Beckman Coulter CEQ 8000 Manual de usuario

A16637-AA

April 2004

Beckman Coulter, Inc.

4300 North Harbor Boulevard, Fullerton, CA 92834-3100

CEQ™ 8000

Genetic

Analysis

System

User’s Guide

publication may be reproduced, transcribed, transmitted, or translated

into any language in any form by any means without the written

permission of Beckman Coulter, Inc.

The software is copyrighted and may not be altered or given to a third

party without the written authorization from Beckman Coulter, Inc.

Table of Contents

CEQ™ 8000 Genetic Analysis System – User’s Guide

Document A16637AA

iii

Foreword ............................................................................................................................... xiii

About this Guide................................................................................................................. xiii

Symbols Used in this Guide ............................................................................................... xiii

Getting Help ........................................................................................................................xiv

System Overview ................................................................................................................1

Purpose of this System ...........................................................................................................1

Functional Description ...........................................................................................................1

Hardware.................................................................................................................................1

Sample Access Cover ....................................................................................................2

Capillary Access Cover .................................................................................................2

Capillary Temperature Control Cover...........................................................................2

Capillary Array..............................................................................................................2

Plenum...........................................................................................................................3

Sample Transport and Plate Holders .............................................................................4

Sample Plate ..................................................................................................................5

Buffer Plate with Buffer Evaporation Cover.................................................................5

Wetting Tray..................................................................................................................5

Gel Waste Bottle............................................................................................................6

Power Switch.................................................................................................................6

Gel Pump/Gel Cartridge Access Cover.........................................................................6

Gel Pump, Gel Cartridge and Gel Pump Plug...............................................................7

Status Indicator Lights...................................................................................................8

Software..................................................................................................................................9

User Interface ................................................................................................................9

Safety Information................................................................................................................11

Safety Symbols............................................................................................................11

Chemical and Biological Safety ..................................................................................13

Electrical Safety...........................................................................................................13

Moving Parts ...............................................................................................................13

Laser Safety .................................................................................................................14

Program Description .....................................................................................................17

Sample Setup Module...........................................................................................................18

Main Window..............................................................................................................19

Menu Bar Options .......................................................................................................22

File Menu ..............................................................................................................23

Edit Menu..............................................................................................................23

View Menu............................................................................................................24

Run Menu..............................................................................................................25

Window Menu ......................................................................................................25

Help Menu ............................................................................................................25

Toolbar Icons...............................................................................................................26

Table of Contents

iv CEQ™ 8000 Genetic Analysis System

Run Module.......................................................................................................................... 29

Main Window ............................................................................................................. 30

Menu Bar Options....................................................................................................... 31

File Menu ............................................................................................................. 32

View Menu ........................................................................................................... 33

Direct Control Menu ............................................................................................ 34

Tools Menu........................................................................................................... 36

Run Menu ............................................................................................................. 38

Log Options Menu................................................................................................ 40

Replenish Menu.................................................................................................... 40

Help Menu............................................................................................................ 45

Toolbar Icons .............................................................................................................. 45

Standard Toolbar .................................................................................................. 46

Direct Control Toolbar ......................................................................................... 47

Data Monitor Toolbar........................................................................................... 48

Log Toolbar.......................................................................................................... 49

Run Sample Toolbar............................................................................................. 49

Sequence Dye Colors Toolbar.............................................................................. 49

Fragment Dye Colors Toolbar.............................................................................. 50

Status Monitor............................................................................................................. 51

Window Selection Tabs .............................................................................................. 53

Data Monitor Window.......................................................................................... 53

Direct Control Window ........................................................................................ 54

Log Window......................................................................................................... 57

Instrument Data Window ..................................................................................... 58

Sequence Analysis Module.................................................................................................. 59

Main Window ............................................................................................................. 60

Menu Bar Options....................................................................................................... 62

File Menu ............................................................................................................. 62

Edit Menu ............................................................................................................. 64

View Menu ........................................................................................................... 65

Tools Menu........................................................................................................... 67

Analysis Menu...................................................................................................... 70

Window Menu...................................................................................................... 73

Help Menu............................................................................................................ 73

Toolbar Icons .............................................................................................................. 73

Standard Toolbar .................................................................................................. 75

Data Toolbar......................................................................................................... 77

Sample View Toolbar........................................................................................... 78

Dye Colors Toolbar .............................................................................................. 79

Batch Control Toolbar.......................................................................................... 80

Sample Plate Toolbar ........................................................................................... 81

Base Sequence Toolbar ........................................................................................ 82

CEQuence Investigator Module ........................................................................................... 85

Main Window ............................................................................................................. 86

Menu Bar Options....................................................................................................... 88

Toolbar Icons .............................................................................................................. 91

Dye Colors Toolbar .............................................................................................. 95

Fragment Analysis Module.................................................................................................. 97

Main Window ............................................................................................................. 98

User’s Guide v

Menu Bar Options .......................................................................................................99

File Menu ..............................................................................................................99

View Menu..........................................................................................................101

Results Menu ......................................................................................................101

Fragments Menu .................................................................................................102

Analysis Menu ....................................................................................................102

Reports Menu......................................................................................................103

Window Menu ....................................................................................................103

Help Menu ..........................................................................................................104

Toolbar Icons.............................................................................................................104

Data Manager Module ........................................................................................................107

Main Window............................................................................................................108

Menu Bar Options .....................................................................................................109

File Menu ............................................................................................................109

Edit Menu............................................................................................................110

View Menu..........................................................................................................111

Tools Menu .........................................................................................................111

Window Menu ....................................................................................................112

Help Menu ..........................................................................................................112

Toolbar Icons.............................................................................................................113

Operating the System .................................................................................................115

Set-Up & Procedures ..........................................................................................................115

Preparing Sample................................................................................................................115

Starting-Up the System.......................................................................................................115

Creating a Database and Project Folder..............................................................................115

Opening the Data Manager Module ..........................................................................115

Creating a Database...................................................................................................116

User’s Database Template ..................................................................................116

Creating and Naming a Project Folder ......................................................................116

Setting up a Sample ............................................................................................................116

Starting the Sample Setup Module............................................................................116

Setting up the Sample Plate.......................................................................................116

Running a Sample...............................................................................................................117

Starting the Run Module ...........................................................................................117

Installing the Capillary Array....................................................................................117

Installing a Gel Cartridge ..........................................................................................117

Installing the Gel Waste Bottle..................................................................................117

Preparing Plates for a Run.........................................................................................117

Setting-Up and Starting the Run................................................................................117

Sample Setup Procedures ...................................................................................................121

Open an Existing Sample Plate .................................................................................121

Creating a New Sample Plate....................................................................................123

Sample Selection ................................................................................................................124

Selecting the Entire Sample Plate..............................................................................124

Selecting the Entire Row...........................................................................................125

Selecting the Entire Column......................................................................................125

Selecting Contiguous Cells........................................................................................126

Select Non-Contiguous Cells ....................................................................................127

View by Sample Name/Subject ID............................................................................127

Table of Contents

vi CEQ™ 8000 Genetic Analysis System

Naming Samples and Assigning Subject IDs ........................................................... 128

Turning Cell Coordinates On or Off .................................................................................. 129

Using Find.......................................................................................................................... 129

Using Replace .................................................................................................................... 129

Using Property Sets............................................................................................................ 131

Assigning Methods ............................................................................................................ 133

Condition Method ..................................................................................................... 134

Creating or Editing a Method............................................................................................. 134

Assigning Analysis Parameter Sets.................................................................................... 135

Assigning a Parameter Set ........................................................................................ 135

Specifying Sample Plate Print Options.............................................................................. 136

Specifying Sample Plate Export Options........................................................................... 136

Import/Export Sample Plate...................................................................................... 136

Import Sample Plate ........................................................................................... 136

Export Sample Plate ........................................................................................... 137

Apply Trimming .......................................................................................... 137

Export Only If Sequence > X nt .................................................................. 137

Reports ............................................................................................................................... 137

Trimming Log........................................................................................................... 137

Lock ................................................................................................................................... 139

View Summary................................................................................................................... 139

Run Procedures .............................................................................................................. 141

Opening the Run Module................................................................................................... 141

Defining System Preferences ............................................................................................. 141

Running a Sample Plate ..................................................................................................... 141

Database Size ............................................................................................................ 141

Gel Re-estimation Feature ........................................................................................ 142

Gel Re-Estimation Procedure............................................................................. 143

Gel Volume Notification........................................................................................... 144

Gel Plug Warning ..................................................................................................... 145

Pausing a Sample Plate Run ..................................................................................... 146

Stopping a Sample Plate Run.................................................................................... 146

Viewing the Last Analysis Performed................................................................................ 147

Sequence Analysis Procedures ......................................................................... 149

Viewing Sample Data......................................................................................................... 149

Sample View Toolbar ............................................................................................... 151

Analyzing Sequence Data .................................................................................................. 151

Editing Sequence Analysis Parameters..................................................................... 151

General Tab ........................................................................................................ 152

Initial Data Detection ......................................................................................... 155

Heterozygote Detection Tab............................................................................... 157

Alignment Template Tab.................................................................................... 159

Advanced Alignment Parameters ................................................................ 161

Quality-based Trimming Tab ............................................................................. 162

Ends To Be Trimmed................................................................................... 163

Trimming Stringency................................................................................... 163

Sequence-based Trimming Tab.......................................................................... 163

User’s Guide vii

Perform Sequence-based Trimming.............................................................164

Vector/Other Sequence Files........................................................................164

Max. # of Vector/Other Sequences ..............................................................164

Min. Substring Length..................................................................................164

Distance from End........................................................................................164

Enable Internal Matches...............................................................................164

Quality Values.....................................................................................................................164

Preventing Alignment Problems................................................................................164

Performing Quality-based Trimming ........................................................................165

Apply Quality Trimming..............................................................................167

Undo .............................................................................................................167

Performing Sequence-based Trimming.....................................................................167

Apply Sequence-based Trimming ................................................................169

Undo .............................................................................................................169

Internal Trimming ........................................................................................170

Batch Trimming.........................................................................................................171

Print Grid Data .............................................................................................173

Export Grid Data ..........................................................................................174

Format Grid ..................................................................................................174

Export Options....................................................................................................175

Apply Trimming...........................................................................................175

Export Only If Sequence > X nt...................................................................175

Editing Bases.............................................................................................................175

Inserting Bases in the Analyzed Data Pane ........................................................175

Inserting Bases in the Base Sequence Pane ........................................................176

Changing Bases in the Analyzed Data Pane .......................................................176

Changing Bases in the Base Sequence Pane.......................................................176

Deleting Bases in the Base Sequence Pane.........................................................177

Specifying Base Grouping ..................................................................................177

Analysis Log..............................................................................................................178

Sequence Result Properties .......................................................................................179

Notes Tab ............................................................................................................180

Property Set Tab .................................................................................................181

Method Tab.........................................................................................................182

Analysis Tab .......................................................................................................183

Alignment Tab ....................................................................................................184

Consumables Tab................................................................................................185

Setting or Changing Display Options.................................................................................185

Setting or Changing Title Properties .........................................................................185

Setting or Changing X Axis Options.........................................................................186

Setting or Changing Y Axis Options.........................................................................186

Setting or Changing Dye Trace Displays ..................................................................187

Setting or Changing Current Trace Displays.............................................................187

Setting or Changing Color Options ...........................................................................187

Changing Raw Data Colors.......................................................................................187

Changing Heterozygote Display Color .....................................................................188

Table of Contents

viii CEQ™ 8000 Genetic Analysis System

Pinning Results ......................................................................................................... 188

Performing a Batch Analysis .................................................................................... 188

Reanalyzing a Batch ................................................................................................. 188

Viewing the Selected Batch Result........................................................................... 189

Skipping the Current Sample Analysis in a Batch.................................................... 189

Skipping the Current Sample Set Analysis in a Batch.............................................. 189

Skipping the Current Sample Plate Analysis in a Batch........................................... 189

Restoring the Original Sequence Data...................................................................... 189

Compare Mode.......................................................................................................... 190

Sequence Result Report ............................................................................................ 192

Displaying Quality Parameters in a Report .............................................................. 195

Magnifying (Zooming) Data..................................................................................... 197

Panning Data............................................................................................................. 197

Synchronizing Result Data with Result Output ........................................................ 197

Aligning Bases and Peaks......................................................................................... 197

Specifying the Base Call Location............................................................................ 198

Using Audio Enable.................................................................................................. 198

Using Audio Playback .............................................................................................. 198

Viewing Color Calibrations ...................................................................................... 198

Computing a New Color Calibration ........................................................................ 199

Sequence Result Export ............................................................................................ 200

Sequence Export File Types............................................................................... 201

Customizing Filename Extension ............................................................................. 204

Exporting Data to a Third Party Package ................................................................. 204

CEQuence Investigator Procedures ................................................................ 207

Creating a Reference File................................................................................................... 207

Selecting a Reference and Sequence Results............................................................ 208

Numbering ................................................................................................................ 210

Viewing Reference Amino Acid Translations.......................................................... 211

Viewing Sequence Traces......................................................................................... 212

Viewing Consensus Amino Acid Translations ......................................................... 212

Quality Values.................................................................................................... 213

Viewing CEQuence Investigator Differences........................................................... 213

Navigation and Editing ...................................................................................................... 214

Complementing the Sequence................................................................................... 215

Flipping the Traces ................................................................................................... 215

Trimming the Bases .................................................................................................. 215

Searching the Consensus for Specific Attributes...................................................... 215

Searching the Consensus for Specific Bases ............................................................ 216

Editing the Consensus............................................................................................... 216

Displaying the Discrepancy Map.............................................................................. 217

Printing and Reports........................................................................................................... 218

Exporting............................................................................................................................ 219

Saving................................................................................................................................. 220

Fragment Analysis Procedures .......................................................................... 221

Performance Safeguards ........................................................................................... 222

Working with Studies in the Fragment Analysis Module......................................... 222

Selecting the Components of the New Study ........................................................... 223

User’s Guide ix

Selecting Raw Data for the New Study ..............................................................224

Selecting Raw Data from the List View tab .......................................................224

Selecting Raw Data from the Plate View tab......................................................225

Selecting an Analysis Parameter Set .........................................................................227

Selecting a Saved Analysis Parameter Set..........................................................228

Analyzing the Data ....................................................................................................228

Selecting Additional Results to be added to the Study..............................................229

Defining Fragment Analysis Parameters...................................................................230

Fragment Analysis Parameters - General tab......................................................231

Fragment Analysis Parameters - Analysis Method tab.......................................233

Fragment Analysis Parameters - Quantitation Tab.............................................235

Fragment Analysis Parameters - STR Locus Tags tab .......................................237

Fragment Analysis Parameters - SNP Locus Tags tab .......................................239

Fragment Analysis Parameters - Advanced Tab.................................................241

STR Locus Tag Editor...............................................................................................244

Locus Tag Editor - STR Locus Tags ..................................................................244

Generating and Viewing the Allele List .............................................................246

Allele List Generation using Interpolation and Linear Regression ...........................247

Interpolation and Linear Regression...................................................................247

STR Locus Tag Editor - Allele ID Criteria.........................................................248

SNP Locus Tag Editor...............................................................................................253

Result Set Filtering ....................................................................................................255

Accessing the Result Set View ...........................................................................255

Applying Exclusion Filters to the Result Set Data .............................................256

Available Exclusion Filters.................................................................................257

Summary Area Information ................................................................................257

Customizing the Result Set View .......................................................................257

Viewing Trace Data...................................................................................................258

Single Sample View............................................................................................258

Copying a Trace..................................................................................................260

Stacked Graph.....................................................................................................261

Overlay Graph.....................................................................................................261

Including Results ................................................................................................261

Excluding Results ...............................................................................................262

Re-Analyze Results ...................................................................................................263

Re-analyzing Results ..........................................................................................263

Fragment Lists ...........................................................................................................265

Accessing the Fragment List View.....................................................................265

Applying Exclusion Filters to the Fragment List................................................266

Available Exclusion Filters.................................................................................266

Customizing the Fragment List...........................................................................266

Manually Selecting Peaks for the Fragment List................................................267

Export Fragment List to CSV File......................................................................267

Show Stacked Graph...........................................................................................267

Including, Excluding & Resetting Peaks in the Fragment List...........................267

Single Sample View............................................................................................267

Bin Analysis ..............................................................................................................268

Setting up a Bin Analysis....................................................................................268

Updating the Locus Tag and Allele List .............................................................271

Reviewing the Source Data.................................................................................272

Table of Contents

x CEQ™ 8000 Genetic Analysis System

Peak Ratios................................................................................................................ 274

Beginning a Peak Ratio Calculation................................................................... 274

Selecting a Reference Trace ............................................................................... 276

Selecting a Reference Peak ................................................................................ 277

Selecting a Test Peak.......................................................................................... 277

AFLP Analysis.......................................................................................................... 278

Using the AFLP feature...................................................................................... 278

Setting the AFLP Analysis Parameters .............................................................. 278

Analysis Parameters ........................................................................................... 279

Starting a New AFLP Analysis .......................................................................... 281

Cluster Analysis Parameters and Display Settings............................................. 282

Customizing the AFLP Table............................................................................. 282

Exporting your Results....................................................................................... 283

LOH Analysis ........................................................................................................... 283

Beginning a LOH Analysis ................................................................................ 283

Customizing the Results List .................................................................................... 286

Selecting Columns for Display........................................................................... 286

Sorting the Results List ...................................................................................... 288

Reporting Results...................................................................................................... 290

Report Templates ............................................................................................... 291

Editing Graph Displays ...................................................................................... 293

Editing X scale type..................................................................................... 293

Editing the X-axis ........................................................................................ 293

Editing the Y-axis ........................................................................................ 294

Editing the Pane Count or the Pane Width......................................................... 294

Editing the number of dyes displayed on the Graph .......................................... 295

Exporting Results...................................................................................................... 296

Text File Format................................................................................................. 296

Features of the Export Options window:............................................................ 297

Text File Format................................................................................................. 298

CEQ File Format ................................................................................................ 298

Exporting Fragment Lists or Genotypes from a Study ............................................. 299

Format Descriptions and Exporting Requirements ............................................ 300

Locus List Editor ................................................................................................ 302

Data Manager Procedures ...................................................................................... 305

User’s Database Template .................................................................................................. 305

Creating a Database............................................................................................................ 305

Creating and Naming a Project Folder............................................................................... 305

Deleting a Project Folder or Database ............................................................................... 305

Renaming a Project Folder or Database............................................................................. 306

Database Size ..................................................................................................................... 306

Shrink Database .................................................................................................................307

Backup Database................................................................................................................ 308

Restore Database................................................................................................................310

Convert Individual IDs to Subject IDs...................................................................... 313

Exporting Database Items .................................................................................................. 314

Sample Plate Result Export ...................................................................................... 314

Exporting from the Data Manager ............................................................................ 316

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