks are present. This mode displays size in basepairs (bp) using a DNA Standard Ladder for calibration. Quantification is achieved via comparison to either an upper or lower marker, or to the DNA Standard Ladder present at a known concentration. Note: The DNA Mode is not recommended for samples containing non-Gaussian peaks, smears or uneven baselines.

ProSize Data Analysis Software User Manual

ProSize Configuration

Global Configuration - Advanced Settings

Table 24 Advanced Settings configuration tab

Settings RNA (Eukaryotic)
RNA (Prokaryotic)
RNA (Plant)
mRNA Small RNA NGS

Description
For analyzing eukaryotic total RNA samples. Calculates size in nucleotides (nt) using a RNA Standard Ladder for calibration. Quantification is achieved via comparison of the lower marker to the RNA Ladder, present at a known concentration. The ratio of the 18S and 28S ribosomal RNA peaks is calculated, as well as the RNA Quality Number (RQN).
For analyzing prokaryotic total RNA samples. Calculates size in nucleotides (nt) using a RNA Standard Ladder for calibration. Quantification is achieved via comparison of the lower marker to the RNA Ladder, present at a known concentration. The ratio of the 16S and 23S ribosomal RNA peaks is calculated, as well as the RNA Quality Number (RQN).
For analyzing plant total RNA samples. Calculates size in nucleotides (nt) using a RNA Standard Ladder for calibration. Quantification is achieved via comparison of the lower marker to the RNA Ladder, present at a known concentration. The ratio of the 18S and 25S ribosomal RNA peaks is calculated, as well as the RNA Quality Number (RQN).
For analyzing messenger RNA (mRNA) samples. Calculates size in nucleotides (nt) using a RNA Standard Ladder for calibration. Quantification is achieved via comparison of the lower marker to the RNA Ladder, present at a known concentration.
For analyzing mico RNA (miRNA) samples. Calculates %microRNA, small RNA (pg/µL), microRNA (pg/µL), and size in nucleotides (nt) using a RNA Standard Ladder for calibration. Quantification is achieved via comparison of the lower marker to the RNA Ladder, present at a known concentration.
For analyzing dsDNA samples containing peaks, smears, non-Gaussian peaks and/or uneven baselines. This mode is commonly used for Next Generation Sequencing (NGS) fragment library analysis. This mode displays size in basepairs (bp) using a DNA Standard Ladder for calibration. Quantification is achieved via comparison to either an upper or lower marker, or to the DNA Standard Ladder present at a known concentration. The Peak Table in this mode displays additional fields for size range, average size, and % CV.

ProSize Data Analysis Software User Manual

ProSize Configuration

Global Configuration - Advanced Settings

Table 24 Advanced Settings configuration tab

Settings Mutation Analysis

Description
This mode is used in combination with TILLING experiments analyzed on the Fragment Analyzer system. Displays size in basepairs (bp) using a DNA Standard Ladder for calibration. Quantification is achieved via comparison to either an upper or lower marker, or to the DNA Standard Ladder present at a known concentration. In this mode, an additional field for % Error is displayed in the Advanced Settings tab. If the size of any two fragments in the sample adds up to the size of the largest detected fragment (assumed to be the intact gene) within the percentage entered in the % Error field, the peaks will be highlighted in the Peak Table and Electropherogram Trace. The user can also select peaks that meet a minimum peak height requirement with Min. RFU.

gDNA Restriction Digest

For analyzing dsDNA samples containing peaks, smears, non-Gaussian peaks and/or uneven baselines. his mode is commonly used for Genomic DNA (gDNA) analysis. This mode displays size in basepairs (bp) using a DNA Standard Ladder for calibration. Quantification is achieved via comparison to a lower marker, or to the DNA Standard Ladder present at a known concentration. The Peak Table in this mode displays additional fields for size range, average size, % CV, and GQN.
For analyzing standard dsDNA fragment (e.g., PCR product) samples where discrete, Gaussian peaks are present. This mode displays size in basepairs (bp) using a DNA Standard Ladder for calibration. Quantification is achieved via comparison to either an upper or lower marker, or to the DNA Standard Ladder present at a known concentration. The mode can be used for analyzing samples cleaved with restriction enzymes.

ProSize Data Analysis Software User Manual

ProSize Configuration

Global Configuration - Peak Analysis

Table 24 Advanced Settings configuration tab

Settings Linear Interpolation
High Sensitivity
Min. RFU Show Marker Information on Peak Table

Description
This mode uses two markers--one above and one below a target fragment. In this case, ladder is not used. The size is based on linear interpolation between the upper and lower marker.
Changes the display (export, .pdf, peak table) to pg/µL instead of ng/µL. For NGS analysis only (and only for very low concentrations).
Allows you to designate the minimum RFU required for integration.
When enabled, the sample Peak Table will display size and concentration information for the lower/upper marker peaks along with all currently integrated sample peaks.

Global Configuration - Peak Analysis
The settings of the Peak Analysis tab allow the user to assign width and height thresholds for selecting and integrating sample peaks; setting data filtering; and selecting the time region to be analyzed (Figure 40). The settings are described in detail in Table 25.

ProSize Data Analysis Software User Manual

ProSize Configuration

Global Configuration - Peak Analysis

Figure 40 Peak Analysis tab in Global Configuration dialog

Table 25 Peak Analysis configuration settings

Settings Peak Analysis Peak Width (sec)
Min. Peak Height (RFU)

Description
Defines the width threshold for peak detection in seconds. Higher settings better define wide peak start/end points; smaller settings better define sharp peak start/end points. Typical values range from 3-5 for sharp peaks or 25-50 for smears. The maximum value for this setting is 50.
Defines the minimum peak height threshold to select a peak for integration in RFUs. Peaks below the set value will not be selected for integration. Typical values for this setting are 20-100 RFU, depending upon the sample signal and application. Note the typical baseline peak-to-peak noise is less than 5 RFU.

ProSize Data Analysis Software User Manual

ProSize Configuration

Global Configuration - Peak Analysis

Table 25 Peak Analysis configuration settings

Settings # Extra Valley Points
Valley to Valley Baseline?

Description
This setting influences the start/end point of baseline integration of peaks and the baseline drawn between peaks. This setting most affects the baseline between two peaks that are not baseline resolved. Higher values draw a straighter baseline between the first peak start point and second peak end point; lower values draw the baseline more to the "valley" between the two unresolved peaks. Note: The Valley to Valley Baseline? setting needs to be enabled for this setting to be active.
This setting influences the start/end point of baseline integration of peaks and the baseline drawn between peaks. This setting most affects the baseline between two peaks that are not baseline resolved. When disabled, will draw a straighter baseline between the first peak start point and second peak end point. When enabled, will draw the baseline more to the "valley" between the two unresolved peaks. In the example below, the Top trace shows the baseline drawn when Valley to Valley Baseline is enabled; the Bottom trace shows the baseline for the same sample when this setting is disabled.

Valley to Valley = Enabled

Valley to Valley = Disabled

ProSize Data Analysis Software User Manual

ProSize Configuration

Global Configuration - Peak Analysis

Table 25 Peak Analysis configuration settings

Settings Filter Selection Filter
# of Points
Analysis Region Start (min) End (min) Manual Baseline Setpoints

Description
Defines the algorithm type to use when filtering the raw data. There are five options for data filtering available to the user: · None: No filtering is performed on the data. · Moving Average: Averages points on either side of a point. · Savitzky Golay: A polynomial regression filter where the coefficients
are calculated using Savitzky and Golay's algorithm. · Binomial:. A polynomial regression filter based on the binomial
coefficients for implementing Gaussian filtering. · Wavelets De-noising: A discrete wavelet transform filter. Note: The recommend filter for most applications is a 3 to 20 point Binomial filter.
Defines the number of points to use in the selected data filter. Maximum points to use for each filter type: · Moving Average: 9 · Savitsky Golay: 9 · Binomial: 20 · Wavelets De-Noising: 4
Defines the start time in minutes for which to integrate and analyze the electropherogram data. This includes both marker and sample peaks. Note: If 0 is entered, will analyze data from start of the electropherogram at 0 min.
Defines the end time in minutes for which to integrate and analyze the electropherogram data. This includes both marker and sample peaks. Note: If 0 is entered, will automatically analyze data to the full end time range of the electropherogram.
Defines the baseline using time points. This function is particularly useful for accurately measuring the total concentration of samples containing smears or uneven baselines.

ProSize Data Analysis Software User Manual

ProSize Configuration

Global Configuration - Peak Analysis

Table 25 Peak Analysis configuration settings

Settings Start (min)
End (min)

Description
Defines the start time in minutes for which to draw the start of the baseline when manually setting the baseline. The Y axis position of the baseline at the entered start point in minutes will be used to form the new baseline start point. Note: If 0 is entered for both the Start (min) and End (min), this function is disabled. When entering values for this function, enter the ending time value in the Start (min) field first. When next entering the start time the end time value will automatically populate the End (min) field.
Defines the end time in minutes for which to draw the end of the baseline when manually setting the baseline. The Y-axis position of the baseline at the entered end point in minutes will be used to form the new baseline end point. Note: If 0 is entered for both the Start (min) and End (min), this function is disabled. When entering values for this function, enter the ending time value in the Start (min) field first. When next entering the start time, the end time value will automatically populate the End (min) field.

ProSize Data Analysis Software User Manual

ProSize Configuration

Global Configuration - Marker Analysis

The settings of the Marker Analysis tab allow the user to select the criteria for choosing the lower and/or upper peak, used to normalize each capillary by migration time and concentration (Figure 41). The settings are described in detail in Table 26.
In ProSize, the user can independently set different peak parameters for the lower/upper markers from those set for the sample peak parameters in Peak Analysis tab. This is useful if, for example, the sample contains broad smearing peaks as compared to markers, which are typically sharp Gaussian peak profiles.
The user can select a lower/upper marker pair (Use Both Markers), or use only a lower marker (Use Lower Marker Only) or upper marker (Use Upper Marker Only) for data normalization by selecting the appropriate drop-down option.

Figure 41 Marker Analysis tab in Global Configuration dialog
The markers can be selected based on peak width and peak height thresholds, and also by time range in the Marker Information section (Figure 42).

ProSize Data Analysis Software User Manual

ProSize Configuration

Global Configuration - Marker Analysis

NOTE
90

Figure 42 Marker Analysis tab showing marker selection options
The lower marker can be selected as the First Peak, or second peak (F + 1), third peak (F + 2), etc. up to the sixth peak above a threshold value in RFU units. Similarly, the upper marker can be selected as the Last Peak, or L 1, L 2, etc. Markers can also be selected based upon a time window, by selecting Between from the drop-down and entering a time in X min +/- Y min. For example, the right image of Figure 42 shows lower marker selection criteria of a peak > 200 RFUs between 8 12 min (10 min +/- 2 min).
The Min. Peak Height (RFU) setting under Marker Peak Analysis sets the primary height threshold for the markers. The height threshold values for each individual marker shown under Marker Information must therefore be equal to or greater than the Min. Peak Height (RFU) value.

The settings of the Marker Analysis tab are described in Table 26.

Table 26 Marker Analysis configuration settings

Settings Marker Peak Analysis Peak Width (sec)

Description
Defines the width threshold for peak detection and peak start/end point in seconds. Higher values better define wide peak start/end points; smaller settings better define sharp peak start/end points. Typical values are 3-5 for marker peaks.

ProSize Data Analysis Software User Manual

ProSize Configuration

Global Configuration - Marker Analysis

Table 26 Marker Analysis configuration settings

Settings Min. Peak Height (RFU) # Extra Valley Points
Valley to Valley Baseline?
Use Both Markers Use Lower Marker Only Use Upper Marker Only Marker Information Lower Marker
Upper Marker

Description
Defines the minimum peak height threshold to select a marker peak in RFUs. Peaks below the set value will not be selected as markers.
This setting influences the start/end point of baseline integration of the marker peaks and the baseline. This setting most affects the baseline between two peaks that are not baseline resolved. Higher values draw a straighter baseline between the first peak start point and second peak end point; lower values draw the baseline more to the "valley" between the two unresolved peaks. Note: The Valley to Valley Baseline? setting needs to be enabled for this setting to be active.
This setting influences the start/end point of baseline integration of the marker peaks and the baseline. This setting most affects the baseline between two peaks that are not baseline resolved. When disabled, will draw a straighter baseline between the first peak start point and second peak end point. When enabled, will draw the baseline more to the "valley" between the two unresolved peaks.
Normalizes and aligns the CE data to both a selected lower and upper size marker.
Normalizes and aligns the CE data to only the selected lower marker.
Normalizes and aligns the CE data to only the selected upper marker.
This setting determines how the lower marker is selected from the CE data, using the threshold value in RFU as the minimum peak height for selection. The lower marker is selectable from the drop-down control as: · First Peak (F) · F + 1, F + 2, F +3, F + 4, or F +5 · Between: Enter a specific time in X minutes +/- Y min
This setting determines how the upper marker is selected from the CE data, using the threshold value in RFU as the minimum peak height for selection. The upper marker is selectable from the drop-down control as: · Last Peak (L) · L - 1, L - 2, L - 3, L - 4, or L - 5 · Between: Enter a specific time in X minutes +/- Y min

ProSize Data Analysis Software User Manual

ProSize Configuration

Global Configuration - Peak Analysis for Reference Cap

The settings of the Peak Analysis for Reference Cap tab allow the user to assign width and height thresholds for selecting and integrating peaks in the "Reference" capillary, which is the assigned well containing the DNA or RNA Ladder used to calibrate size (Figure 43). The settings are described in detail in Table 27.
This feature enables the user to define a higher threshold for the ladder peaks as compared to the sample peaks.

Figure 43 Peak Analysis for Reference Cap tab in Global Configuration dialog

Table 27 Peak Analysis for Reference Cap configuration settings

Settings Peak Analysis Peak Width (sec)

ProSize Data Analysis Software User Manual

ProSize Configuration

Global Configuration - Size Calibration

Table 27 Peak Analysis for Reference Cap configuration settings

Settings Min. Peak Height (RFU) # Extra Valley Points
Valley to Valley Baseline?

Description
Defines the minimum peak height threshold to select a peak for integration in RFUs. Peaks below the set value will not be selected for integration. Typical values for this setting when working with a DNA or RNA Ladder 100-200 RFU, depending upon the ladder concentration and application.
This setting influences the start/end point of baseline integration of peaks and the baseline drawn between peaks. This setting most affects the baseline between two peaks that are not baseline resolved. Higher values draw a straighter baseline between the first peak start point and second peak end point; lower values draw the baseline more to the "valley" between the two unresolved peaks. Note: The Valley to Valley Baseline? setting needs to be enabled for this setting to be active.
This setting influences the start/end point of baseline integration of peaks and the baseline drawn between peaks. This setting most affects the baseline between two peaks that are not baseline resolved. When disabled, will draw a straighter baseline between the first peak start point and second peak end point. When enabled, will draw the baseline more to the "valley" between the two unresolved peaks.

Global Configuration - Size Calibration

NOTE

The settings of the Size Calibration tab allow the user to select the proper "Reference" well containing a DNA or RNA Ladder for calibration of sample size; to enter in the proper size and number of fragments (including lower/upper markers) in the calibration ladder; and to apply a fit algorithm for plotting the size vs. migration time data from the ladder (Figure 44). The settings are described in detail in Table 28.
In ProSize, the user can calibrate the size of sample fragments either by running a standard DNA or RNA ladder in a well of the sample plate (recommended), or by importing a previously calculated and exported size calibration file (*.scal extension).
If an imported size calibration is used in ProSize, the same Separation Gel, capillary array dimensions, injection/run voltage, and lower/upper markers should be used for both the analyzed data and the imported calibration to ensure maximum sizing accuracy.

ProSize Data Analysis Software User Manual

ProSize Configuration

Global Configuration - Size Calibration

To properly calibrate the size, the size ladder must be bracketed by the same lower/upper markers used with the samples. In some cases, the lower or upper marker may co-migrate with a ladder peak; this is acceptable for sizing calibration. In some application kits (for example, RNA and Genomic DNA kits), only a lower marker is used; the data is hence normalized using the lower marker only compared to the respective DNA or RNA Size Ladder.

Figure 44 Size Calibration tab in Global Configuration dialog
It is important to ensure the sample containing the size calibration ladder ("Reference" well) has all ladder fragments integrated correctly with no additional peaks integrated prior to analyzing the remaining samples.
The size of each fragment to be included in the DNA/RNA Ladder (including lower/upper markers) is entered directly or imported into the Calibration Ladder field. To directly enter a value, click in the field and enter the size value.
A context menu is available when selecting the Calibration Ladder table. It offers options such as inserting or deleting fragments in the table; clearing all entries; and importing or exporting ladder information files (*.txt extension).

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ProSize Configuration Global Configuration - Size Calibration

In most cases, predefined ladder information files optimized for different Agilent reagent kits are available and are loaded as part of the Global Configuration. The ladder files are labeled according to the particular kit number, for example, DNF-910 - 35-1500 100 bp ladder.txt. These ladder files are available from Agilent upon request if needed. The preloaded ladder files are located in the Ladders folder under C:\ProSize data analysis software.

Table 28 Size Calibration configuration settings

Settings Calibration Ladder
Calibration Ladder > Insert Before Calibration Ladder > Delete Current Selection Calibration Ladder > Clear All Calibration Ladder > Import
Calibration Ladder > Export
Default Ladder Well

Description
Lists size of DNA/RNA fragments contained in calibration ladder in tabular format. The size of each fragment (include lower/upper markers) is entered directly into the table or imported by using the context menu (right-click in the Calibration Ladder table).
Inserts a blank cell above the currently selected cell in the calibration ladder table. Note: Click in the respective table cell before using this option.
Deletes the currently selected cell in the calibration ladder table. Note: Click in the respective table cell before using this option.
Deletes all cells in the calibration ladder table.
Imports a previously exported and saved sizing ladder information file. When selected, a file browser menu will open to navigate and select the desired file (*.txt extension). Standard ladder information files are located in the C:\ProSize data analysis software\Ladders folder.
Exports the current sizing ladder table information (*.txt extension). When selected, a file browser menu will open to navigate and save the desired filename and location. When exporting ladder information files, it is highly recommended to save the created files to a common directory, located in the C:\ProSize data analysis software\Ladders folder.
Defines the well of the sample plate, which contains the size calibration ladder. From the drop-down list, select the proper row (A-H) and column (1-12) location containing the size calibration ladder.

ProSize Data Analysis Software User Manual

ProSize Configuration

Global Configuration - Quantification

Table 28 Size Calibration configuration settings

Settings Curve Fitting
Use Imported Ladder Profile Ladder Profile

Description
· Point to Point Fits the size calibration plot of Size (bp or nt) vs. Time (sec) with a Point to Point fit (recommended), drawing a straight line between each point of the curve. · Polynomial Fits the size calibration plot of Size (bp or nt) vs. Time (sec) with a polynomial fit. Enter the order value (from 3 to 5) in the Order field.
Imports a previously exported size calibration file (*.scal extension) to use for size calibration.
Displays file path for the imported size calibration file. To load an imported file, click . A file browser menu will open to navigate and select the desired file (*.scal extension). Note: When importing a size calibration file, the same conditions (separation gel, capillary array length, lower/upper markers, CE separation voltage) should be used. Differences between the analyzed and imported conditions will adversely affect the sizing accuracy.

Further information on the Size Calibration function is described in chapter Chapter 5, "ProSize Size Calibration Screen".

Global Configuration - Quantification
The settings of the Quantification tab allow the user to assign how the sample is quantified in the ProSize software (by markers or ladder); to define the working concentration of the marker or ladder reference; and to apply a dilution factor for calculating the final sample concentration (Figure 45). The settings are described in detail in Table 29.
There are two different methods by which the sample concentration may be quantified in the ProSize software:

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ProSize Configuration Global Configuration - Quantification
· Use Lower/Upper Marker: This method compares the sample peak or sample smear area to either the lower or upper marker peak area. The peak areas are taken as corrected peak area (area/migration time), with each data point collected being divided by its migration time. This method relies on a marker of known concentration, typically mixed directly with the sample.
· Use Ladder: This method compares the sample peak or sample smear area to that of a RNA or DNA ladder of known total concentration. The ratio of the RNA/DNA ladder to the lower marker is calculated; then the ratio of the lower marker to the sample is calculated to determine the final sample concentration. This method relies on a RNA/DNA ladder of known concentration, typically mixed directly with the sample.
An advantage of the Use Ladder quantification method is that it can correct for any systematic errors in sample preparation. As long as the sample and ladder are prepared using the same diluent marker solution, any bias from pipetting or preparation is accounted for as long as the RNA/DNA Ladder is pipetted and mixed the same as the sample.
Use Ladder is the default Quantification method for all quantification analysis kits. For the various modes, the user may select from either the Use Lower/Upper Marker or the Use Ladder method. Refer to the corresponding User Manual for each specific Agilent reagent kit for suggested settings.
The most accurate means of quantification involves diluting the sample in a diluent containing marker of known concentration (internal standard). Approximate concentration or relative concentration differences between samples can also be obtained by diluting a sample in a diluent matrix (e.g., 1X TE buffer) that matches that of markers prepared in a separate marker plate, and injected under identical conditions (voltage/time).

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Global Configuration - Quantification

Figure 45 Quantification tab in Global Configuration dialog (Left: Use Ladder selected; Right: Use Upper Marker selected)

Table 29 Quantification configuration settings

Settings Use Lower Marker Use Upper Marker Use Ladder

Description
Uses the ratio of the sample corrected peak area to the lower marker corrected peak area for calculation of sample peak/smear quantification.
Uses the ratio of the sample corrected peak area to the upper marker corrected peak area for calculation of sample peak/smear quantification.
Compares the total corrected peak area of the ladder to the lower marker. The result will be ratioed to the corrected peak area of sample compared to its respective lower marker peak.

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Global Configuration - Smear Analysis

Table 29 Quantification configuration settings

Settings Final Conc. (ng/µL)
Dilution Factor

Description
The value of final working concentration in ng/mL of the marker or ladder to be used for quantification. Example: In the RNA Kit (DNF-471), the RNA Ladder is diluted 12X (2 µL Ladder + 22 µL diluent) and has a starting concentration of 96 ng/mL. Therefore, the Final Conc. (ng/µL) value is set to 8 ng/µL and the Dilution Factor is set to 12.
The multiplication factor to be applied to the sample or ladder to account for any pre-dilution steps prior to analysis. Note: This value may differ between samples depending upon whether a sample has been pre-diluted prior to analysis. For example, in the HS NGS Fragment Kit, 2 L of sample is diluted into 22 L of diluent/marker solution; a dilution factor of 12 should therefore be used when using the Use Ladder quantification method for the ProSize software to accurately output the starting sample concentration. Note: A Dilution Factor of 1 indicates no dilution of the sample.

Global Configuration - Smear Analysis
The settings of the Smear Analysis tab allow the user to specify a size range(s) for which to integrate and calculate the total concentration in ng/mL. This is useful for example for DNA smears, where knowledge of the concentration of a specific size range within a smear is more important than that of the total smear size range (Figure 46). The settings are described in detail in Table 30.
Several different size ranges can be applied at once to a sample; the calculated total concentration within the specified range is presented in a tabular format that can be exported in a .csv format or printed as a hard copy PDF report.
If one or more selections are entered in the Smear Analysis tab, upon opening the data a Smear Analysis tab will displayed next to the Peak Analysis tab, with each individual result populating a separate line, showing the calculated concentrations for each range.

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Global Configuration - Smear Analysis

Figure 46 Smear Analysis tab in Global Configuration dialog

Table 30 Smear Analysis configuration settings

Settings Start Size (bp)
End Size (bp)
Insert Before
Delete Current Selection Clear All

Description
Defines the lower size threshold (in bp) to include in the calculation of total concentration. Note: If the entered size is in the middle of a peak/smear, the concentration is calculated from that size and higher and will not include the portion of the peak/smear below the size threshold.
Defines the upper size threshold (in bp) to include in the calculation of total concentration. Note: If the entered size is in the middle of a peak/smear, the concentration is calculated from that size and below and will not include the portion of the peak/smear above the size threshold.
Context menu option of the Smear Analysis table. Inserts a blank cell row above the currently selected cell in the table. Note: Click in the respective table cell before using this option.
Context menu option of the Smear Analysis table. Deletes the currently selected cell row in the table. Note: Click in the respective table cell before using this option.
Context menu option of the Smear Analysis table. Deletes all cells in the table.

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Global Configuration - Flag

The settings of the Flag tab allow the user to specify specific criteria (size or concentration range) to be met within the data, and generate a binary output (0 = false, 1 = true) of the results. This is useful for applications (for example, genotyping) where the presence/absence of bands is to be scored (Figure 47). The settings are described in detail in Table 31.
Flag criteria can be set based on size, concentration, peak height and/or corrected peak area. Boolean logic is applied to the criteria for establishing various condition.
If one or more selections are entered in the Flag tab, upon opening the data an additional Flag Analysis tab will be displayed with the Peak Table for each individual results, displaying the flag results in a binary 1/0 format.

Figure 47 Flag tab in Global Configuration dialog

Table 31 Flag configuration settings

Settings Tag Value

Description Allows naming of the flag condition being used. Defines the numerical value of the unit for the flag condition.

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Global Configuration Advanced Flag

Table 31 Flag configuration settings

Settings Unit
Range
Boolean Operation
Flag Analysis Clear All Checkbox

Description
Drop-down list which defines the units of the condition to flagged. The first Unit is always size (bp or nt) based; for the second Unit criteria there are four secondary flagging options: · bp (size) · ng/mL (concentration) · nmole/L (concentration) · RFU (Relative Fluorescence Units)
Defines the numerical range to use for applying the flag condition. The drop-down to the left of this field determines the operation to apply: · ± (will flag the value entered with a plus and minus range) · > (will flag when greater than the range) · < (will flag when less than the range) · = (will flag when equal to the range)
Drop-down list which defines the Boolean operation to apply to the flag condition. There are five options to apply: · ---: No second flag condition is applied. · AND: Both the first and second flag condition must apply to be true · OR: First or second flag condition must apply to be true · AND NOT: The first flag condition must apply and the second must not
apply to be true · NOR: Both the first and second flag condition must not apply to be true
Clears all existing flag conditions from the table.
Clears the corresponding row flag condition entry.

Global Configuration Advanced Flag
The Advanced Flag tab allows the user to define more complex flag conditions. However, only a single set of advanced flag conditions can be applied to all 96 capillaries.
In contrast, Set Individual Parameters dialog allows unique flag conditions for each individual well. A plate map of individual flag conditions can be defined, saved, and re-applied to new plates. An entire plate map of advanced flag conditions may be applied to all 96 capillaries, where each advanced flag condition is different for each capillary.

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Add a Flag Criteria
You can define complex flag criteria with an unlimited number of conditions. A criteria can be set based on size, concentration, peak height, RFU and/or corrected peak area. Boolean logic is applied to the criteria for establishing various conditions. This is useful for applications (for example, genotyping) where a complex flag pattern analysis is required.
1 In the Advanced Flag tab, double-click Add.

The Criteria Input window opens. The settings are described in Table 33.

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2 Enter the flag criteria (size, concentration range, RFU range, mixed fragment criteria, etc.) to be met within the data. This will be used for the result output table.
If one or more selections are entered in the Advanced Flag tab, upon opening the data an additional Advanced Flag Analysis tab will be displayed with the Peak Table.

Saving Formats for Flag Conditions
· If you want an individual flag condition to be applied as one of several flag conditions within a well, save the flag as .template.
· If you want a set of flag conditions to be applied to an individual well (which may contain one or more individual conditions), save the flag as .SCtmpt (where SC is abbreviated for single capillary).
· If you want a set of flag conditions to be applied to an entire plate, where each well may have different flag conditions (which can be applied only in the "Set Individual Parameters" discussed later in this chapter), save the flag as .ACtmpt (where "AC" stands for "all capillaries").
Table 32 describes the settings of the Advanced Flag tab.

Table 32 Advanced Flag configuration settings

Settings Add..... Context menu Load From Template
Save to Template Clear Selected Clear All

Double-click opens the Criteria Input window. Allows to enter a set of flag conditions.
Opens and applies a pre-defined set of flag condition (previously created and saved by user) for an individual well (.SCtmpt). The same set of conditions defined by the .SCtmpt file are applied to all 96 capillaries. Saves the flag template as .SCtmpt for the single well--applied to all 96 capillaries (i.e., the same flag condition is applied to all 96 capillaries). Clears a selected flag condition (which are applied to all 96 capillaries). Clears all flag conditions (for all 96 capillaries).

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Table 33 Criteria Input settings to define a flag condition

Settings Title Tag

Description To name the set of conditions being used (which may include several tags). To name an individual flag condition being used.

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Global Configuration Advanced Flag

Table 33 Criteria Input settings to define a flag condition

Settings Value Unit
Range
Boolean Operation
Accept Load Save Cancel

Description
Defines the numerical value of the unit for the flag condition.
Drop-down list which defines the units of the condition to flagged. The first Unit is always size (bp or nt) based; for the second Unit criteria there are four secondary flagging options: · bp (size) · ng/µL (concentration) · nmole/L (concentration) · RFU (Relative Fluorescence Units)
Defines the numerical range to use for applying the flag condition. The drop-down to the left of this field determines the operation to apply: · ± (will flag the value entered with a plus and minus range) · > (will flag when greater than the range) · < (will flag when less than the range) · = (will flag when equal to the range)
Drop-down list which defines the Boolean operation to apply to the flag condition. There are five options to apply: · ---: No second flag condition is applied. · AND: Both the first and second flag condition must apply to be true · OR: First or second flag condition must apply to be true · AND NOT: The first flag condition must apply and the second must not
apply to be true · NOR: Both the first and second flag condition must not apply to be true
To accept the flag conditions defined for the individual well
To load an individual set of flag conditions (*.template). Note that multiple individual flag conditions can be applied to the same well. For example: 200 bp only; 300 bp AND 500 bp; 600 bp only.
Saves an individual flag condition (*.template). Note that multiple individual flag conditions can be applied to the same well. For example: 200 bp only; 300 bp AND 500 bp; 600 bp only.
Cancels any information entered and closes screen.

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Global Configuration Inclusion Region

The settings of the Inclusion Region tab allow the user to define up to four size inclusion ranges. Only peaks (excluding markers) that fall into the defined range(s) will be displayed. This will affect the screen display, PDF reports and exported data. The Inclusion Region tab is shown in with functions described in Table 34.

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Figure 48 Inclusion Region tab in Global Configuration dialog

Table 34 Inclusion Region configuration settings

Settings Start Size (bp)
End Size (bp)

Description
Defines the lower size boundary (in bp) of the Inclusion Region. Peaks (with the exception of the Lower Marker) smaller than the lower boundary will not be displayed.
Defines the upper size boundary (in bp) of the Inclusion Region. Peaks (with the exception of the Upper Marker) larger than the upper boundary will not be displayed.

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Global Configuration Inclusion Region

Table 34 Inclusion Region configuration settings

Settings Insert Before
Delete Current Selection
Clear All

Description
Context menu option of the Inclusion Region table. Inserts a blank cell row above the currently selected cell in the table. Click in the respective table cell to use this option.
Context menu option of the Inclusion Region table. Deletes the currently selected cell row in the table. Click in the respective table cell to use this option.
Context menu option of the Inclusion Region table. Deletes all cells in the table.

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ProSize Configuration

Set Individual Parameters

After a data file is opened in the ProSize program, the configuration settings can be adjusted for individual samples by the Set Individual Parameters option.
The Set Individual Parameters dialog consist of main tabs that can be adjusted similar to those tabs in the Global Configuration dialog. RNA and Genomic modes also present an extra tab specific to those analyses in the Set Individual Parameters.
Some tabs can be applied to individual samples, while others can only be applied to all samples:
· Advanced Settings
When a file is open and the analysis mode is changed, all samples are changed to the newly selected mode.
· Peak Analysis
When a file is open, configuration settings of individual samples can be edited, or all samples may be changed at once.
· Marker Analysis
When a file is open, configuration settings of individual samples can be edited, or all samples may be changed at once.
· Quantification
When a file is open, configuration settings of individual samples can be edited, or all samples may be changed at once.
· Smear Analysis
When a file is open, configuration settings of individual samples can be edited, or all samples may be changed at once.
· Flag
When a file is open, all samples are changed to the current Flag settings and cannot be individually adjusted.
· DNA Quality Number
When a file is open, configuration settings of individual samples can be edited, or all samples may be changed at once.

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NOTE

· Advanced Flag
When a file is open, configuration settings of individual samples can be edited, or all samples may be changed at once.
· Inclusion Region
When a file is open, configuration settings of individual samples can be edited, or all samples may be changed at once.
· Total RNA Exclusion Region
When a file is open, configuration settings of individual samples can be edited, or all samples may be changed at once.
· Small RNA Region
When a file is open, configuration settings of individual samples can be edited, or all samples may be changed at once.
· Genomic Quality Number
When a file is open, all samples are changed to the current GQN settings and cannot be individually adjusted.
When changing configuration settings in the Set Individual Parameters dialog, the reference ladder well is treated separately from the sample wells. Therefore, if Apply to All is performed on a sample well, the current reference ladder well will not be updated.
If the user chooses Apply to All from the reference ladder well, all sample wells including the ladder well will be updated.

At the bottom of the Set Individual Parameters dialog are two functions for setting and loading the configuration, as shown in Figure 49 and summarized in Table 35.

Table 35 Set Individual Parameters buttons

Button Set as Default Configuration
Load

Description
Sets the default configuration for all files that are subsequently opened (i.e., the Global Configuration). This is useful if you consistently open files of a similar type (i.e., all files are based on the use of a ladder in well "A12" for example.
Opens a file browser menu for loading a previously created configuration file (*.ini extension). Navigate to the desired directory location containing the configuration file, and click OK to load.

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Set Individual Parameters - Advanced Settings

The settings of the tabs in the Set Individual Parameters dialog differ from those in the Global Configuration dialog. The tabs are summarized in the following sections.

Set Individual Parameters - Advanced Settings

NOTE

The settings of the Advanced Settings tab allow the user to assign the data analysis mode for processing data (Figure 49). The Minimum RFU for Signal Processing is used to set a minimum RFU value for integration.
When any change is made to the Mode, all samples are changed, including the reference ladder well.

Figure 49 Advanced Settings tab in Set Individual Parameters dialog
Set Individual Parameters - Peak Analysis
Table 36 describes additional settings of the Peak Analysis tab not available in the Global Configuration dialog. If the user changes a field and/or presses [Enter], the new setting will be applied to the current selected sample well only. The Peak Analysis tab is shown in Figure 50.

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Set Individual Parameters - Peak Analysis

Figure 50 Peak Analysis tab in Set Individual Parameters dialog

Table 36 Additional settings in the Peak Analysis tab

Settings Apply to All

Description
All current settings of just the Peak Analysis tab will be applied to all samples of the loaded data file. If this is applied in the reference ladder well, all samples and the reference ladder will be updated; if this is applied in a sample well, all sample wells but not the reference ladder will be updated.

Note: You should only select this option if you wish to apply all settings of the Peak Analysis tab to all samples. Any previous changes made for the samples of the data file will be overwritten.

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Set Individual Parameters Marker Analysis

Table 36 Additional settings in the Peak Analysis tab

Settings
Apply to Selected Samples

Description
Applies all current settings of just the Peak Analysis tab only to any user specified wells of the sample plate. The Selected Samples plate map menu will be displayed to select the wells of the sample plate to apply the settings to. Select desired wells, rows, or columns, and click OK to apply; click Cancel to abort the operation. If this is applied in the reference ladder well, all samples and the reference ladder will be updated; if this is applied in a sample well, all sample wells but not the reference ladder will be updated. Note: You should only select this option if you wish to apply all settings of the Peak Analysis tab to the selected set of samples. Any previous changes to the selected samples of the data file will be overwritten.

Set Individual Parameters Marker Analysis
Table 37 describes additional settings of the Marker Analysis tab not available in the Global Configuration. If the user changes a field and/or presses [Enter], the new setting will be applied to the current selected sample well only. The Marker Analysis tab is shown in Figure 51.

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Figure 51 Marker Analysis tab in Set Individual Parameters dialog
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Set Individual Parameters Quantification

Table 37 Additional settings of the Marker Analysis tab

Settings Apply to All

Description
All current settings of just the Marker Analysis tab will be applied to all samples of the loaded data file. If this is applied in the reference ladder well, all samples and the reference ladder will be updated; if this is applied in a sample well, all sample wells but not the reference ladder will be updated.

Apply to Selected Samples

Note: You should only select this option if you wish to apply all settings of the Marker Analysis tab to all samples. Any previous changes made for the samples of the data file will be overwritten.
Applies all current settings of just the Marker Analysis tab only to any user specified wells of the sample plate. The Selected Samples plate map menu will be displayed to select the wells of the sample plate to apply the settings to. Select desired wells, rows, or columns, and click OK to apply; click Cancel to abort the operation. If this is applied in the reference ladder well, all samples and the reference ladder will be updated; if this is applied in a sample well, all sample wells but not the reference ladder will be updated. Note: You should only select this option if you wish to apply all settings of the Marker Analysis tab to the selected set of samples. Any previous changes to the selected samples of the data file will be overwritten.

Set Individual Parameters Quantification
Table 38 describes additional settings of the Quantification tab not available in the Global Configuration. If the user changes a field and/or presses [Enter], the new setting will be applied to the current selected sample well only. The Quantification tab is shown in Figure 52.

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Set Individual Parameters Quantification

Figure 52 uantification tab in Set Individual Parameters dialog

Table 38 Additional settings in the Quantification tab

Settings Apply to All

Description
All current settings of just the Quantification tab will be applied to all samples of the loaded data file. If this is applied in the reference ladder well, all samples and the reference ladder will be updated; if this is applied in a sample well, all sample wells but not the reference ladder will be updated.

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Note: You should only select this option if you wish to apply all settings of the Quantification tab to all samples. Any previous changes made for the samples of the data file will be overwritten.
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Set Individual Parameters Smear Analysis

Table 38 Additional settings in the Quantification tab

Settings
Apply to Selected Samples

Description
Applies all current settings of just the Quantification tab only to any user specified wells of the sample plate. The Selected Samples plate map menu will be displayed to select the wells of the sample plate to apply the settings to. Select desired wells, rows, or columns, and click OK to apply; click Cancel to abort the operation. If this is applied in the reference ladder well, all samples and the reference ladder will be updated; if this is applied in a sample well, all sample wells but not the reference ladder will be updated. Note: You should only select this option if you wish to apply all settings of the Quantification tab to the selected set of samples. Any previous changes to the selected samples of the data file will be overwritten.

Set Individual Parameters Smear Analysis
Table 39 describes additional settings of the Smear Analysis tab not available in the Global Configuration. If the user changes a field and/or presses [Enter], the new setting will be applied to the current selected sample well only. The Smear Analysis tab is shown in Figure 53.

Figure 53 Smear Analysis tab in Set Individual Parameters dialog

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Set Individual Parameters Smear Analysis

Table 39 Additional settings in the Quantification tab

Settings Apply to All

Description
All current settings of just the Smear Analysis tab will be applied to all samples of the loaded data file. If this is applied in the reference ladder well, all samples and the reference ladder will be updated; if this is applied in a sample well, all sample wells but not the reference ladder will be updated.

Apply to Selected Samples Display Smear Range

Note: You should only select this option if you wish to apply all settings of the Smear Analysis tab to all samples. Any previous changes made for the samples of the data file will be overwritten.
Applies all current settings of just the Smear Analysis tab only to any user specified wells of the sample plate. The Selected Samples plate map menu will be displayed to select the wells of the sample plate to apply the settings to. Select desired wells, rows, or columns, and click OK to apply; click Cancel to abort the operation. If this is applied in the reference ladder well, all samples and the reference ladder will be updated; if this is applied in a sample well, all sample wells but not the reference ladder will be updated. Note: You should only select this option if you wish to apply all settings of the Smear Analysis tab to the selected set of samples. Any previous changes to the selected samples of the data file will be overwritten.
When enabled, two vertical cursors are displayed in the Electropherogram Trace screen. These cursors define the entered values for the Start Size (bp) and the End Size (bp) for the currently selected smear range. The start and end points of the smear range can be adjusted by clicking on a cursor peak, and dragging the respective cursor left or right; the smear analysis region will be automatically updated to the new cursor location.

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Set Individual Parameters Flag Analysis

Table 40 describes additional settings of the Flag Analysis tab not available in the Global Configuration. If the user changes a field and/or presses [Enter], the new flag setting will be applied to all samples of the sample plate. The Flag Analysis tab is shown in Figure 54.

Figure 54 Flag tab in Set Individual Parameters dialog

cTable 40 Additional Flag Analysis settings

Setting Save Flag Parameters
Load Flag Parameters
Remove All Flag Parameters

Description
When selected, the current Flag Analysis settings will be saved as a file (*.Flag extension) that can be loaded into different data files. A file browser menu will open to navigate and save the desired filename and location; click OK to save the file, or Cancel to discard your changes.
When selected, the previously saved Flag Analysis settings will be loaded from a file (*.Flag extension). A file browser menu will open to navigate and load the desired filename and location; click OK to load the file, or Cancel to discard your changes.
When selected, all settings will be cleared from the tab.

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Set Individual Parameters DNA Quality Number

The settings of the DNA Quality Number tab allows the user to select the Size Threshold (bp) required for the calculation of the DNA Quality Number (Figure 55). Designed as a dynamic, user-defined quality metric for use with the DNA and NGS modes under the Advanced Settings tab. The settings of the DNA Quality Number tab are described in Table 41.

Figure 55 DNA Quality Number tab in Set Individual Parameters dialog

Table 41 Settings in the DNA Quality Number tab

Settings Size Threshold (bp)
Apply to All

Description
Custom defined size threshold that the DNA Quality Number calculation is based upon. The DNA Quality Number (DQN) is calculated based on how much of the sample exceeds the threshold. The DQN is composed of the percentage of DNA above the threshold value divided by 10.
All current settings of just the DNA Quality Number tab will be applied to all samples of the loaded data file. If this is applied in the reference ladder well, all samples and the reference ladder will be updated; if this is applied in a sample well, all sample wells but not the reference ladder will be updated.

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Note: You should only select this option if you wish to apply all settings of the DNA Quality Number tab to all samples. Any previous changes made for the samples of the data file will be overwritten.
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Set Individual Parameters Advanced Flag Analysis

Table 41 Settings in the DNA Quality Number tab

Settings

Description

Apply to Selected Samples

Applies all current settings of just the DNA Quality Number tab only to any user specified wells of the sample plate. The Selected Samples plate map menu will be displayed to select the wells of the sample plate to apply the settings to. Select desired wells, rows, or columns, and click OK to apply; click Cancel to abort the operation. If this is applied in the reference ladder well, all samples and the reference ladder will be updated; if this is applied in a sample well, all sample wells but not the reference ladder will be updated. Note: You should only select this option if you wish to apply all settings of the DNA Quality Number tab to the selected set of samples. Any previous changes to the selected samples of the data file will be overwritten.

The DNA Quality Number tab can be activated or deactivated in the Option dialog. From the menu, select Option, and enable DNA Quality Number (DQN). The dialog and settings of the Options menu are described in Chapter 3, "ProSize Main Screen".

Set Individual Parameters Advanced Flag Analysis
Advanced Flag allows the user to define more complex flag conditions.
The Advanced Flag tab in the Global Configuration dialog allows to apply only a single set of flag conditions to all 96 capillaries. However, here in the Set Individual Parameters dialog, unique flag conditions can be set up for each individual well, and a plate map of individual flag conditions can be defined, saved, and re-applied to new plates.
Add a Flag Criteria
Define complex flag criteria with an unlimited number of conditions. A criteria can be set based on size, concentration, peak height, RFU and/or corrected peak area. Boolean logic is applied to the criteria for establishing various conditions. This is useful for applications (for example, genotyping) where a complex flag pattern analysis is required.

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Set Individual Parameters Advanced Flag Analysis

1 In the Advanced Flag tab, double-click Add (the settings are described in Table 42).

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Set Individual Parameters Advanced Flag Analysis

The Criteria Input window opens.

2 Enter the flag criteria (size, concentration range, RFU range, mixed fragment criteria, etc.) to be met within the data. This will be used for the result output table. The settings are described in Table 43.
If one or more selections are entered in the Advanced Flag tab, upon opening the data an additional Advanced Flag Analysis tab will be displayed with the Peak Table.
If you changes a field and/or click [Enter], the new flag setting will be applied to only that well of the sample plate. To apply the conditions to all wells, select Apply to all in the tab.
Saving Formats for Flag Conditions
· If you want an individual flag condition to be applied as one of several flag conditions within a well, save the flag as .template.
· If you want a set of flag conditions to be applied to an individual well (which may contain one or more individual conditions), save the flag as .SCtmpt (where SC is abbreviated for single capillary).
· If you want a set of flag conditions to be applied to an entire plate, where each well may have different flag conditions (which can be applied only in the "Set Individual Parameters"tab), save the flag as .ACtmpt (where "AC" stands for "all capillaries").

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Set Individual Parameters Advanced Flag Analysis

Table 42 Advanced Flag configuration settings

Settings Add.....
Context menu Load From Template
Save to Template
Clear Selected Clear All Apply to All

Description
Double-click opens the Criteria Input window. Allows to enter a set of flag conditions.
Opens and applies a pre-defined set of flag condition (previously created and saved by user) for an individual well (.SCtmpt). The same set of conditions defined by the .SCtmpt file are applied to all 96 capillaries.
Saves the flag template as .SCtmpt for the single well--applied to all 96 capillaries (i.e., the same flag condition is applied to all 96 capillaries).
Clears a selected flag condition (which are applied to all 96 capillaries).
Clears all flag conditions (for all 96 capillaries).
All current settings of just the Advanced Flag tab will be applied to all samples of the loaded data file. If this is applied in the reference ladder well, all samples and the reference ladder will be updated; if this is applied in a sample well, all sample wells but not the reference ladder will be updated.

Apply to Selected Samples

Note: You should only select this option if you wish to apply all settings of the Advanced Flag tab to all samples. Any previous changes made for the samples of the data file will be overwritten.
Applies all current settings of just the Advanced Flag tab only to any user specified wells of the sample plate. The Selected Samples plate map menu will be displayed to select the wells of the sample plate to apply the settings to. Select desired wells, rows, or columns, and click OK to apply; click Cancel to abort the operation. If this is applied in the reference ladder well, all samples and the reference ladder will be updated; if this is applied in a sample well, all sample wells but not the reference ladder will be updated. Note: You should only select this option if you wish to apply all settings of the Advanced Flag tab to the selected set of samples. Any previous changes to the selected samples of the data file will be overwritten.

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Set Individual Parameters Advanced Flag Analysis

Table 42 Advanced Flag configuration settings

Settings Save Advanced Flag Parameters
Load Advanced Flag Parameters

Description
Saves the current Advanced Flag Analysis settings to a file (*.ACtempt extension) that can be loaded into different data files. A file browser menu will open to navigate and save the desired filename and location; click OK to save the file, or Cancel to abort operation. Note that this saves the entire plate flag map (which may have different flag conditions for each different well). Note: This option saves an entire plate map of different flag conditions to each well.
Loads previously saved Advanced Flag Analysis settings from a file (*.ACtempt extension). A file browser menu will open to navigate and load the desired filename and location; click OK to load the file, or Cancel to abort operation. Note that this applies an entire plate flag map. Note: This option applies an entire plate map of different flag conditions to each well.

Table 43 Criteria Input settings to define a flag condition

Settings Title Tag Value Unit
Range

Description
To name the set of conditions being used (which may include several tags).
To name an individual flag condition being used.
Defines the numerical value of the unit for the flag condition.
Drop-down list which defines the units of the condition to flagged. The first Unit is always size (bp or nt) based; for the second Unit criteria there are four secondary flagging options: · bp (size) · ng/µL (concentration) · nmole/L (concentration) · RFU (Relative Fluorescence Units)
Defines the numerical range to use for applying the flag condition. The drop-down to the left of this field determines the operation to apply: · ± (will flag the value entered with a plus and minus range) · > (will flag when greater than the range) · < (will flag when less than the range) · = (will flag when equal to the range)

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Set Individual Parameters Inclusion Region

Table 43 Criteria Input settings to define a flag condition

Settings Boolean Operation
Accept Load Save Cancel

Description
Drop-down list which defines the Boolean operation to apply to the flag condition. There are five options to apply: · ---: No second flag condition is applied. · AND: Both the first and second flag condition must apply to be true · OR: First or second flag condition must apply to be true · AND NOT: The first flag condition must apply and the second must not
apply to be true · NOR: Both the first and second flag condition must not apply to be true Adding a Boolean operator at the end of a flag condition allows you to enter a continuation of flag conditions-enabling complex combinations.
To accept the flag conditions defined for the individual well
To load an individual set of flag conditions (*.template). Note that multiple individual flag conditions can be applied to the same well. For example: 200 bp only; 300 bp AND 500 bp; 600 bp only.
Saves an individual flag condition (*.template). Note that multiple individual flag conditions can be applied to the same well. For example: 200 bp only; 300 bp AND 500 bp; 600 bp only.
Cancels any information entered and closes the window.

Set Individual Parameters Inclusion Region
The settings of the Inclusion Region tab allow the user to define up to four size inclusion ranges (Figure 56). Only peaks (excluding markers) that fall into the defined range(s) will be displayed. The Inclusion Region will affect the screen display, PDF reports and exported data. Table 44 describes additional settings not displayed in Global Configuration dialog.

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Set Individual Parameters Inclusion Region

Figure 56 Inclusion Region tab in Set Individual Parameters dialog

Table 44 Additional settings in the Inclusion Region tab

Settings Apply to All

Description
All current settings of just the Inclusion Region tab will be applied to all samples of the loaded data file. If this is applied in the reference ladder well, all samples and the reference ladder will be updated; if this is applied in a sample well, all sample wells but not the reference ladder will be updated.

Note: You should only select this option if you wish to apply all settings of the Inclusion Region tab to all samples. Any previous changes made for the samples of the data file will be overwritten.

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Set Individual Parameters Inclusion Region

Table 44 Additional settings in the Inclusion Region tab

Settings Apply to Selected Samples

Description
Applies all current settings of just the Inclusion Region tab only to any user specified wells of the sample plate. The Selected Samples plate map menu will be displayed to select the wells of the sample plate to apply the settings to. Select desired wells, rows, or columns, and click OK to apply; press Cancel to abort the operation. If this is applied in the reference ladder well, all samples and the reference ladder will be updated; if this is applied in a sample well, all sample wells but not the reference ladder will be updated. Note: You should only select this option if you wish to apply all settings of the Inclusion Region tab to the selected set of samples. Any previous changes to the selected samples of the data file will be overwritten.

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About Size Calibration 128
Loading the Size Calibration Curve 129 Viewing and Exporting the Size Calibration (File Open) 135 Identifying and Correcting an Improper Size Calibration 140

This chapter describes the size calibration screen (also referred to as calibration curve screen). The calibration curve screen is used to select the conditions for calibrating the size of unknown sample peaks/smears.

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About Size Calibration

NOTE

In the ProSize software, the user can calibrate the size of sample fragments either by running a standard DNA or RNA ladder in a well of the sample plate (recommended), or by importing a previously calculated and exported size calibration file (*.scal extension).
If an imported size calibration is used in ProSize, the same separation gel, capillary array dimensions, injection/run voltage, and lower/upper markers should be used for both the analyzed data and the imported calibration to ensure maximum sizing accuracy.
To properly calibrate the size, the size ladder must be bracketed by the same lower/upper markers used with the samples. In some cases, the lower or upper marker may co-migrate with a ladder peak; this is acceptable for sizing calibration. In some application kits (for example, RNA and Genomic DNA kits), only a lower marker is used; the data is hence normalized using the lower marker only compared to the respective DNA or RNA Size Ladder.
It is important to ensure the sample containing the size calibration ladder has all ladder fragments integrated correctly with no additional peaks integrated prior to analyzing the remaining samples.
The following sections describe how to load, edit and import/export from the calibration curve screen, and how to identify and correct an improperly calibrated data file.

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Loading the Size Calibration Curve

NOTE

The calibration curve can be defined using two methods:
A: When an unprocessed data file is first opened and a matching configuration is not found, the Global Configuration dialog will appear which is shown in Figure 57. This is the most common way to define the calibration curve.
In most cases, predefined, optimized global configuration files are available to load when processing data collected from different Agilent Reagent Kits. The configuration files are labeled according to the particular kit number, for example, DNF-910 Kit - Fragment Analyzer.ini. These configuration files are available from Agilent upon request if needed. The preloaded configuration files are located in the C:\ProSize data analysis software\Configurations folder.
B: At any time after a file is opened, you can view the Calibration Curve screen by selecting from the main screen, or navigating to Analysis > Show Size Calibration.
The Size Calibration tab in the Global Configuration dialog is shown in Figure 57; the settings are described in Table 45.

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Loading the Size Calibration Curve

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Figure 57 Size Calibration tab in the Global Configuration dialog

Table 45 Size Calibration settings

Settings

Description

Calibration Ladder

Lists size of DNA/RNA fragments contained in calibration ladder in tabular format. The size of each fragment (include lower/upper markers) is entered directly into the table or imported by using the context menu (right-click in the Calibration Ladder table).

Calibration Ladder > Insert Before

Inserts a blank cell above the currently selected cell in the calibration ladder table. Note: Click in the respective table cell to use this option.

Calibration Ladder >

Deletes the currently selected cell in the calibration ladder table. Note:

Delete Current Selection Click in the respective table cell to use this option.

Calibration Ladder > Clear Deletes all cells in the calibration ladder table. All

Calibration Ladder > Import

Imports a previously exported and saved sizing ladder information file. When selected, a file browser menu will open to navigate and select the desired file (*.txt extension). Standard ladder information files are located in the C:\ProSize data analysis software\Ladders folder.

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ProSize Size Calibration Screen

Loading the Size Calibration Curve

Table 45 Size Calibration settings

Settings Calibration Ladder > Export
Default Ladder Well
Curve Fitting
Use Imported Ladder Profile Ladder Profile

Description
Exports the current sizing ladder table information (*.txt extension). When selected, a file browser menu will open to navigate and save the desired filename and location. When exporting ladder information files, it is highly recommended to save the created files to a common directory, located in the C:\ProSize data analysis software\Ladders folder.
Defines the well of the sample plate, which contains the size calibration ladder. From the drop-down list, select the proper row (A-H) and column (1-12) location containing the size calibration ladder.
· Point to Point Fits the size calibration plot of Size (bp or nt) vs. Time (sec) with a Point to Point fit (recommended), drawing a straight line between each point of the curve. · Polynomial Fits the size calibration plot of Size (bp or nt) vs. Time (sec) with a polynomial fit. Enter the order value (from 3 to 5) in the Order field.
Imports a previously exported size calibration file (*.scal extension) to use for size calibration.
Displays file path for the imported size calibration file. To load an imported file, click . A file browser menu will open to navigate and select the desired file (*.scal extension). Note: When importing a size calibration file, the same conditions (separation gel, capillary array length, lower/upper markers, CE separation voltage) should be used. Differences between the analyzed and imported conditions will adversely affect the sizing accuracy.

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5
NOTE NOTE

ProSize Size Calibration Screen Loading the Size Calibration Curve
Define Calibration Ladder
The size of each fragment to be included in the DNA/RNA Ladder (including lower/upper markers) is listed in the Calibration Ladder table (see Figure 57). You can enter directly a value into table or use the Import command of the context menu (right-click in the table). Table 45 describes the commands of the context menu, which include inserting or deleting fragments, clearing all entries, and importing or exporting ladder information files (*.txt extension).
In most cases, predefined ladder information files optimized for different Agilent Reagent Kits are available and are loaded as part of the global configuration. The ladder files are labeled according to the particular kit number, for example, DNF-910-33 - 35-1500bp.txt. These ladder files are available from Agilent upon request if needed. The preloaded ladder files are located in the C:\ProSize data analysis software\Ladders folder.
Define Default Ladder Well
If a size ladder is contained in the sample plate, the sample well for the ladder is displayed in the Default Ladder Well field (see Figure 57, Table 45). Click in the field, and from the drop-down list, select the proper well row (A, B, etc.) and well column (1, 2, 3 etc.) of the well containing the sizing ladder. When processing 12-capillary data, only the well column field (1-12) is selectable.
Define Curve Fitting
The Curve Fitting drop-down menu allows you to define how the size calibration curve is fitted - via a Point to Point or Polynomial Fit (3rd to 5th order) (see Figure 57, Table 45).
It is highly recommended to use Point to Point when fitting the size calibration curve. All standard methods use this setting.
Use Imported Ladder
To use your previously exported size calibration settings, select Use Imported Ladder (see Figure 57). To import your file, select . A file browser menu will open to navigate and select the desired file (*.scal extension) (Figure 57, Table 45).

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Loading the Size Calibration Curve

Peak Analysis for Reference Cap
The default ladder well is considered to be the "Reference capillary". When loading the configurations (for example, for an unprocessed file), note that a separate tab labeled Peak Analysis for Reference Cap is available (Figure 58). This tab enables you to enter independent peak settings to properly identify the DNA/RNA Ladder peaks as compared to the sample peaks, as outlined in Table 46.
Further information on these settings is available in Chapter 4, "ProSize Configuration". When loading or editing the settings of the global configurations, it is important to set these parameters properly to ensure the ladder peaks are selected correctly for size calibration.

Figure 58 Peak Analysis for Reference Cap tab in the Global Configuration dialog

Table 46 Peak Analysis for Reference Cap configuration settings

Settings Peak Analysis Peak Width (sec)

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Loading the Size Calibration Curve

Table 46 Peak Analysis for Reference Cap configuration settings

Settings Min. Peak Height (RFU) # Extra Valley Points
Valley to Valley Baseline?

Description
Defines the minimum peak height threshold to select a peak for integration in RFUs. Peaks below the set value will not be selected for integration.
This setting affects the start/end point of baseline integration of peaks and the baseline drawn between peaks. This setting most affects the baseline between two peaks that are not baseline resolved. Higher values draw a straighter baseline between the first peak start point and second peak end point; lower values draw the baseline more to the "valley" between the two unresolved peaks. Note: The Valley to Valley Baseline? setting needs to be enabled for this function to be active. A typical setting for a DNA Ladder is 3; for a RNA Ladder this is typically set to 0.
This setting affects the start/end point of baseline integration of peaks and the baseline drawn between peaks. This setting most affects the baseline between two peaks that are not baseline resolved. When disabled, will draw a straighter baseline between the first peak start point and second peak end point. When enabled, will draw the baseline more to the "valley" between the two unresolved peaks. This setting is typically enabled for a DNA Ladder and disabled for a RNA Ladder.

Once the proper Calibration Ladder fragments, Default Ladder Well, and Curve Fitting algorithm have been selected (or previous calibration imported) and the correct Peak Analysis for Reference Cap settings have been loaded, along with the other configuration settings, the file is loaded into the ProSize software.

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Viewing and Exporting the Size Calibration (File Open)

Once a data file is loaded into the ProSize software, if the size calibration ladder is contained within the sample plate its well location will be marked with a light blue color (Figure 59, well H12 highlighted with a blue circle). To view the ladder well, click on the respective well in the Plate Map.

NOTE

Figure 59

Main screen: The Size Calibration well H12 is marked in blue in the Plate Map (circled in blue to highlight position of reference capillary). Note: the ladder is selected properly (no error message).

If the number of integrated peaks in the reference well matches the number of fragments in the Calibration Ladder table, the size calibration curve will be fitted with the selected Curve Fitting algorithm and the corresponding calculated sizes will be reported for each sample. The bottom task bar of the ProSize software will display the file path and no errors will be displayed (Figure 59).

It is generally recommended to inspect the size calibration well as well as the calibration curve screen prior to processing or exporting data, to ensure the desired fragments/markers are selected properly.

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Viewing and Exporting the Size Calibration (File Open)

To view the size calibration curve from the main screen of the ProSize software, click in the main screen, or navigate to Analysis > Show Size Calibration. The calibration curve screen will be displayed (Figure 60).

Figure 60 Figure Calibration Curve screen showing properly selected size calibration ladder
The size calibration curve is a plot of Time vs. Size in upper left corner. A proper fit is characterized by the red fit line passing through each blue data point as closely as possible. In most cases, the Point to Point algorithm provides the best fit to the data. However, in some situations the Polynomial algorithm may provide smoother, more continuous fit in the higher size region.
The functions in this screen are the same as in the Size Calibration and Peak Analysis for Reference Cap tabs in the Global Configuration dialog (Figure 57 and Figure 58).
· The Calibration Ladder table and its context menu functions, and the Default Ladder Well, Curve Fitting, and Use Imported Ladder Profile functions are described in Table 45.
· The Peak Analysis panel enables adjustments to be made to the peak integration settings of calibration ladder; these functions are the same as those summarized in Table 46.

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Viewing and Exporting the Size Calibration (File Open)

In the calibration curve screen also an Electropherogram Trace (bottom) of the calibration ladder well is displayed. The toolbar of the trace enables you to zoom, autoscale, copy or view the start/end points of each peak as summarized in Table 47. The functions of the Electropherogram Trace context menu enable you to modify and adjust the selected peaks in the selected reference well as needed to match those present in the Calibration Ladder table (Table 47).
You can export the currently viewed size calibration by selecting Export.

Table 47 Calibration Curve screen additional functions

Item Apply Export Cancel Zoom AutoFit Copy Show Peak Start/End Edge
Accept Change

Description
Applies the current size calibration settings and exit to the main screen of the ProSize software. Any changes made from the initially opened Calibration Curve screen will be applied to the data.
Exports the current size calibration settings and fitted electropherogram/plot, which can be imported to other similarly generated data files (*.scal extension). A file browser menu will open to navigate and save the desired filename and location.
Closes the Calibration Curve screen and exit to the main screen of the ProSize software. Any changes made from the initially opened Calibration Curve screen will not be applied to the data.
Enables zooming-in on any portion of the Electropherogram Trace. To zoom-in, click the icon and position the mouse cursor over desired starting position; click and drag the cursor to the desired location, and release the mouse button.
To autoscale the Electropherogram Trace x-/y-axis view.
Copies an image of the Electropherogram Trace to the clipboard, for pasting in another program such as Microsoft PowerPoint.
Toggle to display or hide the start and end points used for peak integration, shown as vertical orange lines. Displaying the peak integration start/end points can aid in determining if adjustments need to be made to the peak integration to better define the actual peak area. The user can change the peak start/end points by adjusting the Peak Width (sec), or by using the context menu of the Electropherogram Trace such as Split Peak or Move Peak Start/End Points.
Accepts any manual modification to the peak integration (Add Peak, Split Peak, Merge Peaks, Delete Peak, Move Peak Start/End Points). After the manual modification has been made, right-click the Electropherogram trace, and select Accept Change.

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Viewing and Exporting the Size Calibration (File Open)

Table 47 Calibration Curve screen additional functions

Item Cancel Add Peak
Split Peak Merge Peaks

Description
Cancels any manual modification to the peak integration (Add Peak, Split Peak, Merge Peaks, Delete Peak, Move Peak Start/End Points). After the manual modification has been made, right-click the Electropherogram trace, and select Cancel.
To manually add a peak which has not been auto-integrated by the Peak Analysis settings and to define the start and end point for integration. Zoom-in to the region where the peak is to be added. Right-click this region and select Add Peak. Two red vertical cursors will appear; the left cursor defines the new peak start point and the right cursor the new peak end point. Drag each cursor to the desired position, then right-click, and select Accept Change to add the peak for integration. Note: The Add Peak function will not add a peak that is located outside the lower/upper marker window (or before lower marker if using only lower marker).
To manually split a peak which is currently integrated into two peaks and to define where the split occurs. Zoom-in to the region where the peak is to be split. Right-click this region, and select Split Peak. A red vertical cursor will appear. Drag the cursor to the desired location, then right-click, and select Accept Change to split the peak into two peaks for integration.
To merge any number of peaks and integrate as a single peak. Zoom-in to the region where the peak is to be split. Right-click this region, and select Merge Peaks. Two red vertical cursors will appear; the left cursor defines the left most peak to merge and right cursor defines the right most peak to merge. Drag the cursor within that peak's start/end point region to merge, then right click, and select Accept Change to merge the peaks into a single peak for integration.

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Viewing and Exporting the Size Calibration (File Open)

Table 47 Calibration Curve screen additional functions

Item Delete Peak
Move Peak Start/End Points

Description
To manually delete a peak which has been integrated. Zoom-in to the region where the peak is to be deleted. Right-click this region, and select Delete Peak. A red vertical cursor will appear. Drag the cursor to the desired peak location, then right-click, and select Accept Change to delete the peak from the integration.
To change the currently positioned start/end integration points of a peak. Zoom-in to the region where the peak is located. Place the cursor between the start and end points of the integration. Right-click and select Move Peak Start/End Points. Two red vertical cursors will appear at the current start/end points. Drag the cursors to the desired locations, then right-click and select Accept Change to apply the new start/end point positions to reintegrate the peak. Note: This function will not change the baseline; only the start and end points are affected. To change the baseline, the user must adjust the via the Peak Width (sec), Valley to Valley, or Manual Baseline Setpoints tools.

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ProSize Size Calibration Screen

Identifying and Correcting an Improper Size Calibration

When performing the size calibration, the ProSize software will attempt to fit the integrated peaks in the size calibration well to the fragments listed in the size Calibration Ladder table. If the number of integrated peaks in the ladder well does not match the number of fragments in the size Calibration Ladder table (either less or more peaks integrated), a red flashing error message will appear at the bottom of the main screen: Warning: Mis-match between detected peaks and ladder assignment! No sizing calibration curve is established.
An example of less integrated peaks than calibration fragments is shown in Figure 61 and Figure 62 an example of too many integrated peaks is shown in Figure 63 and Figure 64. In each case, you need to adjust the Peak Analysis settings either in the main screen configuration tab, or in the calibration curve screen, to select the proper number of peaks in the size calibration ladder well (including lower/upper markers) to achieve the proper size calibration. Once the proper number of peaks is selected, click the Apply button. The bottom task bar of the ProSize software will then display the file path with no errors.

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Figure 61

Main screen showing improperly fitted size calibration (not enough integrated peaks). Solution to correct: In the Peak Analysis table lower the Min. Peak Height (RFU) threshold to integrate missing peak (circled in blue).

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ProSize Size Calibration Screen

Identifying and Correcting an Improper Size Calibration

Figure 62 Calibration Curve screen showing improperly fit size calibration from Figure 61.

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ProSize Size Calibration Screen

Identifying and Correcting an Improper Size Calibration

Figure 63

Main screen showing improperly fitted size calibration (too many integrated peaks, circled in blue). Solution to correct: increase the Min. Peak Height (RFU) threshold to no longer integrate extra peaks.

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Identifying and Correcting an Improper Size Calibration

Figure 64 Calibration Curve screen showing improperly fit size calibration from Figure 63

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ProSize Overlay Samples

Overlay Options 145
Quick Overlay 145 Overlay Samples Screen 146

This chapter gives an overview of the possible ways to compare samples in the ProSize software.

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Overlay Options

There are two options for comparing samples: The Quick Overlay and the Overlay Samples screen.
Quick Overlay
1 Right-click the sample on the gel image you want to overlay.

The overlay images appear in the electropherogram area.

2 Be sure to select the desired Normalize icon (for example, Normalize to Lower Marker) to get an accurate representation of how the samples compare with each other.
3 To end the Quick Overlay view, double-click on any lane of the gel image.

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ProSize Overlay Samples

Overlay Samples Screen

The Overlay Samples screen is used to overlay and generate comparisons, in digital gel view and electropherogram view, of selected samples from the currently opened data file and optionally from different data files (when using the same markers and experimental conditions), and generate reports of the overlaid data.
Once the Overlay Samples screen is opened, all results are handled and saved as Projects. Before you can export data or generate a .pdf report, the results of the overlay plots must be saved as a Project. Previously saved projects can be opened via the menu Project tab.
The following sections describe how to open, select data to overlay, adjust/annotate overlaid data, open additional data files, and copy/export information from the Overlay Samples screen.
Opening the Overlay Samples Screen
1 To view the Overlay Samples screen from the main screen, select or navigate to Analysis > Overlay Samples.
The Overlay Samples screen is displayed (Figure 65). Table 48 summarizes the top menu bar functions.

Figure 65 Overlay Samples screen (no samples selected)

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Overlay Samples Screen

Table 48 Overlay Screen top menu functions

Menu Item Project...Load Project...Save
Project...Save As
Project...Create View Data File

Description
Opens a previously saved overlay project.
Saves the current overlay project. Note: The user must save an overlay project before the export or "generate report" icons are active.
Saves an additional copy of the overlay project without replacing the original. Note: The user must save an overlay project before the export or "generate report" icons are active.
Creates a new overlay project. This re-opens the overlay file with a blank screen.
To temporarily exit the overlay screen to view the data file. To return to the overlay screen, select Project....Return to Project.

Table 49 summarizes the available functions in the screen prior to opening any sample wells. A Plate Map menu is visible in the top left corner for selection of samples to overlay; the sample filename is listed at the top of the menu.

Table 49 Overlay Samples screen functions (no samples selected)

Menu Item Open File
Exit Overlay Samples Default Layout Image Layout Trace Layout

Description
When selected, a file browser window opens for locating and opening an additional raw data file (*.raw extension) for data analysis comparison to the currently opened data file. Note: If multiple files are to be opened for comparison in the Overlay Samples screen, to generate a meaningful comparison, the additional opened files should: · Be previously processed in the ProSize software (i.e., markers
selected, size calibrated). · The same lower/upper markers must be used. · Similar experimental conditions (separation gel, array length,
separation voltage) must be used.
Closes the Overlay Samples screen and returns to the main screen of the ProSize software.
Shows an "overlay of samples" using both the electropherogram and digital gel view in the same window)(Figure 68).
Shows an "overlay of samples" using a digital gel image window only (Figure 69).
Shows an "overlay of samples" using an electropherogram trace window only (Figure 70).

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Overlay Samples Screen

Table 49 Overlay Samples screen functions (no samples selected)

Menu Item Generate Report
Export Data
Plate Map

Description
Generates a PDF report of the Overlay Samples (see Figure 71). Each file will be color coated. Report options are discussed in detail in Chapter 8, "Generating Reports from ProSize". Data will be color coded per separation. Note: A project must be saved before the program allows you to generate a report.
Exports data from the Overlay Samples (see Figure 72). Export options are discussed in detail in Chapter 7, "Exporting Data from ProSize". Data will be color coded per separation. Note: A project must be saved before the program allows you to export data.
Used to select a sample well(s) for comparison. To select well(s), click the well to add to the overlay; click a second time to remove (in any order). To add/remove a column, click the corresponding column number (1, 2, etc.). To add/remove a row, click the corresponding row letter (A, B, etc.). To select/remove all wells of the plate, select the well in the upper left corner of the plate. The sample filename is displayed at the top of the Plate Map.

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Overlay Samples Screen

Table 49 Overlay Samples screen functions (no samples selected)

Menu Item Sample ID navigation

Description
When selected, a Sample ID list will slide out (Below), enabling the User to select the samples for overlay by the Sample ID previously entered into the instrument software. To select a sample, click the sample name; that well will be highlighted. To remove a well, click it a second time. To clear all wells, right-click and select Clear All. To close the Sample ID navigation, click the arrow at the bottom right of the panel.

Information

When selected, a window opens displaying the instrument software user interface, containing experimental information and notes fields.

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Overlay Samples Screen

Once sample wells are selected in the Overlay Samples screen, they are displayed in order of selection from left to right on the digital gel view and from bottom to top on the electropherogram trace view (Figure 66). If 24 samples are selected the Sample ID will be shown above the gel view, if more than 24 samples are selected the Sample ID will not be shown.
In the digital gel view, each sample/lane selected is annotated at the top by well ID and sample name; the currently opened data file is labeled with a (1) to signify it is the first file open. Additional data files if opened will have their samples/lanes labeled with (2), (3), etc. The side axes for the digital gel image are labeled by size, corresponding to the size calibration ladder. On the right side of the digital gel image, a slider bar is present allowing the adjustment of the intensity/contrast of the image by left clicking and dragging the bar.
In the electropherogram trace overlay, each sample is displayed in a different color with matching color annotation to the right of the overlay by well ID and sample name; the currently opened data file is labeled with a (1) to signify it is the first file open. Additional data files if opened will have their samples/lanes labeled with (2), (3), etc. The x-axis is labeled by Size and the y-axis by RFU in the trace overlay. On the right side of the electropherogram trace overlay, a slider bar is present to adjust the vertical spacing between traces by left clicking and dragging the bar.

Figure 66 Overlay Samples screen (24 samples selected)

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Overlay Samples Screen

Display, Annotation and Export Options in the Overlay Samples Screen
Toolbar functions are available in the Overlay Samples screen for both the digital gel view and electropherogram trace overlays to adjust the display, annotate samples, and export results. These functions are summarized in Table 50.
To access the toolbar functions of the digital gel view overlay, click the corresponding icons (Figure 67). For the electropherogram trace overlay, in addition to the toolbar, a context menu is also available.

Figure 67 Overlay Samples screen (digital gel image toolbar and electropherogram trace with context menu)

Table 50 Overlay Samples toolbar and context menu commands (samples selected)

Menu Item Digital Gel Image Toolbar Copy Full Gel Image

Description
Copies an image of the current 24 lane digital gel view overlay to the clipboard, for pasting in another program such as Microsoft PowerPoint. Any zoom, image normalization, or annotation will be copied in the image. The well ID and sample name will be copied on the top of each lane, and the y-axis will correspond to the currently selected size range obtained from the size calibration ladder in the copied image.

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Overlay Samples Screen

Table 50 Overlay Samples toolbar and context menu commands (samples selected)

Menu Item Show/Hide Cursor
Zoom AutoFit Auto Intensity Normalize Intensity to Lower Marker
Normalize Intensity to Upper Marker
Gel Image Color

Description
Displays a horizontal line cursor across the digital gel image overlay, annotated by size (bp or nt) corresponding to the current position. The cursor can be dragged to any position along the gel image by clicking the cursor and holding the mouse button. When the cursor is active and displayed, a similar vertical cursor annotated by size is displayed in the electropherogram trace overlay, whose movement corresponds to that in the gel image. To deactivate the cursor, click the icon a second time.
Enables zooming in the y-axis (Size) of the digital image overlay. To zoom-in: Place the mouse over the trace, click and drag it outward to expand a box area to define the zoom region. Release the mouse button to apply. The zoomed image can be copied to the clipboard.
To autoscale the digital gel image overlay y-axis view for all 24 currently viewed lanes. The autoscaled image will display the gel from the selected lower marker to upper marker, or if only a lower marker is used from the lower marker to the end of the separation window.
To autoscale the intensity of the digital gel image overlay. The viewed intensity across the image will reflect the actual observed fluorescence signals from each lane.
To normalize the intensity of the digital gel image overlay lower marker for each lane to the same value, adjusting the relative intensities of all bands in each lane accordingly. The viewed intensity across the image will as a result reflect the fluorescence signals from each lane after normalization to the lower marker, as is done when calculating the concentration of samples using the lower marker.
To normalize the intensity of the digital gel image overlay upper marker for each lane to the same value, adjusting the relative intensities of all bands in each lane accordingly. The viewed intensity across the image will as a result reflect the fluorescence signals from each lane after normalization to the upper marker, as is done when calculating the concentration of samples using the upper marker.
Changes the color scheme for the Digital Gel View, the gel image in the Overlay Samples screen and any exported or printed report. Options for the gel image color: · White on Black (white bands on black background) · Black on White (black bands on white background) · Green on Black (green bands on black background) · Red on Black (red bands on black background) · Pseudo Color (green to red bands on blue background)

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Overlay Samples Screen

Table 50 Overlay Samples toolbar and context menu commands (samples selected)

Menu Item

Description

Electropherogram Trace Overlay Toolbar

Zoom

Enables zooming in the x-and y-axis of the Electropherogram Trace. To zoom-in: Place the mouse over the trace, click and drag it outward to expand a box area to define the zoom region. Release the mouse button to apply. The zoomed image can be copied to the clipboard. The zoom region will be preserved when viewing other samples in the Plate Map. To undo the zoom, use the Autoscale function.

Drag

Enables dragging in the x-and y-axis of the Electropherogram Trace.

To drag: Place the mouse over the trace, click and drag it in any

direction, and release the mouse.

Autoscale

To autoscale the Electropherogram Trace x-/y- axis display. The autoscaled image will display the full trace from the start to the end of the separation when using the Time Scale and Size Scale display modes; when using the Uniform Size Scale, the autoscaled display will start at 0 bp. Note: The y-axis scale will automatically zoom to the highest detected sample peak, regardless of the marker peak heights. To view the entire y-axis scale including markers, go to Option, and select Autoscale as the Display Mode.

Copy

Copies an image of the current view of the Electropherogram Trace to the clipboard, for pasting in another program, such as Microsoft PowerPoint. Any zoom, annotation, baseline and/or peak start/end point displayed will be copied in the image. The well ID and sample name will be copied in the top left of the trace, and the x-/y-axis will correspond to the currently selected view in the copied image.

Units

Displays a menu for changing the peak annotation of the Electropherogram Trace. Only integrated peaks are annotated. Units for the peak annotation: · None · Peak ID (labels in order as 1, 2, etc.) · Migration Time (min:sec) raw migration time · Peak Height (in RFUs) · Corrected Peak Area (Peak Area/Migration Time) · Size (in bp or nt) · Average Size (in bp or nt) · Concentration (in ng/µL)

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ProSize Overlay Samples

Overlay Samples Screen

Table 50 Overlay Samples toolbar and context menu commands (samples selected)

Menu Item Create Annotation

Edit Annotations

To move the annotation to the desired position on the trace: Click the annotation and drag it to the desired position. An arrow will appear at the opposite end of the annotation upon dragging. Multiple annotations can be created by repeatedly selecting Create Annotation. Any created annotations will be copied to the clipboard with the Copy function.
Opens the Annotation Editor window. Allows the user to: · Edit the annotation text by typing in the text field. · Change the color of the annotation by left clicking on the
annotation color. · Delete one annotation by selecting Delete next to each annotation,
or delete all annotations by selecting Delete All.

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Select Apply to confirm your settings.

Electropherogram Trace Overlay Context Menu

Copy Image to Clipboard

Copies an image of the current view of the Electropherogram Trace to the clipboard, for pasting in another program, such as Microsoft PowerPoint. Any zoom, spacing, or annotation displayed will be copied in the image. This function operates similar to the Copy icon function.

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Overlay Samples Screen

Table 50 Overlay Samples toolbar and context menu commands (samples selected)

Menu Item Export Data to Clipboard
Export Data to Excel
Create Annotation

Description
To copy the electropherogram x-data (time; in seconds, starting with the lower marker) and y-data (RFUs) in a .csv format that can be pasted directly into common spreadsheet programs such as Microsoft Excel. The x-data is copied into the first column, and the y-data into the second column of the file from left to right starting with the bottom- most sample upon pasting. The columns are annotated starting with Plot 0, 1, 2, etc. in order. To export, right-click to display the context menu and select Export Data to Clipboard. Then open spreadsheet, position in desired cell and paste into the program; process and save as desired.
This function requires Microsoft Excel to be installed on the computer to function properly. Selection will automatically open Microsoft Excel and copy each overlaid electropherogram x-data (time; in seconds, starting with the lower marker) and y-data (RFUs) in a .csv file format. The x-data is copied into the first column, and the y-data into the second column of the file from left to right starting with the bottommost sample upon pasting. The columns are annotated starting with Plot 0, 1, 2, etc. in order. To export, right-click to display the context menu, and select Export Data to Excel. The file can then be processed and saved as desired.
To create customized annotation in the Electropherogram Trace display. In the Add Annotation dialog, the user can type desired annotation into the field. Click OK and the annotation will be displayed in the Electropherogram Trace window.

Clear All Annotations

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Overlay Samples Screen

Displaying Multiple Files in the Overlay Samples Screen
Several different data files can be simultaneously opened and compared using the Overlay Samples screen in ProSize.
To compare different data sets, several criteria should be met when overlaying multiple files:
· The data must use the same size lower/upper marker combination. For RNA analysis, the same lower marker should be used.
· Any additional data files opened in the Overlay Samples screen should be previously processed, with the lower/upper markers correctly selected for the data and the size calibration correctly performed. No additional processing can be done on the opened comparison files; only adjustments to the intensity/spacing can be made.
· The experimental conditions should ideally be the same or similar for both sets of data, using the same separation gel, array length, separation voltage, and/or assay type (for example, NGS Kit).
With the currently open data file displayed in the Overlay Samples screen, select to open a second data file. A file browser menu will be displayed for locating
and opening the additional raw data file (*.raw extension). Once opened, a second Plate Map will be displayed for the second data file below the original file, labeled at the top with the filename (Figure 68).
All functions apply the same to the second opened data file compared the first; to close the second data file at any time, click in the upper right corner of the Plate Map.

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Overlay Samples Screen

Figure 68 Overlay Samples screen displaying two different data files open

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Overlay Samples Screen

Alternate Views in the Overlay Samples Screen
Several different views can be displayed in the Overlay Samples screen as described in Table 49. Figure 69 shows the Image Layout view; Figure 70 shows the Trace Layout view. The functions in these screens are the same as those in the Default Layout view.

Figure 69 Overlay Samples screen displaying Image Layout view

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Overlay Samples Screen

Figure 70 Overlay Samples screen displaying Image Layout view

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ProSize Overlay Samples

Overlay Samples Screen

Figure 71 Overlay Samples PDF Report showing the first page of overlayed traces page, Traces Summary page, and Electropherogram/Peak Table page

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Overlay Samples Screen

Figure 72 Export folder showing files generated from the Overlay Samples screen

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Overlay Samples Screen

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Exporting Data from ProSize

Export Data Utility 164
Export Data Window Settings 164 Examples of Exported Data 168

This chapter provides an overview of the options available for exporting processed data from the ProSize software. The following sections describe how to open the Export Data window; provide an outline of various data export options; describe how to perform data export; and give examples of exported data.

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Export Data Utility

Once the data is opened and processed within the ProSize program, the measured/calculated information can be exported in common formats for storage in a common database or for use in other programs. Export Data is used to export information from the ProSize program.
Export Data Window Settings
To export sample results from the ProSize software: 1 From the main screen, select , or navigate to File > Export Data.
The Export Data window will be displayed (Figure 73).

Figure 73 Export Data window

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Export Data Window Settings

Table 51 summarizes the available settings in the Export Data window.

Table 51 Export Data settings

Settings Sample Option

Description
Determines which samples of the data file will have information exported. There are two options: All Samples (results for all samples of data file exported); and Selected Samples. The Selected Samples option will open the Selected Samples window.

Export All Peak Table Standard or Alternate Smear Analysis Table
Flag Analysis Criteria Table Standard or Alternate
Advanced Flag Criteria Table

Select the respective wells, columns and/or rows of the sample plate, and click Apply. To abort the operation and export all samples, click Cancel.
Exports all fields of the Export Data utility for the data file.
Standard: Exports the Peak Table for all selected samples as a single .csv file, listed in order of sample well (Figure 75). Alternate: Exports the Peak Table for all selected samples as a single .csv file, listed in rows (Figure 76).
Exports the Smear Analysis table for all selected samples as a single .csv file, listed in order of sample well (Figure 77). If no data is present in the Smear Analysis table, this function will be disabled.
Standard: Exports the Flag Analysis table for all selected samples as a single .csv file, listed in order of sample well (Figure 78). The output will be in a binary 0/1 format, with each column labeled by the respective Flag condition. Alternate: Export the Flag Analysis table for all selected samples as a single .csv file, listed in order of sample well) (Figure 79). The output will list the values within the set criteria of the Flag analysis by row. If no data is present in the Flag Analysis table, this function will be disabled.
Exports the Advanced Flag Analysis table for all selected samples as a single .csv file, listed in order of sample well (Figure 80). The output will lists the values for the configurations of the Advanced Flag analysis by row.

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Export Data Window Settings

Table 51 Export Data settings

Settings Electropherogram Exports as CSV File File Export Option
X Axis Scale Option
Size Calibration Data Exports Selected Samples Digital Gel Image Image Format for Electropherogram and Gel
Show Annotation Export File Path

Description
Exports electropherogram data in a .csv file format.
Determines how electropherogram .csv data is exported. When Single File is selected, will export all electropherograms together in a single file, with the x-axis in the first column and each sample result's y-axis data in separate columns increasing from left to right (Figure 81). When Separated Files is selected, will export each electropherogram as a separate file, with the x-axis in the first column and the y-xis data in the second column (Figure 82).
Determines how electropherogram .csv data is formatted in x-axis. When Size Scale is selected, will export x-axis data as size, starting with 0 (Figure 81). When Time Scale is selected, will export x-axis as time, starting with 0 seconds (Figure 82).
Exports the Size Calibration information as a .csv file, with column 1 as Ladder Size and column 2 as Time (sec) (Figure 83).
Exports a digital gel image overlay of the selected samples as an image file, in the format specified (.bmp, .jpg, or .png).
Determines which image file format is used for exported electropherogram images and digital gel image overlay (Figure 84). There are three different image file format options: · BMP (.bmp extension) · JPEG (.jpg extension) · PNG (.png extension)
Shows any annotation(s) made to the sample wells on Digital Gel Image.
Determines the file path for saving exported data. The default directory is the same folder that contains the .raw data file (recommended). To select an alternative directory, click the folder icon. In the file browser menu, navigate to the desired directory, and save the file.

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Export Data Window Settings

Table 51 Export Data settings

Settings Export

Description
Exports the data file with the settings made. In the dialog Export Complete, select Open Folder Now to open the directory. Click Close to close without opening the export folder.

Cancel

Cancels the export operation and returns to the main screen of the ProSize software.

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Examples of Exported Data

When exporting data from ProSize, the exported files will be named by the .raw file name followed by an extension dependent upon the information exported (Figure 74 and Table 52).

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Figure 74 Exported data showing file name conventions

Table 52 Exported data naming conventions

Exported Item Peak Table Size Calibration Smear Analysis Flag Analysis Result Advanced Flag Analysis Result
Selected Samples Gel Image

Description
Filename Peak Table Example: 2013 07 15 18H 29M Peak Table
Filename Size Calibration Example: 2013 07 15 18H 29M Size Calibration
Filename Smear Analysis Result Example: 2013 07 15 18H 29M Smear Analysis Result
Filename Flag Analysis Result Example: 2013 07 15 18H 29M Flag Analysis Result
Filename Advanced Flag Analysis Result Example: 2013 05 08 16H 02M Advanced Flag Analysis Result
Filename Gel Example: 2013 07 15 18H 29M Gel

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Examples of Exported Data

Table 52 Exported data naming conventions

Exported Item Individual Electropherogram Gel Image
Individual Electropherogram Data (CSV)

Description
Filename <Well ID> <Sample Name> Example: 2013 07 15 18H 29M H1 SampH1
· If exported as one file: Filename Electropherogram Example: 2013 07 15 18H 29M Electropherogram
· If exported as individual files: Filename <Well ID> <Sample Name> Example: 2013 07 15 18H 29M H1 SampH1

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Examples of Exported Data

The following figures show examples of typical exported data formats: · Peak Table

Figure 75 Exported Peak table - Standard file format

Figure 76 Exported Peak Table - Alternate file format

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Exporting Data from ProSize Examples of Exported Data
· Smear Analysis
When multiple Smear Analysis conditions are entered for a sample, separate lines will be created in the exported table. The DNA Quality Number (DQN) along with the associated size threshold are also shown in the exported Smear Analysis data. More information is available in Chapter 4, "ProSize Configuration".

Figure 77 Exported smear Analysis file format

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Exporting Data from ProSize

Examples of Exported Data

· Flag Analysis Criteria Table

Figure 78 Exported Flag Analysis - Standard file format

Figure 79 Exported Flag Analysis - Alternate file format

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Examples of Exported Data

· Advanced Flag Analysis Criteria Table

Figure 80 Exported Advanced Flag Analysis
· Electropherogram CSV Single File Size Scale

Figure 81 Exported Electropherogram CSV Single File Size Scale file format

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Examples of Exported Data

· Electropherogram CSV Separated Files Time Scale

Figure 82 Exported Electropherogram CSV Separated Files Time Scale file format
· Size Calibration

Figure 83 Exported Size Calibration file format

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Examples of Exported Data

· Selected Samples Gel Image

Figure 84 Exported Selected Samples Gel Image file format

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Exporting Data from ProSize

Examples of Exported Data

· Individual Electropherogram Image

Figure 85 Exported Individual Electropherogram Image file format

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Generating Reports from ProSize

Generate a Report 178
Generate a report of sample results 178
Examples of Generated Reports 182

This chapter provides an overview of the options available for generating reports from the ProSize software. The following sections describe how to open the Generate Report window; provide an outline of various report generation options; describe how to perform the report generation; and give examples of reported data.

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Generate a Report

Generate a report of sample results
The ProSize software can generate PDF formatted reports for convenient viewing of processed data, reporting detailed information for each sample analysis. The Generate Report command is used to generate PDF formatted results. 1 From the main screen, select , or navigate to File > Generate Report.
The Generate PDF window opens (Figure 86). Table 53 summarizes the available settings of this window.

Figure 86 Generate PDF window

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Generate a report of sample results

Table 53 Generate PDF settings

Settings Sample Option

Description
Determines which samples of the data file will be included in the report. All selected samples will be saved in a single PDF report file; each sample will be printed on a separate page of the report. There are two options: All Samples (results for all samples of data file exported); and Selected Samples. The Selected Samples option will open the Selected Samples window.

Run Summary Method Detail

Select the respective wells, columns and/or rows of the sample plate, and click Apply. To abort the operation and export all samples, click Cancel.
A summary of run information will be printed on the first page of PDF report including Filename and Data Path; date Created; # of Capillaries; Array Serial #; capillary Effective Length; Array Usage Count; Version # of software; Device Serial # (Figure 87). If this is not selected, these fields will be blank.
Details of the performed experimental method will be printed on the first page of the PDF report (from the user interface Information file ( in ProSize Plate Map)). If not selected, these fields will be blank. An example for the Fragment Analyzer is shown below: Method Information: Method Name: DNF-493-33 HS Large Fragment.mthds Gel Prime: No Full Conditioning: Yes Gel Prime to Bufer: Yes Gel Selection: Gel 1 Perform Prerun: 6.0 kV, 30 sec. Rinse: No Marker 1: No Rinse: Tray: 3, Row: H, # Dips: 1 Sample Injection: 5.0 kV, 30 sec. Separation: 6.0 kV, 50.0 min. Tray Name: Tray-1 Analysis Mode: NGS NOTE Testing

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Generate a report of sample results

Table 53 Generate PDF settings

Settings Traces Summary
Peak Table

Description
The Trace Summary page will be printed with up to 12 electropherograms per page (Figure 88). Traces will show the Sample ID information for each trace. If not selected, these field will be blank.
For all selected samples, the Peak Table will be printed on each sample result page in the PDF report (Figure 89). If not selected, this field will be blank. Select to define the reported items and their order in the table. You can save these settings for future use.

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Electropherograms Flag Analysis Advanced Flag Analysis Smear Analysis

For all selected samples, the Electropherogram Trace will be printed on each sample result page in the PDF report. Shown are the peaks with the currently selected annotation and x-axis scale in ProSize at the time of printing (Figure 89). The digital gel image will be displayed to the right, labeled by size. If not selected, this field will be blank.
For all selected samples, a Flag Analysis results table will be printed on a separate page in the PDF report, with one page per Flag condition (Figure 90). The output will be in a binary 0/1 format, with each page column labeled by the respective Flag condition. If no Flag Analysis table is present for the data file, this option will be disabled.
For each sample well defined, the Advanced Flag Analysis results will be printed on a separate page in the PDF report (Figure 91). The output will list the values within the set criteria of the Advanced Flag analysis.
For each selected sample, the Smear Analysis results will be printed underneath the Peak Table. The color-coded vertical smear range lines and Average Size (bp) will be also reported (Figure 89). If no Smear Analysis table is present for the data file, this option will be disabled.
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Generate a report of sample results

Table 53 Generate PDF settings

Settings Gel Image Calibration Curve
Show Integration Parameters Information
Show Annotation Logo
Generate PDF report

Description
Prints a digital gel overlay image of the selected samples on the first page of the PDF report (Figure 87). If not selected, this field will be blank.
Prints a separate page at the end of the PDF report showing the Size Calibration curve including the well location and fit type used (Figure 92). If not selected, this page will be not printed.
Prints a detailed summary of the individual integration parameters for each selected sample at the bottom of each sample result page in the PDF report (Figure 89). If not selected, this field blank will be blank.
Shows the annotation(s) made to individual sample electropherograms. If not selected, no annotation for samples will be shown.
Allows to import a custom logo (which will print at the top of each page). This is for customers that want their company logo to be printed on each page.
Generates the configured report. A file browser menu will open to navigate and save the file in the desired directory. The default directory is that containing the .raw data file. In the Message dialog, click OK to open the report; click Cancel to close without opening the report.

Cancel

Abort the report generation and return the main screen of the software.

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Examples of Generated Reports

Figure 87 through Figure 92 below show examples of the PDF report pages generated by the ProSize software version 3.0. The header of each page lists the .raw data file name and the page number; the footer contains software version and copyright information as well the date and time of report generation.

Figure 87 Page 1 of PDF report (all fields shown; 12 samples were selected)

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Examples of Generated Reports

Figure 88 Trace Summary of PDF report (all fields shown; 12 samples were selected). Note: When reporting total RNA data, the RQN is reported.

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Examples of Generated Reports

Figure 89

Individual result page of PDF report (all fields shown). Note: When reporting total RNA data, the rRNA peaks are highlighted and the rRNA ratio and RQN are reported. Not shown: When reporting Genomic Data, the GQN will be reported.

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Examples of Generated Reports

Figure 90 Example Flag Analysis table in PDF report Figure 91 Example Advanced Flag Analysis table in PDF report
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Examples of Generated Reports

Figure 92 Example Size Calibration page in PDF report

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ProSize View Capillary Positions

About View Capillary Positions 188
Open the View Capillary Positions Window 189 View Capillary Positions Window Settings 191 Manually Resetting Capillary Positions 194

This chapter briefly covers the tools and functions of the View Capillary Positions window.

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ProSize View Capillary Positions

About View Capillary Positions

The ProSize software performs an automated capillary alignment procedure when reading in .raw data files. This ensures that the capillary locations on the CCD detector selected for data analysis are of maximum fluorescence intensity for providing the best possible signal to noise ratio.
View Capillary Positions is used to examine the capillary array alignment and the assigned locations used for data analysis. This option is typically not used in routine use since the locations are automatically assigned; it serves rather as a diagnostic/troubleshooting tool.

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Open the View Capillary Positions Window

1 From the main screen of the ProSize software program, select Analysis > View Capillary Positions.
The View Capillary Positions window opens (Figure 93). Table 54 summarizes the available functions in this window.

Figure 93 View Capillary Positions utility
The View Capillary Positions window contains two sections:
· A plot of Capillary Positions (Pixels) vs. Signal Distribution (Intensity), which shows a summation of fluorescence intensity for each capillary.
· The Capillary Positions table listing the pixel location for each of the 12, 48, or 96 capillaries in the 12-capillary, 48-capillary, or 96-capillary array, respectively.
In Figure 93, a proper alignment is shown where the red Current Positions symbols are centered on the peaks corresponding to each capillary. The algorithm automatically detects the peak locations and sets the capillary positions. If no fluorescence is detected in a particular capillary, the algorithm will

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NOTE

ProSize View Capillary Positions Open the View Capillary Positions Window
fill the gap using the spacing between capillaries as a reference. In this way, the positions of all capillaries are indexed properly, even if no data was generated by some capillaries of the array.
It is important when installing a new capillary array to perform a run with samples (for example, DNA Ladder or marker plate), followed by performing an optical alignment using the collected data file (i.e., Read Raw optical alignment function). This is to ensure that the assigned instrument capillary locations are close to actual positions. For further information, refer to the Capillary Alignment section of the System Manual.
During typical operation, no modifications should need to be made to the capillary positions. In rare occurrences, it may be necessary to adjust the positions slightly for one or two capillaries, or to manually adjust positions as described below. provides information on how to change a location and save the changes. Only changes necessary to center the red Current Positions symbols on any misaligned capillaries should be performed, to avoid introducing artifacts into the data.

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View Capillary Positions Window Settings

Table 54 summarizes the settings of View Capillary Positions window.

Table 54 Capillary Positions window functions

Menu Item Capillary Positions

Description
Lists the pixel locations used on the CCD detector for data analysis. There are either 12, 48 or 96 cells, each corresponding to a capillary of the array. The table is labeled by row and well position.

Reset Method
Peak Width Undo

To view a particular capillary position, click the cell in the table, or click on a peak in the plot of Capillary Positions (Pixels) vs. Signal Distribution (Intensity) to view the corresponding assigned well. A red vertical cursor will be displayed on the plot to indicate the detector position.
Performs an automatic alignment algorithm to reset the capillary locations. Any manual changes made to the pixel positions will be overwritten. A blue threshold line will appear (see Figure 94). This line can be moved up or down to select an appropriate threshold for automatically determining capillary location.
Two methods for determining capillary location can be used: · Close Proximity (default): This setting will use the original instrument
alignment file to determine where to locate the capillary positions, filling any gaps in signal as needed. · Ignore Original: If the Close Proximity method fails to locate the correct capillary locations, this will attempt to assign capillary positions based on the signal positions; press Reset and use the threshold cursor and Peak Width settings to adjust assignments.
Provides a width threshold for selecting peaks in the plot of Capillary Positions (Pixels) vs. Signal Distribution (Intensity); a higher value selects wider peaks while a smaller value selects narrow peaks. The recommended value is 3.
To undo the last manual adjustment operation to the Capillary Positions table.

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View Capillary Positions Window Settings

Table 54 Capillary Positions window functions

Menu Item Zoom
Autoscale Pan
Accept Cancel

Description
Enables zooming in the x- and y-axis of the plot of Capillary Positions (Pixels) vs. Signal Distribution (Intensity). To zoom-in: Place the mouse over the plot, click and drag it outward to expand a box area to define the zoom region. Release the mouse button to apply. . To undo the zoom, use the Autoscale function.
To autoscale the plot of Capillary Positions (Pixels) vs. Signal Distribution (Intensity).
Enables panning of the plot of Capillary Positions (Pixels) vs. Signal Distribution (Intensity). Move the image around with the mouse cursor: Place the mouse over the trace and click; drag the cursor to shift the current view. This is most often used in combination with the Zoom function.
Accepts the current Capillary Positions table locations for the capillary array to use for data analysis and return to the main screen of the ProSize program.
Cancels any manual modification to the Capillary Positions table locations for the capillary array. It closes the View Capillary Positions window and return to the main screen of the ProSize program.

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View Capillary Positions Window Settings

Figure 94 Capillary Positions window after pressing Reset. A blue horizontal cursor can be used to select the threshold for locating capillary positions.

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Manually Resetting Capillary Positions

In very few situations, it may be necessary to manually relocate the capillary positions in the View Capillary Positions window. This is usually the result of a combination of the two following conditions:
· The instrument optical alignment (i.e., capillary positioning) is not set properly. When the set capillary locations are far from the actual locations, the ProSize software will have difficulty locating them automatically, especially for 96-capillary data. Therefore is it very important to ensure the capillary array locations have been reset and properly aligned after changing a capillary array.
· A capillary within the array has no detectable fluorescent signal, either due to no sample or marker being loaded into the particular sample well, or because the capillary is plugged.
Ensuring the capillary array is properly aligned will in most all cases enable automatic detection of capillary locations, even if some capillaries do not generate signal. In the event, it is necessary to manually adjust or reset a capillary location, the user should follow the steps outlined below.
Figure 95 shows the View Capillary Positions window for a file where a capillary signal is missing (118 pixels), and as a result of a slight misalignment of the instrument optical alignment the software has mistakenly assigned a capillary to pixel 495 and not assigned a location to the second capillary from the right (circled). In this instance, it is not possible to adjust the threshold to successfully select the proper capillaries.

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Manually Resetting Capillary Positions

Figure 95 View Capillary Positions window where capillary is not assigned properly (circled in red)

Figure 96 View Capillary Positions window showing misaligned capillary not reset by threshold
In Figure 96 it is shown that clicking Reset and using the threshold will not correctly reassign the capillaries due to the low signal from pixel 118.
To manually delete the improper capillary position, click the mouse over the location, and click [Delete] on the keyboard (Figure 97).

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Manually Resetting Capillary Positions

Figure 97 Manually deleting pixel 495 by selecting it, and click [Delete]
Note in that Figure 98 pixel 495 has now been removed from the Capillary Positions table and a blank cell is present in H12.

Figure 98 Alignment after manually deleting pixel 495
Next, to manually add a location, click on the adjacent capillary location to where the user wishes to add the capillary, and insert it by clicking [Insert] on the keyboard. Click the keyboard arrows to move the newly added vertical red cursor to the desired location, in this case pixel 684 (Figure 99).

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Manually Resetting Capillary Positions

Once the proper positions have been adjusted, press Accept to accept the current Capillary Positions table locations for the capillary array and return to the main screen of the ProSize program. All subsequent data analysis will use the newly saved capillary locations.

Figure 99

Alignment after clicking [Insert] at the rightmost position and using the keyboard arrow to move the vertical cursor to pixel 684. Capillary positions are now correctly located. Press Accept to save.

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ProSize Troubleshooting Guide

ProSize Software Error Messages 199

This chapter gives an overview of troubleshooting the ProSize software.

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ProSize Software Error Messages

The following table lists some potential error messages or issues that may be encountered when using the ProSize software and how to correct them.
· When multiple causes and corrective actions are listed, the items are presented from most likely to the least likely.
· If all corrective actions are employed and a problem still persists, contact your corresponding Agilent Service Representative for additional help.
Further information regarding processing data within can be found in the ProSize Tutorial Videos under Help > Tutorial Videos.
For hardware related issues, please refer to the individual Reagent Kit User Manual, or the instrument System Manual.

Table 55 ProSize software error messages

Problem

Cause

Corrective Action

When processing data in main screen, a red flashing message appears on the bottom toolbar: Warning: Mis-match between detected peaks and ladder assignment! No sizing calibration curve is established.

1 The number of peaks integrated in the sample well assigned for size calibration does not match the number of fragments in calibration ladder.
2 Wrong sample well selected for size calibration ladder.

1 Verify the correct number of peaks are integrated, adjust as necessary. Markers should be included in the calibration ladder table.
2 Verify correct sample well location of size calibration ladder; reassign in Size Calibration menu.
For more information, refer to Chapter 5, "ProSize Size Calibration Screen".

When generating a PDF report, the following message is displayed:

1 Another PDF report is open with the 1 Close any additional open files and

same name.

repeat the operation.

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Table 55 ProSize software error messages

Problem
When attempting to integrate a smear, the baseline does not properly follow the trace. See example below:

Cause

Corrective Action

1 The Peak Width (sec) setting is too low.

1 Increase the Peak Width (sec) setting until baseline draws properly.
2 Use the Manual Baseline Setpoints function to set the baseline from outside of the lower/upper marker peak region. See Table 25 for further information.

Peaks are observed in the Electropherogram Trace view but no corresponding band is seen in the Digital Gel View. Example:

1 The Peak Width (sec) setting is too high.
2 The function Hide/Show Non-Integrated Peaks on the Digital Gel Image toolbar is set to hide non-integrated peaks.

1 Decrease the Peak Width (sec) setting until peaks are integrated properly.
2 Toggle the icon to Show Non-Integrated Peaks.

In RNA mode, the RQN number is stated as NaN.

1 The concentration of the sample is too low for the software to accurately calculate the RQN number.

1 Increase the injection time of the sample method to inject more sample.
2 Increase the concentration of the sample added to the Diluent Marker.

The lower and/or upper markers do not properly line up between lanes in the Digital Gel Image display.

1 The Min. Peak Height (RFU) threshold is set too high, preventing the marker peaks from being selected.
2 Extra peaks (for example, primer/dimer) that are migrating close to the marker are being assigned as the lower marker.

1 Adjust/decrease the Min. Peak Height (RFU) threshold in the Marker Analysis tab of the Set Individual Parameters menu; apply to individual sample or all samples as needed.
2 Adjust/increase the Min. Peak Height (RFU) threshold in the Marker Analysis tab of the Set Individual Parameters menu; apply to individual sample or all samples as needed.

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Batch Processing Using ProSize

Batch Processing 202
Perform a Batch 202

This chapter explains how to perform a batch process and provides an outline of the various exporting options.

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Batch Processing

The ProSize software can batch process a list of runs within a folder, and send the parameters/reports to an output folder. If an output folder is not designated, the data is exported and placed back into the original data folders. To successfully apply the batch processing routine, the name of the Run Method in the instrument software must exactly match the configuration method in ProSize.
Perform a Batch
1 From the menu, select Batch Processing > Batch Data Process.

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Perform a Batch

The Batch Data Processing window opens (Figure 100). Table 56 summarizes the available options in this window.

Figure 100 Batch Data Processing window. With open file selection browser to define folder.

Table 56 Batch Data Processing window options

Item Folder Selection Export Path (If empty, uses data path)
Export File Options Start

Description
Allows you to select the folder location with data files to be batch processed. After locating the folder, select Current Folder to set the folder location.
Allows you to select a folder location for exported data. After locating the folder, select Current Folder to set the folder location. Note: If this field is left empty, the program will export the data to the data folders in Folder Selection above.
Allows you to define an export option available in data export (PDF, peak table, flag analysis, etc.).
Starts the batch processing routine.

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11
NOTE

Batch Processing Using ProSize Perform a Batch

Table 56 Batch Data Processing window options

Item Exit
View Error Log

Description
Terminates the batch processing routine and closes the window.
Opens an Error Log dialog which allows you to quickly determine errors associated with batch processing (Figure 101).

To initiate batch processing:

1 In the Batch Data Processing window, under Folder Selection, select and navigate to your folder with the input files.

2 Click Current Folder to accept the folder location.

3 Under Export Path, select files.

and navigate to a folder location for the exported

4 Click Current Folder to accept the folder location.

If no folder is selected, the program will export the data back into the original data folders.

5 Select Start to initiate the process. Once the batch processing is finished, the Error Log window shows the final processing status for each file.
To display the batch processing status of the files:
1 Select View Error Log within the Batch Data Processing window. Or From the menu, select Batch Processing > View Error Log.

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Perform a Batch

The Error Log window opens (Figure 101). Table 57 describes the output errors associated with this window.

Figure 101 Error Log window
2 To view the specific error for a file, click the respective file. A dialog opens and displays information about the error.
3 To open the file in ProSize, close the dialog and double-click on the file. Detailed information for the error is provided.
A folder with the .txt files of all errors is generated and located under C:ProSize data analysis software/Error Log (Figure 102).

Figure 102 Generated error text files in C:ProSize data analysis software/Error Log
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Table 57 Error messages generated by the batch process routine

Error class Critical Error Input Error Generation Error
Individual Error

Description
1 ProSize cannot find the configuration file (there is no file with the exact same name as the method file).
2 A configuration file exists, but ProSize was not able to apply the sizing calibration.
1 The user has specified a table (such as Flag Analysis or Smear Analysis), but there are no parameters set up in ProSize to generate these reports. The user must go to the Set Individual Parameters screen and set up the appropriate features (Flag, Smear, Advanced Flag, etc.).
1 The user has specified a table (such as Flag Analysis or Smear Analysis), but there are no parameters set up in ProSize to generate these reports. The user must go to the Set Individual Parameters screen and set up the appropriate features (Flag, Smear, Advanced Flag, etc.).
2 The user is using a mode such as DNA, and has requested a Quality table, which is only available in the RNA mode.
1 The markers are too broad (FWHM is to large). 2 There are no peaks.

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ProSize Enhanced Security Features

Activate Enhanced Security Feature 208
Activate Enhanced Security Feature in the System Controller Software 208 Activate Enhanced Security Feature in ProSize 208 Use eSignature 213 View Event Report 217 View Data Event Log 219

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ProSize Enhanced Security Features

Activate Enhanced Security Feature

ProSize data analysis software has been updated to include enhanced security and integrity features. To use all enhanced security functions, a .db3 run file from enhanced security is required to run the controller software version 1.2, or higher.
Activate Enhanced Security Feature in the System Controller Software
1 From the menu, select Admin > Configuration. 2 Select Login Required and Electronic Records. 3 Select Save prior to leaving the configuration screen.
The program will notify you that ProSize versions 3.0, or higher, is required to read the database files created by the enhanced security enabled software. All runs performed will now be stored in an encrypted database that can be read only by ProSize version 3.0, or higher.
Activate Enhanced Security Feature in ProSize
1 From the menu, select Administration > Configuration.

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Activate Enhanced Security Feature in ProSize

The ProSize Data Analysis Software Login Configuration window opens (Figure 103).

Figure 103 Login Configuration window
2 To enable the enhanced security features, select Login required, and afterwards Enable Enhanced Security.
3 Define the other settings as necessary. Table 58 describes the settings shown in the ProSize Login Configuration window.

Table 58 ProSize Login Configuration settings

Settings Login required Enable enhanced security Auto logoff time: (minutes): Minimum password length:
Password expiration time (months):

Description
When selected, ProSize requires a user login. It does not enable enhanced security compliance.
Enables enhanced security. To enable this option Login required must be enabled.
Designates how long the system is idle prior to auto-logoff.
Sets minimum character length for the user passwords. An entry of zero indicates no minimum password length.
Determines how often a password must be changed by the user. An entry of zero indicates no Password expiration time.

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Activate Enhanced Security Feature in ProSize

Table 58 ProSize Login Configuration settings

Settings Allowed login attempts
Previous unique passwords

Description
Set the number of allowed login attempts by the user. A value of zero indicates no maximum number of login attempts.
Sets the number of passwords that must be unique for a single user. A value of zero indicates no requirements related to previous unique passwords.

4 Click Accept. You will be prompted to setup the administrator account.

Figure 104 Message to setup an administrator account

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Activate Enhanced Security Feature in ProSize

5 Select OK to set up the administrator account.
The User Account Setup window opens (Figure 105). The user settings are described in Table 59.

Figure 105 User Account Setup window

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Table 59 User account settings User settings Name User ID Password Password (Repeat) Email Access Level
eSignature Level
Active? Receive email alerts

Description
Enter name of the user.
Enter a user ID.
Choose a password. Note: Passwords must follow the requirements specified in the Login Configuration window (Figure 103).
Re-enter selected password to confirm accuracy.
Enter an email to be associated with the account. Note: This is required only if the user wants to receive e-mail alerts when an invalid login is attempted.
Select one of the three access levels: Administrator: Unrestricted access, able to change all user information and modify login settings. Access to all analysis functions. Advanced User: Able to set a default data file path, configuration settings, language, capillary array format, and display mode. Access to all analysis functions. User: The most restricted level with access to analysis functions but none of the advanced features the Advanced Users and Administrators have access to.
Select one of the three eSignature levels: Not Allow: Specified user is not allowed to apply or remove an eSignature. eSign & un-Sign: Specified user is able to apply and remove an eSignature. eSign Only: Specified user may only apply an eSignature.
If selected, the account is active.
Email will be sent upon termination of ProSize due to a failed user login (I.e. a failed login attempt).

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Use eSignature

Once a signature is applied to a data file, the further analysis of the data file is disabled.
To eSign a data file
You must have the appropriate eSignature level (eSign & un-Sign, or eSign only) to access the eSign menu command (for details, refer to Table 59).
1 Go to eSignature > eSign.

Figure 106 eSignature drop-down menu
The ProSize Data Analysis Software e-Sign Logon window opens (Figure 107).

Figure 107 e-Sign Logon window
2 Enter your user credentials (UserID and password).

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Use eSignature

3 Define a Meaning. From the drop-down list, select Author, Review, or Approve. 4 Under Description, enter relevant notes or comments (optional).
To un-Sign a data file
An eSigned data file (.db3 file) can be un-signed again, allowing further analysis. You must have the appropriate eSignature level (eSign & un-Sign) to access
the un-Sign menu comand (for details, refer to Table 59). 1 Go to eSignature > un-Sign.

Figure 108 eSignature drop-down menu
The ProSize Data Analysis Software e-Sign Logon window opens (Figure 109).

Figure 109 e-Sign Logon window
2 Provide all necessary information. Enter your user credentials (UserID and password), a meaning, and a description (optional).

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Use eSignature

To View eSignature Records
The eSignature Records window provides information of all eSignature events associated with the data file. 1 From the menu, select eSignature > eSignature Records.

Figure 110 eSignature drop-down menu
The eSignature Records window opens (Figure 111). A description of the fields is shown in Table 60.

Figure 111 eSignature records for a data file

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Table 60 Fields shown in the eSignature records window

Field eSignature UserID
Description
Date

Description
Displays if it is an eSign or an un-Sign event.
Displays the userID of the user who applied or removed an eSignature.
Displays any notes the user made concerning the application or removal of an eSignature.
Displays the Date and Time of all events down to the second.

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ProSize Enhanced Security Features

View Event Report

The Event Report window tracks changes made by all registered users in ProSize for the entire history of ProSize enhanced security enabled software (unless this file has been archived).
1 From the menu, select Administration > Event Report.

Figure 112 Administration drop-down menu
The Event Report window opens (Figure 113). The report table is described in the Table 61. Menu items shown in the window are described in Table 62.

Figure 113 Event Report window

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View Event Report

Table 61 Fields shown in the Event Report window

Field UserID
Computer Name Event Time Operation
File Path
Description

Description
Displays the UserID of the user who applied or removed an eSignature.
Name of the computer the event occurred on.
Date and Time of all events, recorded to the second.
The type of event performed by the user, for example, Logon Action, File Open, etc.
Displays the file paths relevant to the event performed by the user.
Relevant description about the event performed by the user.

Table 62 Menu items of the Event Report window

Menu item Archive
Export to CSV PDF Close Range
Export Data to Clipboard Export Data to Excel

Description
Allows the user to save entries as an archive or to open archived entries (this transfers entries of the current database to a new, archived database).
Exports Event Report entries to a .csv file.
Generates a PDF report of all Event Report entries.
Closes Event Report window.
User selects a period of time to view entries: · Last 24 Hours · Last Week · Last Month · Last Year · Beginning of the Year · All
Right-click in the window to open the context menu. Copies Event Report entries to the clipboard.
Right-click in the window to open the context menu. Exports Event Report entries to Microsoft Excel.

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View Data Event Log

The Data Event Log window tracks all changes made to a .db3 file (i.e. a specific data file) reporting User ID, Computer Name, Event Time, Operation and Description.
1 Open a .db3 file.
2 From the menu, select Administration > Data Event Log.

Figure 114 Access to Data Event Log
The Data Event Log window opens (Figure 115). The menu items shown in the window are described in Table 63.

Figure 115 Data Event Log window

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View Data Event Log

Table 63 Menu items of the Data Event Log window

Menu item Export to CSV PDF Close Range
Export Data to Clipboard Export Data to Excel

Description
Exports Data Event Log entries to a .csv file.
Generates a PDF report of all Data Event Log entries.
Closes Data Event Log window.
User selects a period of time to view entries: · Last 24 Hours · Last Week · Last Month · Last Year · Beginning of the Year · All
Right-click in the window to open the context menu. Copies Data Event Log entries to the clipboard.
Right-click in the window to open the context menu. Exports Data Event Log entries to Microsoft Excel.

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In This Book
This manual contains information about the ProSize data analysis software. The manual describes the following: · System overview, · Requirements and installation instructions, · Software main screen and menus, · Data configuration, · Comparing samples, · Data export, · Report generation, · TroubleShooting, · Batch processing, · Enhanced software features.
www.agilent.com © Agilent Technologies, Inc. 2020
Published in Germany 02/2020 Document No: D0002111 Rev. A

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