sizing analysis module peak scanner software · a.0 05 february 2018 new document. material...

For Research Use Only. Not for use in diagnostic procedures.

Sizing Analysis Module Peak Scanner™

SoftwareUSER GUIDE

Publication Number MAN0017576

Revision A.0

Manufacturer: Life Technologies Corporation | 200 Oyster Point Blvd | South San Francisco, CA 94080 | USA

The information in this guide is subject to change without notice.

DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL,INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOURUSE OF IT.

Revision history: Pub. No. MAN0017576

Revision Date DescriptionA.0 05 February 2018 New document. Material represents the Help content in the Sizing

Analysis Module Peak Scanner™ Software.

Important Licensing Information: This product may be covered by one or more Limited Use Label Licenses. By use of this product, you accept theterms and conditions of all applicable Limited Use Label Licenses.TRADEMARKS: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.

©2018 Thermo Fisher Scientific Inc. All rights reserved.

Contents

■ CHAPTER 1 About the Sizing Analysis Module PeakScanner™ Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Compatible instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

■ CHAPTER 2 Workflows . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Sizing Analysis Module Peak Scanner™ Software Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Data analysis workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

■ CHAPTER 3 Create a project and import samples . . . . . . . . . . . . . . . . . . . . 9

Create a demo project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Create your own project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Other ways to create projects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10Create from the DataConnect window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11Create from My Apps in the Home window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Share an object . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

■ CHAPTER 4 Review and analyze samples in the Setup screen . . . . . 13

Review samples after first import . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Analyze samples using default analysis settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Reanalyze samples using custom settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Add/remove samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Search tables and lists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Apply a different size standard to the project or a sample . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Edit a sample name and type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Customize the columns in the sample or sizing table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Sizing Analysis Module Peak Scanner™ Software User Guide 3

■ CHAPTER 5 Examine low quality samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

View data collection settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Examine EPT and raw data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Modify the default analysis settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Adjust Quality Flag ranges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Apply a different size standard to the project or a sample . . . . . . . . . . . . . . . . . . . . . . . . . . . 18Select a single size standard for a project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Select a size standard for individual samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Create a new size standard definition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

■ CHAPTER 6 Review the plot and peak information in theResults screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Review results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Review and edit peaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21Sort and filter peaks in the sizing table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21Edit peaks in the sizing table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21Edit peaks using Peak Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Zoom on the electropherogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Modify size matches using the Size Match Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Customize plot data and views . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26Select dye data to display . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26View dyes in combined or separate plots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27View raw and/or analyzed data in the plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28Customize the Plot View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29Set plot Zooming and Scaling Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31Customize labels on the plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32Show/Hide the Sizing Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

■ CHAPTER 7 Export size standard, sample information,and results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Export size standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Export the sample table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Export Sizing Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Export Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Export Summary Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

■ APPENDIX A Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

Contents

4 Sizing Analysis Module Peak Scanner™ Software User Guide

■ APPENDIX B Software screen descriptions . . . . . . . . . . . . . . . . . . . . . . . . . . 38

Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

Parts of the Setup screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

Sample table fields . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

Parts of the Results screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

Parts of the electropherogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

Sizing table fields . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

■ APPENDIX C Reference information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

Size Standard selected by the software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

Analysis status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

Analysis settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

Peak detection overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48Baseline Window size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49Smoothing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51Polynomial Degree and Peak Window Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51Slope Thresholds for Peak Start/End parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

Sizing overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55Size matching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55Size-calling curve generation and size calling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56Sizing Quality (SQ) status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

■ Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Related documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Contents


About the Sizing Analysis ModulePeak Scanner™ Software

Sizing Analysis Module Peak Scanner™ Software is a DNA sizing module available onThermo Fisher Cloud.

Use this software to perform DNA fragment analysis, separate a mixture of DNAfragments according to their sizes, provide a profile of the separation, and preciselycalculate the sizes of the fragments.

The software allows you to view, edit, analyze, print, and export fragment analysisdata generated using Applied Biosystems™ genetic analyzers.

Compatible instruments

Capillary Electrophoresis System Supported softwareversion(s)

Fileextension

Applied Biosystems™ SeqStudio™ GeneticAnalyzer (3200)

v1.0 or later FSA

Applied Biosystems™ 3500/3500xL GeneticAnalyzer

Applied Biosystems™ 3130/3130xl GeneticAnalyzer

Applied Biosystems™ 3730/3730xl DNAAnalyzer

1


Workflows

Sizing Analysis Module Peak Scanner™ Software Workflow

Chapter 3, “Create a project and import samples“

▼

Chapter 4, “Review and analyze samples in the Setup screen“

▼

Chapter 5, “Examine low quality samples“

▼

Chapter 6, “Review the plot and peak information in the Resultsscreen“

▼

Chapter 7, “Export size standard, sample information, andresults“

2


Data analysis workflow

The workflow shown below is an example approach for data analysis.

Chapter 4, “Review and analyze samples in the Setup screen“

“Review samples after first import“ on page 13

(If needed) “Add/remove samples“ on page 15

“Analyze samples using default analysis settings“ on page 14

▼

(If needed)

“View data collection settings“ on page 17

“Examine EPT and raw data“ on page 17

“Modify the default analysis settings“ on page 17

“Apply a different size standard to the project or a sample“ onpage 15

“Reanalyze samples using custom settings“ on page 15

▼

Chapter 6, “Review the plot and peak information in theResults screen“

“Customize plot data and views“ on page 26

“Review and edit peaks“ on page 21

(If needed) “Modify size matches using the Size MatchEditor“ on page 24

▼

Chapter 7, “Export size standard, sample information, andresults“

Chapter 2 WorkflowsData analysis workflow2


Create a project and import samples

Create a demo project

IMPORTANT! We recommend that new users create a Demo project to quickly learnhow to use the module.

1. In the Thermo Fisher Cloud Dashboard, go to the Home page, click Tutorials,then click Create a Demo project.

2. At the bottom of the Tutorials screen, click , then click OK.

3. Click Recent projects and files, then click PeakScanner_DEMO_Project.

Note: It may take several minutes for the Demo project to appear in the Recentprojects and files screen.

4. (If needed) Review the Getting Started pop-up window, then click OK.

5. Proceed to Chapter 4, “Review and analyze samples in the Setup screen“.

3


Create your own project

You must have saved FSA files from an instrument run before you can create a projectin the module. The FSA files can be analyzed or unanalyzed.

1. In the Thermo Fisher Cloud Dashboard, click Create project.

2. Enter a project name, select a save location, then click Create.

3. (If needed) Review the Getting Started pop-up window, then click OK.

4. In the Manage Data Files window, select an option to import FSA files:

1. Import From My Computer—Imports FSA files from your computer.2. Import from Thermo Fisher Cloud—Imports FSA files from the Thermo

Fisher Cloud. The Cloud contains Demo files from Thermo Fisher Scientificand any files that you have previously uploaded in Data Manager.

3. Import From Dropbox—Imports FSA files from Dropbox.

5. The imported files are displayed in the table. In the navigation bar, click theSizing Analysis Module Peak Scanner™ Software icon

Common Callouts and Arrows

1. Copy-paste a callout or arrow to use in this SVG.

Note: If you need more advanced callouts or arrows use the TechComm_Inkscape_Callout&Arrow_Libary.

3. Delete this text, this rectangle, and unused callouts, arrows, or other SVG elements before adding this SVG to the repository.

2. Edit number and/or line-length, as needed.

1 1

1

1 .

6. In the Import Project Settings dialog box, select an option:• Yes—Imports size standard, analysis settings, table settings and plot settings

from an existing project. Use this option to import a custom size standardfrom a previous project.No—Uses the default system settings.

Other ways to create projects

You must have saved FSA files from an instrument run before you can create a projectin the module. The FSA files can be analyzed or unanalyzed.

Chapter 3 Create a project and import samplesCreate your own project3


1. In the Thermo Fisher Cloud Dashboard, click in the navigation bar to openthe DataConnect screen.

2. Browse to and select the trace files of interest, then click






1 1

1

1

.

3. In the Create Project dialog box, enter a project name, select a save location, thenclick Create.

4. Proceed with step 3 in “Create your own project“ on page 10.You can import additional trace files, or skip “Create your own project“ onpage 10.






1 1

1

1

1

2

3

1 Files icon—Click to open the DataConnect window2 Project folders and trace files3 Create project link—Click to open the Create Project dialog box

1. In the Thermo Fisher Cloud Dashboard, click in the navigation bar to openthe Home screen.

2. In the My Apps pane, click Common Callouts and Arrows





1 1

1

1 .

3. In the Open Project dialog box, select Create New Project, enter a project name,select a save location, then click OK.

4. Proceed with step 3 in “Create your own project“ on page 10.






1 1

1

1

1

2

1 Home icon—Click to open the Home window2 Module icon in the My Apps pane—Click to open the Peak Scanner™ module

Create from theDataConnectwindow

Create from MyApps in the Homewindow

Chapter 3 Create a project and import samplesOther ways to create projects 3


Share an object

• To Share a single file, folder, or project with another Thermo Fisher Cloud user:1. Click (Home) if necessary to return to the platform, then click (Files).

2. In Files, select the object to be shared, then click Share with people.

3. Enter the email address of the user(s) with whom you want to share theselected object, separating each email address by a comma. Click Add.

4. Click Confirm.The user(s) will receive a notification and email that you have shared anobject with them and the shared object will appear in their Home Folder.

Chapter 3 Create a project and import samplesShare an object3


Review and analyze samples in theSetup screen

Review samples after first import

When you import samples, the Setup screen is displayed (see “Parts of the Setupscreen“ on page 39) and shows data collection results with an analysis status ofUnanalyzed or Reanalyze.

1. Review information that is imported for the project.• The total number of samples in the project is color-coded red to indicate that

samples have not been analyzed.• A size standard is selected for the project (see “Size Standard selected by the

software“ on page 45).• Analysis status for the project (see “Analysis status“ on page 46).• The analysis and sizing ranges from the data collection analysis settings are

shown.• Size Quality and Offscale overviews are shown. Color-coding is based on

the imported data collection analysis settings.

Note: The Quality Flags Size Quality and Offscale bar shows a gray portionfor any imported samples that are unsized.

4


2. Review the following information for the samples in the sample table.

Parameter Description

Status • for SeqStudio™ (3200) and 3500 FSA files

• for files analyzed using the current analysissettings. Analyze samples after the first import toapply current settings.

• for 31xx FSA files[1]

SQ • , , or for SeqStudio™ and 3500 FSA files, basedon the Quality Flags setting from the data collectionanalysis settings.

• Blank for 31xx FSA files[1]

OS • or for SeqStudio™ and 3500 FSA files, based onthe Quality Flags setting from the data collectionanalysis settings.

• Blank for 31xx FSA files[1]

Size Standard See “Size Standard selected by the software“ on page 45

[1] If 31xx FSA files were previously analyzed using the Peak Scanner™ desktop software, the imported Status, SQ and OS parameters will be the same as those for SeqStudio™ (3200) and 3500 FSA files.

Note: This table describes the parameter fields following initial import. Theseparameters will update as the user applies modified analysis settings to theproject.

3. As needed:• Click the Sample Type column header to sort and view control samples as

needed.• “Customize the columns in the sample or sizing table“ on page 16.• “Search tables and lists“ on page 15.

Analyze samples using default analysis settings

1. In the Setup screen, select one or more samples to analyze.

2. Click Analyze.

3. Review results in the Setup screen, then:

• If the sample quality is , proceed to Chapter 7, “Export size standard,sample information, and results“.

• If the sample quality is or , or analysis fails, proceed to:– “Reanalyze samples using custom settings“ on page 15– Chapter 6, “Review the plot and peak information in the Results screen“

Chapter 4 Review and analyze samples in the Setup screenAnalyze samples using default analysis settings4


Reanalyze samples using custom settings

1. Perform any of the following tasks as needed:• “Modify the default analysis settings“ on page 17• “Edit a sample name and type“ on page 16• “Apply a different size standard to the project or a sample“ on page 15• “Modify size matches using the Size Match Editor“ on page 24• “Adjust Quality Flag ranges“ on page 18

2. Click Reanalyze.

Add/remove samples

In the Setup screen, select the appropriate option:.

Option Description

Add more files 1. Click Actions

2. Click Add More Files

3. Select the appropriate import option.

Remove files 1. Select the samples to remove in the sample table.

2. Click Actions.

3. Click Remove Files.

Search tables and lists

Click in the upper-right corner of a table or list to search the contents of that tableor list.

Click Clear All to clear previous search results.

Apply a different size standard to the project or a sample

You can do any of the following:• “Select a single size standard for a project“ on page 19• “Select a size standard for individual samples“ on page 19• “Create a new size standard definition“ on page 20

Chapter 4 Review and analyze samples in the Setup screenReanalyze samples using custom settings 4


Edit a sample name and type

Edit a sample name and type in the sample table.• Edit the sample name—Click the sample name.• Edit the sample type—Click the sample type, then select an option from the

dropdown list.– Sample– Positive Control– Allelic Ladder– Primer Focus– Negative Control

Note: The software designates Positive Control, Allelic Ladder, Primer Focus,Negative Control sample types as controls, and does not apply additionalprocessing to these samples. The user has the option to arrange control plots ontop during review of sample plots in the Results screen. See Chapter 6, “Reviewthe plot and peak information in the Results screen“.

Customize the columns in the sample or sizing table

Customize the columns displayed in the sample table.• Sort the columns in the table—Double-click a column header.• Edit the order of columns in the table—Click a header, then drag and drop in

the desired order.• Show/Hide columns in the table—Click at the top right corner of the table,

click Show/Hide Columns, then select the columns to show or hide.Click Restore to default to undo previous Show/Hide selections.

Chapter 4 Review and analyze samples in the Setup screenEdit a sample name and type4


Examine low quality samples

View data collection settings

1. In the Setup screen, open the Sample Details window using one of the followingmethods:

• Double-click, or right-click on the sample file.• Click Actions, then click View Sample Details.

2. Click Information to review the following data collection settings for a sample.

3. Click Close.

Examine EPT and raw data

Review the EPT (ElectroPherogram Telemetry) plot to identify instrumentperformance issues that can affect size quality.

Review the raw data to evaluate anomalies, the causes of poor size-calling, and todetermine the start and stop points for analysis. The start point for data analysisoccurs after the primer peak and before the first sizing peak. The stop point foranalysis occurs after the last sizing peak.

1. Open the sample details window using one of the following methods:

• Double-click, or right-click on the sample file.• Click Actions, then click View Sample Details.

2. Click EPT Data to view the EPT plot.

3. Click Raw Data to view the raw data plot.

4. Click Close.

Modify the default analysis settings

1. In the Setup screen, click Settings.

2. Modify the settings as needed (see “Analysis settings“ on page 46 ).

3. Click Reanalyze all files with settings.

4. Click Done.

5


To restore project analysis settings to the default settings, click Settings4Restore todefault.

Adjust Quality Flag ranges

1. In the Setup screen, click Settings.

2. Click Quality Flags.

3. Adjust the low, medium and high Quality Flag ranges as appropriate. See “SizingQuality (SQ) status“ on page 62.






1 1

1

1

1

Figure 1 Quality Flag ranges

1 Drag to adjust to quality flag ranges.

Apply a different size standard to the project or a sample

You can do any of the following:• “Select a single size standard for a project“ on page 19• “Select a size standard for individual samples“ on page 19• “Create a new size standard definition“ on page 20

Chapter 5 Examine low quality samplesAdjust Quality Flag ranges5


See “Create a new size standard definition“ on page 20 to create a custom sizestandard for your project.

1. In the Setup screen, click Size Standard.

2. Select an option to apply a size standard:

Option Action

Factory default SizeStandard

1. Select a factory default Size Standard from thedropdown list.

Import Size Standard(.xml)—Import a sizestandard file from yourcomputer.

1. Scroll to the bottom of the dropdown list, thenselect Import Size Standard (.xml).

2. Browse to and select a size standard, then clickOpen.

Note: Imported size standards only appear in theproject in which they were imported.

3. Click Reanalyze all files with this size standard.

4. Click Apply.

The software allows you to apply different size standards to, then analyze individualsamples in a project. When you analyze the project, only the size standard that isselected for the project is used, and size standards that are selected for individualsamples are overwritten.

1. From the Setup screen, select the samples to which you want to apply a specificsize standard.

2. Click Size Standard, then select the appropriate option to apply a size standard.See “Apply a different size standard to the project or a sample“ on page 15.

3. Deselect Reanalyze all files with this size standard.

4. Click Apply.

Note: The new size standard is not displayed in the Size Standard column of thesample list until the samples are analyzed.

5. In the Setup screen, click Reanalyze.

Select a singlesize standard for aproject

Select a sizestandard forindividual samples

Chapter 5 Examine low quality samplesApply a different size standard to the project or a sample 5


Customized size standards only appear in the project in which they were created. See “Export size standard“ on page 35 and “Create your own project“ on page 10 forinformation about exporting and importing a previously defined custom sizestandard when creating your project.


2. Create a new size standard using one of the following options:

Option Action

Customize a factory defaultSize Standard

1. Select a Size Standard from the dropdown list.

2. Select Clone size standard to make changes.

3. Edit the numeric values for the size standarddefinition. Separate size peak values withcommas.

Manually create acustomized Size Standard

1. Select Enter new Size Standard from thedropdown list.

2. Enter the following information:

• Name

• Color

• Description

• Numeric values for the size standarddefinition. Separate size peak values withcommas.

3. (Optional) Select Reanalyze all files with this size standard.

4. Click Apply.

Create a new sizestandarddefinition

Chapter 5 Examine low quality samplesApply a different size standard to the project or a sample5


Review the plot and peakinformation in the Results screen

Review results

1. Click the Results tab at the top of the screen.

2. Perform any of the following tasks as needed:• Chapter 5, “Examine low quality samples“• “Customize labels on the plot“ on page 32• “Zoom on the electropherogram“ on page 23• “Edit peaks using Peak Editor“ on page 22• “Edit peaks in the sizing table“ on page 21• “Customize the columns in the sample or sizing table“ on page 16• “Sort and filter peaks in the sizing table“ on page 21• Click Controls on top.

Review and edit peaks

Editing peaks allows you to redefine, add, delete, merge, and split peaks, which maybe necessary with suboptimal peak resolution.

Task Action

Sort Click a dye color in the Peak overview panel to sort the sizing tableby dye color.

Filter1. Click in the upper‑right corner of the sizing table.

2. Click Filter Peaks, the select the appropriate option from thedropdown list.

3. Click Apply.

1. Select the peak or peaks to edit using one of the following methods:• In the electropherogram, place the pointer over a peak, then click to select it.

Repeat to select additional peaks.• In the Sizing table, click a row.

6

Sort and filterpeaks in the sizingtable

Edit peaks in thesizing table


2. Select the appropriate action:

Option Description

Deletepeaks

Allows you to remove extraneous peaks that may result fromdye‑labeled primers, contaminants, or nonspecific PCR amplification.Information that is associated with a particular peak is deleted from thesizing table, but the peak is still displayed in the plot view.

Mergepeaks

Allows two individual peaks to be treated as a single peak. The new"bin" is automatically determined. This function is typically used foroffscale peaks, where overloading of sample results in peak splitting.

Split peak Allows you to separate a single peak into two peaks. The new bins areautomatically determined for both peaks by the software. This option iscommonly used when two peaks are not sufficiently resolved.

1. In the Results screen, select a sample to review.

2. Click Actions.

3. Click Turn on Peak Editor.

4. In the Peak Editor window, select the dye color of the peaks you want to edit.

5. In the electropherogram, select individual peak or peaks to edit using one of thefollowing methods:

• Place the pointer over a peak, then click to select it. Repeat to selectadditional peaks.

• Click and drag the pointer to select a range of peaks.

6. Select then apply appropriate action:

Option Description

Delete peaks Allows you to remove extraneous peaks that may results fromdye‑labeled primers, contaminants, or nonspecific PCR amplification.Information that is associated with a particular peak is deleted fromthe sizing table, but the peak is still displayed in the plot view.

Add missingpeaks

Allows you to add and enter into the sizing table peak informationthat was not included in the original analysis.

Merge peaks Allows two individual peaks to be treated as a single peak. The new"bin" is automatically determined. This function is typically used foroffscale peaks, where overloading of sample results in peak splitting.

Split peak Allows you to separate a single peak into two peaks. The new bins areautomatically determined for both peaks by the software. This optionis commonly used when two peaks are not sufficiently resolved.

Edit peaks usingPeak Editor

Chapter 6 Review the plot and peak information in the Results screenReview and edit peaks6


Zoom on the electropherogram

To set zoom and scale defaults, see “Set plot Zooming and Scaling Options“ onpage 31.

Action Icon Description

Turn panning on/off

Pan over the plot. Click, then drag to move overthe plot.

Panning cannot be selected simultaneously withrectangular selection.

Turn rectangularselection on/off

Zoom in on a rectangular region of the plot. Click,then drag to select a rectangular region.

Rectangular selection cannot be selectedsimultaneously with panning.

Zoom in x-axisClick to zoom in on the x-axis.

Alternatively, zoom in on the x-axis by clickingdirectly on the value in the axis.

Zoom out x-axisClick to zoom out on the x-axis.

Double-click on the x-axis to reset x-axis zoom.

Reset zoom to fit Reset the plot view, panning and rectangularselection options to default settings.

Turn synchronized zoomon

Synchronize zooming when viewing multiple plots.

Icon indicates synchronized zoom is currentlyturned off.

Turn synchronized zoomoff

Turn off synchronize zooming when viewingmultiple plots.

Icon indicates synchronized zoom is currentlyturned on.

Zoom in y-axis None Zoom in on the y-axis by clicking directly on thevalue in the axis.

Zoom out y-axis None Double-click on the y-axis to reset y-axis zoom.

Chapter 6 Review the plot and peak information in the Results screenZoom on the electropherogram 6


Modify size matches using the Size Match Editor

If size standard peaks are not correctly detected by the software, you can modify thesize matches, then apply the modified size standard definition to one or all samples inthe project.

1. In the Results screen, select a sample from the sample list.

2. Click Actions4Size Match Editor to view the peak assignments for the sizestandard peaks in the selected sample.

3. Examine all size standard peaks to ensure that all peaks are present, peaks arelabeled correctly, and the sizes match the fragment sizes in the size standarddefinition file used for the sample.

Chapter 6 Review the plot and peak information in the Results screenModify size matches using the Size Match Editor6


4. Change size values or delete peaks as needed, then auto-adjust size matches. Thisfunction is useful if a peak is incorrectly labeled by the software.

Task Action

Change a size value 1. Click the size value in the list or click a peak, then selecta different value.

2. Select the peak, then click (auto adjust).

For example, changing the 110 peak to 100, then clicking moves the 110 size label to the current 100 peak, moves the120 size label to the current 110 peak, and so on.

Delete a peak 1. Click a peak, then click Delete.

2. Select the same peak, then click

For example, deleting the 110 peak, then clicking movesthe 110 size label to the current 120 peak, moves the 120 sizelabel to the current 130 peak, and so on.

5. Click to recalculate the SQ using the new sizes.

6. Click Override SQ.The SQI (Sizing Quality Invalid) indicator will be displayed

• At the top right of the Results screen for any sample that is analyzed withthe size standard—Displays SQI

• In the Setup screen sample table —Displays in the SQI field.

7. (Optional) To create a new size standard definition for the project using themodified size matches, click Reanalyze all files with this size standard.

8. Click Done.

See “Sizing Quality (SQ) status“ on page 62.

Chapter 6 Review the plot and peak information in the Results screenModify size matches using the Size Match Editor 6


Customize plot data and views

1. In the Results screen, click Plot Settings to display the plot settings selectorscreen.

2. Review the default dye display settingsand change as needed.

3. To display only selected dyes:

• Click the square of the dye color to hide.• Click Deselect all, then click the square of the dye color to display.

Select dye data todisplay






1 1

1

1

Chapter 6 Review the plot and peak information in the Results screenCustomize plot data and views6



2. Select an option from the Plot Display dropdown list:

Option Description

Combine Dyes(Default)

All dye data for the selected sample or samples is presentedin one plot.

Separate Dyes Data for each dye color for the selected sample or samples ispresented in a separate plot.

3. Close the dialog window.

View dyes incombined orseparate plots

Chapter 6 Review the plot and peak information in the Results screenCustomize plot data and views 6



2. In Show Data In, select one of the following options:• Analyzed (Default)

Note: Reset plot Zoom and Scaling if you change the view from Scan toBasepairs. See“Set plot Zooming and Scaling Options“ on page 31.

• Raw• Analyzed + Raw

3. Close the dialog window.

View raw and/oranalyzed data inthe plot




2. In the Plot View section, click Options.

Customize thePlot View



3. Customize the plot view using the following options:

Setting Description

Plot Layout OptionsChange plot layout options to viewcontrols, samples or separate dye plotswithin the electropherogram window.

Plot Header Options

Select up to 3 parameters to display inthe plot heading:

• Sample Name (Default)• File Name (Default)• Sample type

• R2 Value

• Sizing Quality

• Sizing Quality Invalidated (SQI) Flag

Plot Display

Display the following:

• Sizing Curve

• Peak Position

• Offscale Indicator (Default)

4. Click Back or close the dialog window.




2. In the Zooming + Scaling Options section, click Options.

3. Customize zooming and scaling using the following options:

Setting Description

Zoom Plots Select the X‑ and y‑axis displays start andstop point.Default settings are from 0–100,000.

Axis Settings Scale the x‑axis and y‑axis. Possibleparameters include:

• Individually (Default)• To Maximum Value

• To Value

Scale dyes Select the scale for individual dyes.

4. Click Back.

Set plot Zoomingand ScalingOptions



Labels on the plot are turned on by default.


2. In the Label section, click Options.

Customize labelson the plot



3. Customize the labels on the plot using the following options:

Setting Description

Show Labels For Select the type of peaks to label:

• Selected peak (Default)• All Peaks

• Specified Range

Label Display Select parameters to display in the plotheading. Possible parameters include:

• Area (Default)• Height (Default)• Size (Default)• Data Point (Default)• Comments

(Optional) Select abbreviated labelprefixes.

Text size Select the text size for the labels.

Visual Style Select one of the following:

• Dye Color—(Default) Label outline isthe same color as the dye peak.

• Black and White— Label outline isblack.

4. Click Back or close the dialog window.



The Sizing table is displayed by default in the Results screen.


2. Toggle the Sizing Table View on or off.

Show/Hide theSizing Table



Export size standard, sampleinformation, and results

Export size standard


2. Select a size standard from the dropdown list, then click Export Current SizeStandard (.xml).

3. In the Export To pane, click one of the following:

• My Computer• Thermo Fisher Cloud

4. Click Export.

Export the sample table

1. In the Setup screen, select samples in the sample table to export.

2. Select Actions4Export Sample Table.



4. Click Export.

Export Sizing Table

1. In the Results screen, click Actions44Export Sizing Table to export the sizingtable to a tab-delimited CSV file.



3. Click Export.

7


Export Plot

1. In the Results screen, click Actions44Export Plot to export theelectropherogram to a PNG or JPEG image.



3. Click Export.

Export Summary Report

1. In the Results screen, select samples in the Sample List .

2. Select Actions4Export Summary Report

3. Select the contents to include in the Summary Report.



5. Click Export.

Chapter 7 Export size standard, sample information, and resultsExport Plot7


Troubleshooting

Troubleshooting

Observation Possible cause Action

Project status is , but thesample SQ column containsa result

Samples have beenimported, but have not beenanalyzed. The SQ result isimported from the FSA fileand is determined by thedata collection settings.

Analyze samples.

SQ column is blank The imported FSA file doesnot contain an SQ value.

Analyze samples.

SQ is or The software is unable toproperly assign sizestandard peaks.

See “Modify size matchesusing the Size MatchEditor“ on page 24.

A


Software screen descriptions

Setup

The Sample Setup screen displays a list of all sample files in the project and providesQC flag, run, and data collection information for each file.

How to ... Learn more about...

“Analyze samples using default analysis settings“ onpage 14 “Compatible instruments“ on page 6

“Reanalyze samples using custom settings“ onpage 15 “Data analysis workflow“ on page 8

Chapter 5, “Examine low quality samples“ “Parts of the Setup screen“ on page 39

“View data collection settings“ on page 17 “Sample table fields“ on page 40

“Modify the default analysis settings“ on page 17 “Analysis status“ on page 46

“Customize the columns in the sample or sizingtable“ on page 16 “Size Standard selected by the software“ on page 45

“Select a single size standard for a project“ on page 19 “Sizing Quality (SQ) status“ on page 62

“Select a size standard for individual samples“ onpage 19

“Create a new size standard definition“ on page 20

“Edit a sample name and type“ on page 16

“Examine EPT and raw data“ on page 17

“Add/remove samples“ on page 15

“Export the sample table“ on page 35

B


Parts of the Setup screen

31

6

2 54

9 10 118

7

Figure 2 Setup screen1 Files—Total number of samples in the project. Color‑coded to indicate analysis status of the project.2 Size Standard—Size standard that is selected for the project.3 Status (project)— project analysis status indicates that all samples files have been analyzed in the Sizing Analysis

Module Peak Scanner™ Software using the current settings. , , indicate that all samples are not analyzed usingthe current settings and lists the number of files that are unanalyzed, analyzed, or have been changed (reanalyze).

4 Analysis Settings—Peak detection, sizing, and quality settings that are specified for all samples in the project.5 Quality Flags—Bar graph that shows the overall status of Size Quality and Offscale flags.6 Sample table menu—Search samples and select the information to display in the sample table.7 Sample table—Lists all samples in the project.8 Status (sample)— indicates that a sample file has been analyzed in the Sizing Analysis Module Peak Scanner™

Software using the current settings. Updating the size standard or analysis settings changes the sample analysis statusto Reanalyze. Unanalyzed is displayed after first import for sample files that do not contain information from thedata collection software.

9 SQ—Sizing Quality indicator. , , or as determined by the ranges set in analysis settings. Place the cursor overthe indicator to display the Sizing Quality value. SQ field is blank after first import for FSA files that do not contain an SQvalue.

10 SQI—Sizing Quality Invalid indicator. Displays if the size standard for the sample has been edited and overridden.11 OS—Offscale indicator. Displays if no peaks in the sample are offscale.

Appendix B Software screen descriptionsParts of the Setup screen B


Sample table fields

Column name Description

Status

Indicates the analysis status of individual samples. Possible statesinclude:

• Unanalyzed — The sample has not been analyzed.

• Analyzed — The sample is analyzed using the currentanalysis settings.

• Reanalyze — The sample needs to be reanalyzed using thecurrent analysis settings.

Size Quality (SQ) pass, check, or fail. The sizing quality is defined in theQuality Flags tab in analysis settings and is user editable. To viewthe numerical value of the SQ flag, hover the pointer over the flagicon.

See “Sizing Quality (SQ) status“ on page 62 and “Modify thedefault analysis settings“ on page 17.

Sample Name The name of the sample is derived from the sample name tag usedin the sample file; user editable.

Sample Type

Indicates the sample type:

• Sample

• Positive Control

• Allelic ladder

• Primer Focus

• Negative Control

This field is user editable.

Instrument Model Indicates the instrument model on which the experimental runwas performed.

Plate ID Indicates the plate ID associated with sample.

Well Indicates the well number of the sample.

UD1 A user-defined field where you can make notes or commentsabout the samples.

Size Standard Indicates the size standard applied to the current analysissettings.

Appendix B Software screen descriptionsSample table fieldsB


Column name Description

Sizing QualityInvalidated (SQI)

indicates that the Size Standard peaks definition for thesample was manually edited by the user and, therefore, the SQautomatically generated by the software is invalid.

Offscale

Indicates if the sample contains offscale peaks. If a green checkmark is displayed, the fluorescence signal was greater than themaximum readable signal on the Genetic Analyzer. To correctoffscale data, adjust the amounts of labeled fragments. Thesoftware cannot correct for offscale data. Although offscalesamples can still be analyzed, their peak sizes may not beaccurate.

Results

How to ... Learn more about...

Chapter 5, “Examine low quality samples“ “Data analysis workflow“ on page 8

“View raw and/or analyzed data in the plot“ on page 28 “Parts of the Results screen“ on page 42

“Edit peaks using Peak Editor“ on page 22 “Parts of the electropherogram“ on page 43

“Edit peaks in the sizing table“ on page 21 “Sizing table fields“ on page 44

“Modify size matches using the Size Match Editor“ onpage 24 “Peak detection overview“ on page 48

“Zoom on the electropherogram“ on page 23

“Set plot Zooming and Scaling Options“ on page 31

“Customize labels on the plot“ on page 32

“Customize plot data and views“ on page 26

“Customize the columns in the sample or sizingtable“ on page 16

Chapter 7, “Export size standard, sample information,and results“

Appendix B Software screen descriptionsResults B


Parts of the Results screen

The Results screen displays a list of all sample files in the project, theelectropherogram plot and the sizing table.






1 1

1

1

5

2 3

68

4

9

7

1

Figure 3 Results screen1 Sample list2 Sample search and filter menu–Search samples and select 1 or more samples to display in the plot and sizing table.3 Zoom tools4 Electropherogram5 Edit peaks menu–Delete, add, or merge peaks selected from the sizing table.6 Peak search and filter menu– Search peaks and select the information to display in the sizing table.7 Sizing table8 Currently selected sample9 Peak overview–Displays the number of peaks for each dye color. Click a dye color to sort the sizing table by dye color.

Appendix B Software screen descriptionsParts of the Results screenB


Parts of the electropherogram






1 1

1

1

5

1

11

8 7 6

9

32

410

1 File name—File name of the sample that is displayed in the electropherogram.2 Zoom controls—Controls zooming options for the electropherogram.3 Sample name—Name of the sample that is displayed in the electropherogram.4 Peak Position—Black markers indicate the peak start, peak end, and peak apex. Turn on

peak position markers in Plot Settings.5 Sizing Curve—Used to determine the size of unknown fragments in an experimental

sample. Display the sizing curve in Plot Settings.6 Labels on Plot—Labels display area (A), height (H), size (S), and data point (D) information

for individual peaks. Customize labels on Plot in Plot Settings.Multiple peaks in the same region are indicated by circles under the x-axis. The numberedcircle indicates the total number of peaks in the region. Color circles indicate the numberof peaks of that dye color in the region. Zoom in on the x-axis to view individual peaklabels.

7 Resize the electropherogram window - vertical—Click, then drag to vertically to resize theelectropherogram window.

8 Scroll bar—Scroll back and forth to view the x-axis.9 Resize the electropherogram window - horizontal—Click, then drag to horizontally to

resize the electropherogram window.10 Y-axis—Click directly on the axis to zoom.11 Sample selection—Sample that is displayed in the electropherogram.

Appendix B Software screen descriptionsParts of the electropherogram B


Sizing table fields

Column Description

Sample File Name Indicates the sample file name.

Dye Color Indicates the peak dye color. Possible states are blue,green, yellow, red, and orange.

Dye, Sample Peak Assigns a name to each peak. The format is color, peaknumber.

Sample Name[1] Indicates the sample name.

Size Indicates the peak size based on base pairs.

Height Indicates the peak height.

Area (Data Point) Indicates the peak area based on scan number.

Area (Base Pairs) Indicates the peak area based on base pairs.

Data Point Indicates the peak data point or scan number.

Begin Point (Data Point) Indicates the begin point of the peak based on the scanposition.

End Point (Data Point) Indicates the end point of the peak based on the scanposition.

Begin Point (Base Pairs) Indicates the begin point of the peak based on base pairs.

End Point (Base Pairs) Indicates the end point of the peak based on based on basepairs.

Comments[1] Displays user comments.

[1] To display this column, see “Customize the columns in the sample or sizing table“ on page 16.

Appendix B Software screen descriptionsSizing table fieldsB


Reference information

Size Standard selected by the software

When samples are first imported, the software selects a size standard for the projectbased on the data imported in the FSA file.

Instrument Size Standard datain FSA file Size Standard selection

SeqStudio™ GeneticAnalyzer

Name and values

Factory default—If a matching sizestandard name and value is found, thesoftware selects it for the project.

Custom—If a matching size standard nameand value is not found, the software createsa custom size standard, then selects it forthe project.

3500 GeneticAnalyzer

3130 GeneticAnalyzer

Name[1]

Factory default—If a matching sizestandard name is found, the softwareselects it for the project.

GS500-250LIZ—If a matching size standardname is not found, the software selectsGS500-250LIZ (the first size standard in thefactory default list) for the project.

3730/3730xl DNAAnalyzer

[1] Size Standard names are not included in all FSA files

C


Analysis status

Icon Analysis status Description

Reanalyze

The sample has not been analyzed using the currentanalysis setting. Reanalyze to apply current settings.

Note: SeqStudio™ (3200) and 3500 FSA files display after being imported because these files include

the Size Standard, Size Quality, and peak informationfrom the data collection software analysis.

Unanalyzed

Individual sample—The sample has not beenanalyzed.

Note: 31xx and 3700xx FSA files display afterbeing imported because these files do not includeinformation from the data collection softwareanalysis.

Project—One or more of the samples in the projecthave not been analyzed using the current analysissettings.

Analyzed

Individual sample—The sample has been analyzedusing the current analysis settings.

Project—All of the samples in the project have beenanalyzed using the current analysis settings.

Analysis settings

Setting Description

Analysis Range • Full Range—(default) To analyze the entire scan region ascollected by the genetic analysis instrument, including theprimer peak.

• Specificed Range—To analyze only data points within aspecified range. Enter Start Point in data points after theprimer peak and before the first required size standard peak.Enter a Stop Point after the last required size standardfragment. Start and Stop points may vary from instrument toinstrument and platform to platform. View raw data todetermine the appropriate analysis range.Data points outside the specified analysis range are ignored.

Note: Ensure the Analysis Range contains all size standardfragments included in the Sizing Range.

Appendix C Reference informationAnalysis statusC


Setting Description

Sizing Range The size range (in base pairs) appropriate for the kit you areusing:

• Full Range for the software to analyze fragments of all sizesin the Analysis Range.

• Partial Range for the software to analyze only fragmentswithin a specified range. Enter a Start Size and a Stop Sizeappropriate for the size standard used.

Peak amplitude threshold The peak height threshold (RFU) for peak detection for each dyecolor. Peaks below the threshold are still displayed in theelectropherogram plots but are not detected or labeled.

Note: Use the same peak amplitude thresholds in secondaryanalysis software.

Quality Flags The pass/fail range for the Size Quality status.

See “Sizing Quality (SQ) status“ on page 62.

Linear Migration The range in basepairs for fragments that migrate linearly.

Baseline Window Specify a window to adjust the baseline signals of all detected dyecolors to the same level for an improved comparison of relativesignal intensity.

See “Baseline Window size“ on page 49.

See “Effects of varying Baseline Window size“ on page 50.

Minimum Peak Half Width Specify the minimum full peak width at half maximum PeakHeight required for peak detection. The range is 2 to 99 datapoints.

Polynomial Degree Polynomial Degree cannot be greater than Peak Window Size.

Adjust to affect the sensitivity of peak detection. You can adjustthis parameter to detect a single base pair difference whileminimizing the detection of shoulder effects and/or noise.

See “Polynomial Degree and Peak Window Size“ on page 51.

Peak Window Size Enter a window width in data points for peak detection sensitivity.If more than one peak apex is within the window, all are labeledas a single peak.

See “Polynomial Degree and Peak Window Size“ on page 51.

Peak Slope Start The peak starts when the first derivative (slope of the tangent) inthe beginning of the peak signal before the inflection pointbecomes equal to or exceeds the Peak Start value. This thresholdis set to 0 by default, which means that the peak will normallystart at the leftmost point where the slope of the tangent isclosest to 0° (horizontal line). A value other than 0 moves the peakstart point toward its center. The value entered must be non-negative.

See “Slope Thresholds for Peak Start/End parameters“ onpage 52.

Appendix C Reference informationAnalysis settings C


Setting Description

Peak Slope End The peak ends when the first derivative (slope of the tangent) inthe end of the peak signal after the inflection point becomes equalto or exceeds the Peak End value. This value is set to 0 by default,which means that the peak will normally end at the rightmostpoint where the slope of the tangent is closest to 0° (horizontalline). A value other than 0 moves the peak end point toward itscenter. The value entered in this field must be non-positive.

Peak Smoothing Select an option to smooth the outline of peaks and reduce thenumber of false peaks detected:

• None (default) to apply no smoothing. Best if the data displaysharp, narrow peaks of interest.

• Light to provide the best results for typical data. Lightsmoothing slightly reduces peak height.

• Heavy for data with very sharp, narrow peaks of interest.Heavy smoothing can significantly reduce peak height.

Note: The peak heights that are listed in the sizing table matchthe peak heights that are shown in the electropherogram whensmoothing is applied.

See “Smoothing“ on page 51.

Size Calling Method • Local Southern—(default) Determines the fragment sizesusing the reciprocal relationship between fragment lengthand electrophoretic mobility.

• Global Southern—Compensates for standard fragments withanomalous electrophoretic mobility (similar to least squaresmethods).

• Least Square Second Order—Uses regression analysis tobuild a bestfit size calling curve.

• Least Square Second Order—Uses regression analysis tobuild a bestfit size calling curve.

• Cubic Spline Interpolation—Forces the sizing curve throughall the known points of the selected size standard.

See “Size Calling - Local Southern Method“ on page 57.

See “Size Calling – Global Southern Method“ on page 58.

See “Size Calling – Least Squares Method“ on page 59.

See “Size Calling – Cubic Spline Interpolation Method“ onpage 61.

Size Standard Normalization For 3500 samples only, select to apply the normalization factorfrom the data collection software.

Peak detection overview

Parameters set in the analysis settings determine how the raw data are baselined andsmoothed, and how peaks are detected.

Appendix C Reference informationPeak detection overviewC


The Baseline Window size parameter controls baselining for a group of peaks.

The software determines a reference baseline value for each data point. In general, thesoftware sets the reference baseline to be the lowest value that it detects in a specifiedwindow size (in data points) centered on each data point.

A small baseline window relative to the width of a cluster, or grouping of peaksspatially close to each other, can result in shorter peak heights.

Larger baseline windows relative to the peaks being detected can create an elevatedbaseline, resulting in peaks that are elevated or not resolved to the baseline.

Guidelines for the Baseline Window size parameter

Choose a value that encompasses the width in data points of the peaks being detectedwhile preserving a qualitatively smooth baseline.

The trade-off for a smoother baseline that touches all peaks is a reduction in peakheight.

Baseline Windowsize

Appendix C Reference informationPeak detection overview C


Effects of varying Baseline Window size

The baseline window size determines the size of the window. Increasing the windowsize will decrease the baselining effect.

The following figure shows a sample with different reference baselines (zero in theanalyzed electropherogram) that result from different baseline window size settings:

• The red trace shows a baseline derived from an extreme baseline window sizevalue of 2801. At this setting, the reference baseline does not touch all peaks andelevates peak heights.

• The blue trace shows a baseline derived from the default value of 51 data points.• The black trace shows a baseline that is derived from an extreme baseline

window size value of 5 data points. At this setting, the reference baseline tracksthe peaks, significantly reducing peak height.






1 1

1

1

Figure 4 Baseline Window example



This parameter smooths the outline of peaks, and reduces the number of false peaksthat are detected.

Smoothing is performed before peak detection and can be set to:

Option Description

None Applies no smoothing. Select for slowerruns with very broad peaks, or to avoid the

detection of sharp edges.

Light Provides the best results for typical data.Light smoothing slightly reduces peak

height.

Heavy Select for data with very sharp, narrowpeaks of interest. Heavy smoothing can

significantly reduce peak height.






1 1

1

1

Figure 5 Smoothing example

Polynomial Degree and Peak Window Size settings affect the peak detectionsensitivity. You can adjust these parameters to detect a single base pair differencewhile minimizing the detection of shoulder effects and/or noise (see Table 1 .

The peak window size functions with the polynomial degree to set the sensitivity ofpeak detection. The peak detector calculates the first derivative of a polynomial curvefitted to the data within a window that is centered on each data point in the analysisrange.

Using curves with larger polynomial degree values allows the curve to more closelyapproximate the signal and, therefore, the peak detector captures more of the peakstructure in the electropherogram.

Smoothing

PolynomialDegree and PeakWindow Size



The peak window size sets the width (in data points) of the window to which thepolynomial curve is fitted to data:

• Higher peak window size values smooth out the polynomial curve, which limitsthe structure being detected.

• Smaller window size values capture more of the peak structure.

Table 1 Change peak detection sensitivity using Polynomial Degree and Peak WindowSize

Function Polynomial Degree value Peak Window Size value

Increase sensitivity Higher Lower

Decrease sensitivity Lower Higher

The Slope Threshold for Peak Start and Slope Threshold for Peak End parametersadjust the start and end points of a peak.

The values assigned to these parameters can be used to better position the start andend points of an asymmetrical peak, or a poorly resolved shouldering peak to moreaccurately reflect the peak position and area.

In general, from left to right, the slope of a peak increases from the baseline up to theapex. From the apex down to the baseline, the slope decreases negatively until itreturns to zero at the baseline.






1 1

1

1

Figure 6 Peak slope

If either of the slope values you enter exceeds the slope of the peak being detected, thesoftware overrides your value and reverts to zero.

Guidelines for Slope Threshold Peak Start and Peak End parameters

• For typical or symmetrical peaks, use a value of zero.• For asymmetrical peaks, select values other than zero to better reflect the

beginning and end points.• A value of zero does not affect the sizing accuracy or precision of an

asymmetrical peak.

Slope Thresholdsfor Peak Start/Endparameters



Using Slope Threshold Peak Start and Peak End parameters

Note: The size of a detected peak is the calculated apex between the start and endpoints of a peak. Peak size does not change based on start and end settings.

To move the… Then… Example

Start point of a peak closerto its apex

Change the Slope Thresholdfor Peak Start value fromzero to a positive number.






1 1

1

1

End point of a peak closer toits apex

Change the Slope Thresholdfor Peak End value to a more

negative number.






1 1

1

1



Slope Threshold example — Asymmetrical peak

Figure 7 shows the initial electropherogram analyzed with value of 0 for Peak Startand Peak End. Note the asymmetrical peak with a noticeable tail on the right side.






1 1

1

1

Figure 7 Electropherogram showing an asymmetrical peak

After reanalysis with a value of –35.0 for the Slope Threshold for Peak End, the endpoint that defines the peak moves closer to its apex, thereby removing the tail(Figure 8). Note that the only change to tabular data is the area (peak size and heightare unchanged).






1 1

1

1

Figure 8 Electropherogram showing the effect of changing the slope threshold for peakend



Sizing overview

During sizing, the software:• Performs size matching of the internal size standard in all samples against the

size standard definition selected in the software.• Generates a size-calling curve.• Sizes DNA sample peaks.• Assesses the sizing quality.

During size matching, the software matches the size standard fragments from theelectropherogram to the list of fragment sizes in the size standard definition specifiedin the software.

Size matching uses ratio matching, based on relative height and distance ofneighboring peaks. It then derives quality values statistically by examining thesimilarity between the theoretical (from the size standard definition) and actual(observed) fragment patterns.

The software ignores anomalous peaks that do not match the expected patterns. Thesoftware constructs a best-fit curve using the data points of each size standardfragment detected. A comparison between the sizes calculated from the best-fit curveand the matched peaks from the size standard definition file using the array ofnumbers is performed. Size-matching (and subsequent size calling) fails if significantdifferences in peak patterns are found, if no match can be made based on the expectedpatterns, or if all peaks are not found.

Because the software uses ratio-matching (looks for the expected number of allelesand expected peak patterns instead of specific data points), it is not necessary todefine new size-standard definitions due to migration shifts.

The size matching and size-calling curve generation algorithm:• Determines the expected peak sizing and height ratios — Uses the list of sizes

from the Size Standard definition file.

Note: The values that are used are for example only and do not indicate typicalsize-standard values.






1 1

1

1

Size matching

Appendix C Reference informationSizing overview C


• Evaluates peaks in the size standard data— Ignores peaks that do not meet theexpected pattern (dotted peak).






1 1

1

1 • Plots the size–calling curve — Uses peaks that meet the expected pattern.






1 1

1

1

To generate the size-calling curve, the software plots the actual data points of the sizestandard against the expected size of each size standard peak. The size-calling methoddetermines how the size-calling curve is generated and used to size each sample.

During size matching and size calling:• 2 size-calling curves are generated for each sample:

– Black: A best-fit second order curve, regardless of the size-calling methodthat is selected.

– Red: A curve based on the size-calling method that is selected in the analysismethod.

• The data points of non-size-standard peaks are plotted against the size-callingcurve.

• Peaks are sized according to the size-calling method that is selected in theanalysis method.

Size-calling methods are:• Local Southern• Global Southern• Least Squares (2nd Order or 3rd Order)• Cubic Spline Interpolation

Size-calling curvegeneration andsize calling

Appendix C Reference informationSizing overviewC


Size Calling - Local Southern Method

The Local Southern method determines the sizes of fragments by using the reciprocalrelationship between fragment length and mobility, as described by E. M. Southern(1979).






1 1

1

1Figure 9 Local Southern method

Local Southern method equation

The equation attempts to describe the reciprocal relationship between the mobility, m,and the length, L0, of the standard fragments.

L = [c/(m – m0)] + L0

How the Local Southern method works

This method, which is similar to the Cubic Spline method, uses the four fragmentsclosest in size to the unknown fragment to determine a best-fit line value. Only theregion of the size standard near the fragment of unknown length is analyzed.

Note: Size estimates may be inaccurate if any of the standard fragments runanomalously.

In the Local Southern method:

1. The fitting constants of the curve are calculated for each group of threeneighboring points on the standard.

2. A curve is then created using three standard points (two points below and onepoint above the fragment), then a fragment size is determined.

3. Another curve is created using an additional set of three points (one point belowand two points above the fragment), to assign another value.

4. The two curves are averaged to determine the unknown fragment length.



Size Calling – Global Southern Method

The Global Southern method is similar to the Least Squares method in that itcompensates for standard fragments that may run anomalously. The method creates abest-fit line through all the available points, and then uses values on that line tocalculate the fragment values.






1 1

1

1

Figure 10 Global Southern method

Global Southern method equations

Table 2

Equation Description

L = [c ⁄ (m – m0)] + L0 Attempts to describe the reciprocalrelationship between the mobility, m, andthe length, L0, of the standard fragments.

Σi{Li – [c ⁄ ((mi – m0) + L0)]}2 The fitting constants L0, m0, and c arecalculated by a least-squares fit to

minimize the left side quantity.

How the Global Southern method works

All points in the standard are weighted equally, and the curve is not constrained to gothrough any specific point. The software can analyze a large range of fragment sizeswith this method. For best results, use a standard that brackets all the fragments ofinterest.



Size Calling – Least Squares Method

Both Least Squares methods (2nd-Order and 3rd-Order) use regression analysis tobuild a best-fit size-calling curve. This curve:

• Produces the minimum additive distance from the curve to the plotted datapoints.

• Compensates for any fragments that may run anomalously.

Consequently, this method typically results in the least amount of deviation for all thefragments, including the size standards and the samples.

Depending on whether you choose the 2nd- or 3rd-Order Least Squares Method in theAnalysis Parameters dialog box, the resulting size curve is either a quadratic or acubic function. The software uses the known standard fragments and the associateddata points to produce a sizing curve based on Multiple Linear Regression.

How the Least Squares method works

The following figures show that in nearly all instances the mobility of an individualDNA fragment is coincident with the best curve fit of the entire data set. Stateddifferently, the mobility of most DNA fragments is strictly length dependent. Thismethod automatically compensates for fragments that run anomalously.

To generate the Least Squares curve, the software:• Plots the known fragment sizes (bp) versus data points.• Generates a best-fit curve using regression analysis.• To generate the Least Squares curve, the software:

– Plots the known fragment sizes (bp) versus data points.– Generates a best-fit curve using regression analysis.– Applies the following calculation to determine the size in data points of the

unknown fragments:Y = Ax3 + Bx2 +Cx + Dwhere:

– Y = size (bp)– x = datapoint– A = First order coefficient– B = Second order coefficient



– C = Third order coefficient– D = Zeroth Coefficient or constant






1 1

1

1

Figure 11 2nd‑Order Least Squares size‑calling curve (quadratic)






1 1

1

1

Figure 12 3rd‑Order Least Squares size‑calling curve (quadratic)



Size Calling – Cubic Spline Interpolation Method

The Cubic Spline method, which is similar to the Local Southern method, forces thesizing curve through all the known points of the selected size standard. Although thisenforcement produces exact results for the values of the standards themselves, it doesnot compensate for standard fragments that may run anomalously.






1 1

1

1

Figure 13 Cubic Spline Interpolation method

Possible local sizing inaccuracy

Mobility of any DNA fragment can be affected by its sequence, and by secondary andtertiary structure formation. If any internal size standard fragment has anomalousmobility, the Cubic Spline method may exhibit local sizing inaccuracy.

For example, assume that a standard fragment is close in molecular length to anunknown sample fragment. Assume further that the standard fragment runsanomalously. The Cubic Spline method assigns the official value to this standardfragment, even though it may be slightly incorrect. The size of the unknown fragmentis then likely to be calculated incorrectly as well.

Note: This method does not determine the degree of sizing accuracy error.



The Sizing Quality assessment evaluates the quality of the size standard profile withineach sample (SQ) and allows you to flag samples with size standards that have poorpeak resolution. You can adjust the ranges that correspond to the High, Medium, andLow Quality ranges in the Analysis Settings. See “Adjust Quality Flag ranges“ onpage 18.

Acronym Full name Function/Rule(s) Flag Indicator

SQ Sizing Quality Evaluates the similaritybetween the fragmentpattern for the sizestandard dye that isspecified in the sizestandard definition andthe actual distribution ofsize standard peaks inthe sample.

SQ is within theuser-definedHigh Qualityrange

(Default = 0.75to 1.0)

SQ is within theuser-definedMedium Qualityrange.

(Default = 0.26to 0.74)

SQ is within theuser-definedLow Qualityrange

(Default = 0.0 to0.25)

Sizing Quality (SQ)status



Documentation and support

Related documentation

Document Publication number Description

DNA Fragment Analysis byCapillary ElectrophoresisUser Guide

4474504

http://tools.thermofisher.com/content/sfs/manuals/4474504.pdf

Describes how to plan,conduct, and troubleshootfragment analysisapplications.

Customer and technical support

Visit thermofisher.com/support for the latest in services and support, including:• Worldwide contact telephone numbers• Product support, including:

– Product FAQs– Software, patches, and updates– Training for many applications and instruments

• Order and web support• Product documentation, including:

– User guides, manuals, and protocols– Certificates of Analysis– Safety Data Sheets (SDSs; also known as MSDSs)

Note: For SDSs for reagents and chemicals from other manufacturers,contact the manufacturer.

Limited product warranty

Life Technologies Corporation and/or its affiliate(s) warrant their products as set forthin the Life Technologies' General Terms and Conditions of Sale found on LifeTechnologies' website at www.thermofisher.com/us/en/home/global/terms-and-conditions.html. If you have any questions, please contact LifeTechnologies at www.thermofisher.com/support.






http://thermofisher.com/support

http://www.thermofisher.com/us/en/home/global/terms-and-conditions.html

http://www.thermofisher.com/us/en/home/global/terms-and-conditions.html

http://www.thermofisher.com/support

thermofisher.com/support | thermofisher.com/askaquestion

thermofisher.com

5 February 2018

http://thermofisher.com/support

mailto:thermofisher.com/askaquestion

http://thermofisher.com

sizing analysis module peak scanner software · a.0 05 february 2018 new document. material...

Documents