Thermo Fisher Scientific Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Applied Biosystems 3130/3130xl Genetic Analyzers

Getting Started Guide

Preparing the

Instrument

Performing

Spatial

Calibration

Performing

Spectral

Calibration

Autoanalysis and

Sequencing

Analysis Software

Autoanalysis and

SeqScape

Software

Autoanalysis and

Fragment

Analysis

Running the

Instrument

© Copyright 2007, 2010 Applied Biosystems. All rights reserved.

Information in this document is subject to change without notice. Applied Biosystems assumes no responsibility for any errors that may appear in this document. This document is believed to be complete and accurate at the time of publication. In no event shall Applied Biosystems be liable for incidental, special, multiple, or consequential damages in connection with or arising from the use of this document.

NOTICE TO PURCHASER:

This instrument is Authorized for use in DNA sequencing and fragment analysis. This authorization is included in the purchase price of this instrument and corresponds to the up-front fee component of a license under process claims of U.S. patents and under all process claims for DNA sequence and fragment analysis of U.S. patents now or hereafter owned or licensable by Applied Biosystems for which an Authorization is required, and under corresponding process claims in foreign counterparts of the foregoing for which an Authorization is required. The running royalty component of licenses may be purchased from

Applied Biosystems or obtained by using Authorized reagents purchased from Authorized suppliers in accordance with the label rights accompanying such reagents. Purchase of this instrument does not itself convey to the purchaser a complete license or right to perform the above processes. This instrument is also licensed under U.S. p atents and apparatus and system claims in foreign counterparts thereof. No rights are granted expressly, by implication or by estoppel under composition claims or under other process or system claims owned or licensable by Applied Biosystems. For more information regarding licenses, please contact the Director of Licensing at Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.

Applied Biosystems 3130 Genetic Analyzer:

Covered by one or more of U.S. Pats. Nos. RE37,606; RE37,941.

Applied Biosystems 3130xl Genetic Analyzer:

Covered by one or more of U.S. Pats. Nos. RE37,606; RE37,941.

NOTICE TO PURCHASER

The purchase price of this Applied Biosystems 3130/3130xl Genetic Analyzer includes a grant of a limited, non-transferable license under U.S. p atent claims and method claims of its foreign counterparts, and element claims of its foreign counterparts, to use this particular instrument for electrophoresis methods employing fluorescence as a means of detection. No other licenses or rights are hereby conveyed either expressly, by implication, or estoppel including, but not limited to, any claims to a composition.

The Applied Biosystems 3130 and 3130xl Genetic Analyzers includes patented technology licensed from Hitachi, Ltd. as part of a strategic partnership between Applied Biosystems and Hitachi, Ltd., as well as patented technology of Applied Biosystems.

Trademarks

Applied Biosystems, AmpFlSTR, BigDye, Cofiler, GeneMapper, Identifiler, Profiler, Profiler Plus ID, SeqScape, SGM Plus, SNaPshot, and StockMarks are registered trademarks and AB (Design), Applera, Hi-Di, KB, POP, POP-4, POP-6, POP-7, SEfiler, and Yfiler are trademarks of Appli ed Biosystems or its subsidiaries in the U.S. and/or certain other countries.

Dell is a registered trademark of Dell Computer Corporation.

Microsoft, Windows, and Windows XP are registered trademarks of the Microsoft Corporation in the United States and other countries.

Oracle is a registered trademark of the Oracle Corporation.

All other trademarks are the sole property of their respective owners.

Part Number 4352715 Rev. D

06/2010

Applied Biosystems 3130/3130xl Genetic Analyzers

Getting Started Guide Workflow

Chapter 1

Preparing the

Instrument

Start the system

Chapter 2

Performing a

Spatial Calibration

Start a spatial calibration run

Start Data Collection software

Check polymer block, select capillary array, and polymer

Prepare buffer and fill reservoirs

Evaluate spatial calibration profile

Accept or reject spatial calibration

Chapter 3

Performing a

Spectral Calibration

Prepare spectral calibration chemistry

Create a spectral instrument protocol

Perform a spectral calibration

Evaluate the spectral calibration data

Activate a spectral calibration

Chapter 4

Chapter 5

Chapter 6

Chapter 7

Autoanalysis and

Sequencing Analysis

Software

Create protocols for automated sequencing analysis

Create a

Sequencing Analysis

Plate Record

Autoanalysis and

SeqScape

®

Software

Create protocols for automated

SeqScape Software analysis

Create a

SeqScape Software Plate

Record

Autoanalysis and

Fragment Analysis

Create protocols for automated fragment analysis

Create a GeneMapper

Software

Plate Record

®

Running the

Instrument

Prepare samples and plate assemblies

Place Plate onto

Autosampler

Link the Plate Schedule a run Run the instrument

Set up for continuous operation

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

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iv

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Contents

Preface ix

How to Use This Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

How to Obtain More Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x

How to Obtain Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

Safety xiii

Safety Conventions Used in This Document . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiv

Symbols on Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv

Safety Labels on Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvi

General Instrument Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvii

Chemical Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xviii

Chemical Waste Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xx

Electrical Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxi

Physical Hazard Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxi

Laser Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxi

Workstation Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxii

Safety and Electromagnetic Compatibility (EMC) Standards . . . . . . . . . . . . . . . . xxiii

Chapter 1

Preparing the Instrument 1

Instrument Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Application Summary Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Barcode Readers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Starting the 3130/3130xl Genetic Analyzer System . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Data Collection Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Preparing the Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Preparing Buffer and Filling Reservoirs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Chapter 2

Performing a Spatial Calibration 21

Spatial Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Creating a Spatial Calibration File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Evaluating a Spatial Calibration File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Examples of Spatial Profiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

v

Chapter 3

Performing a Spectral Calibration 27

Spectral Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Preparing the Spectral Calibration Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

Loading Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

Creating a Spectral Instrument Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Performing a Spectral Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

Evaluating the Spectral Calibration Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

Activating a Spectral Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

Examples of Passing Sequencing Spectral Calibrations . . . . . . . . . . . . . . . . . . . . . 49

Examples of Passing Fragment Analysis Spectral Calibrations . . . . . . . . . . . . . . . . 51

Chapter 4

Autoanalysis and

Sequencing Analysis Software 53

Sequencing Analysis Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

About Plate Records and Sequencing Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

Creating Protocols for Automated Sequencing Analysis . . . . . . . . . . . . . . . . . . . . . 59

Analysis Protocol for Sequencing Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

Results Group for Sequencing Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

Filling Out a Sequencing Analysis Plate Record . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

Chapter 5

Autoanalysis and SeqScape Software 81

SeqScape Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

About Plate Records and Seqscape Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

Creating Protocols for Automated SeqScape Software Analysis . . . . . . . . . . . . . . . 88

Filling Out a SeqScape Software Plate Record . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

Chapter 6

Autoanalysis and Fragment Analysis 105

GeneMapper Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106

About GeneMapper Software Plate Records . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108

Creating Protocols for Automated Fragment Analysis . . . . . . . . . . . . . . . . . . . . . . 112

Creating a GeneMapper Software Plate Record . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

Chapter 7

Running the Instrument 127

Preparing Samples and Plate Assemblies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128

Linking and Unlinking a Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128

Running the Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130

Controlling the Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

Set Up for Continuous Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134

Working with Data in The Run History View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

Viewing Data from a Completed Run in the Data Collection Software . . . . . . . . . . 146

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Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Viewing Autoextraction Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147

Using Autoanalysis Manager Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155

Viewing Analyzed Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160

Appendix A

Plate Mapping 161

Injection Scheduling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161

Capillary Array Map . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161

96-Well Plate Mapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162

384-Well Plate Mapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163

Index 3

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

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Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Preface

How to Use This Guide

Purpose of This

Guide

The Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide provides step-by-step instructions for preparing and analyzing a sample. It is designed to help you quickly learn how to use the Applied Biosystems 3130/3130xl Genetic Analyzers.

Audience

This manual is written for principle investigators and laboratory staff who are planning to operate and maintain the Applied Biosystems 3130/3130xl Genetic Analyzers.

Assumptions

This guide assumes you have the following background:

• Familiarity with Microsoft

®

Windows

®

XP operating system.

• Knowledge of general techniques for handling DNA samples and preparing them for electrophoresis.

• A general understanding of hard drives, data storage, file transfers, and copying and pasting.

If you want to integrate the 3130/3130xl Genetic Analyzers into your existing laboratory data flow system, you need networking experience.

Text Conventions

This guide uses the following conventions:

• Bold indicates user action. For example:

Type 0, then press Enter for each of the remaining fields.

• Italic text indicates new or important words and is also used for emphasis. For example:

Before analyzing, always prepare fresh matrix.

• A right arrow bracket (>) separates successive commands you select from a dropdown or shortcut menu. For example:

Select File > Open > Spot Set.

Right-click the sample row, then select View Filter > View All Runs.

User Attention

Words

Two user attention words appear in Applied Biosystems user documentation. Each word implies a particular level of observation or action as described below:

• Note – Provides information that may be of interest or help but is not critical to the use of the product.

• IMPORTANT! – Provides information that is necessary for proper instrument operation, accurate chemistry kit use, or safe use of a chemical.

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

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x

Preface

How to Obtain More Information

Examples of the user attention words appear below:

Note:

The size of the column affects the run time.

IMPORTANT!

To verify your client connection to the database, you need a valid Oracle user ID and password.

Safety Alert

Words

Safety alert words also appear in user documentation. For more information, see “Safety

Alert Words” on page xiv .

How to Obtain More Information

Related

Documentation

The following related document is shipped with the system:

• Applied Biosystems 3130/3130xl Genetic Analyzers Maintenance, Troubleshooting,

and Reference Guide (P/N 4352716) - Contains information on maintaining the instrument and computer system, troubleshooting tables to diagnose performance failures and advance reference content.

• Applied Biosystems 3130/3130xl Genetic Analyzers Site Preparation Guide

(P/N 4352717) - Contains the space, environmental, and electrical requirements to support the 3130/3130xl Genetic Analyzer system.

• Applied Biosystems 3730/3730xl DNA Analyzers and Applied Biosystems

3130/3130xl Genetic Analyzers AB Navigator Software Administrator Guide

(P/N 4359472) - Contains information and procedures for the administrator maintaining the computer system and software files of the Applied Biosystems

3130/3130xl Genetic Analyzers.

• Applied Biosystems 3130/3130xl Genetic Analyzers Quick Reference Card

(P/N 4362825) - Contains a flowchart on how to run your samples and instrument, a table of maintenance tasks, and a Data Collection software reference guide.

Note:

For additional documentation, see “How to Obtain Support” on page xi

.

Send Us Your

Comments

Applied Biosystems welcomes your comments and suggestions for improving its user documents. You can e-mail your comments to:

[email protected]

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Preface

How to Obtain Support

How to Obtain Support

For the latest services and support information for all locations, go to

http://www.appliedbiosystems.com

, then click the link for Support.

At the Support page, you can:

• Search through frequently asked questions (FAQs)

• Submit a question directly to Technical Support

• Order Applied Biosystems user documents, MSDSs, certificates of analysis, and other related documents

• Download PDF documents

• Obtain information about customer training

• Download software updates and patches

In addition, the Support page provides access to worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities.

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

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Preface

How to Obtain Support

xii

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Safety

This safety section describes the following:

Safety Conventions Used in This Document . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiv

Symbols on Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv

Safety Labels on Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xvi

General Instrument Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvii

Chemical Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xviii

Chemical Waste Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xx

Electrical Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxi

Physical Hazard Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xxi

Laser Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xxi

Workstation Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxii

Safety and Electromagnetic Compatibility (EMC) Standards . . . . . . . . . . . . . . . . . . xxiii

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

xiii

Safety

Safety Conventions Used in This Document

Safety Conventions Used in This Document

Safety Alert

Words

Four safety alert words appear in Applied Biosystems user documentation at points in the document where you need to be aware of relevant hazards. Each alert word–IMPORTANT, CAUTION, WARNING, DANGER–implies a particular level of observation or action, as defined below:

Definitions

IMPORTANT!

– Indicates information that is necessary for proper instrument operation, accurate chemistry kit use, or safe use of a chemical.

– Indicates a potentially hazardous situation that, if not avoided, may result in minor or moderate injury. It may also be used to alert against unsafe practices.

– Indicates a potentially hazardous situation that, if not avoided, could result in death or serious injury.

– Indicates an imminently hazardous situation that, if not avoided, will result in death or serious injury. This signal word is to be limited to the most extreme situations.

Except for IMPORTANTs, each safety alert word in an Applied Biosystems document appears with an open triangle figure that contains a hazard symbol. These hazard

symbols are identical to the hazard icons that are affixed to Applied Biosystems

instruments (see

“Safety Symbols” on page xvi

).

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Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Safety

Symbols on Instruments

Examples

The following examples show the use of safety alert words:

IMPORTANT!

You must create a separate a Sample Entry Spreadsheet for each 96-well microtiter plate.

The lamp is extremely hot. Do not touch the lamp until it has cooled to room temperature.

CHEMICAL HAZARD. Formamide. Exposure causes eye, skin, and respiratory tract irritation. It is a possible developmental and birth defect hazard.

Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves.

ELECTRICAL HAZARD. Failure to ground the instrument properly can lead to an electrical shock. Ground the instrument according to the provided instructions.

Symbols on Instruments

Electrical

Symbols on

Instruments

The following table describes the electrical symbols that may be displayed on

Applied Biosystems instruments.

Symbol Description

Indicates the On position of the main power switch.

Indicates the Off position of the main power switch.

Indicates the On/Off position of a push-push main power switch.

Indicates a terminal that may be connected to the signal ground reference of another instrument. This is not a protected ground terminal.

Indicates a protective grounding terminal that must be connected to earth ground before any other electrical connections are made to the instrument.

Indicates a terminal that can receive or supply alternating current or voltage.

Indicates a terminal that can receive or supply alternating or direct current or voltage.

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

xv

Safety

Safety Labels on Instruments

Safety Symbols

The following table describes the safety symbols that may be displayed on

Applied Biosystems instruments. Each symbol may appear by itself or in combination

with text that explains the relevant hazard (see “Safety Labels on Instruments” on page xvi

). These safety symbols may also appear next to DANGERS, WARNINGS, and

CAUTIONS that occur in the text of this and other product-support documents.

Symbol Description

Indicates that you should consult the manual for further information and to proceed with appropriate caution.

Indicates the presence of an electrical shock hazard and to proceed with appropriate caution.

Indicates the presence of a hot surface or other high-temperature hazard and to proceed with appropriate caution.

Indicates the presence of a laser inside the instrument and to proceed with appropriate caution.

Indicates the presence of moving parts and to proceed with appropriate caution.

Safety Labels on Instruments

The following CAUTION, WARNING, and DANGER statements may be displayed on

Applied Biosystems instruments in combination with the safety symbols described in the preceding section.

English

CAUTION Hazardous chemicals. Read the

Material Safety Data Sheets (MSDSs) before handling.

CAUTION Hazardous waste. Read the waste profile (if any) in the site preparation guide for this instrument before handling or disposal.

CAUTION Hazardous waste. Refer to

MSDS(s) and local regulations for handling and disposal.

WARNING Hot lamp.

WARNING Hot. Replace lamp with an

Applied Biosystems lamp.

CAUTION Hot surface.

DANGER High voltage.

Francais

ATTENTION Produits chimiques dangeureux.

Lire les fiches techniques de sûreté de matériels avant la manipulation des produits.

ATTENTION Déchets dangereux. Lire les renseignements sur les déchets avant de les manipuler ou de les éliminer.

ATTENTION Déchets dangereux. Lire les fiches techniques de sûreté de matériels et la régulation locale associées à la manipulation et l'élimination des déchets.

AVERTISSEMENT Lampe brûlante.

AVERTISSEMENT Composants brûlants.

Remplacer la lampe par une lampe

Applied Biosystems.

ATTENTION Surface brûlante.

DANGER Haute tension.

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Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Safety

General Instrument Safety

English

WARNING To reduce the chance of electrical shock, do not remove covers that require tool access. No user-serviceable parts are inside.

Refer servicing to Applied Biosystems qualified service personnel.

DANGER Class 3B laser radiation present when open and interlock defeated. Avoid direct exposure to laser beam.

DANGER Class 3B laser radiation when open. Avoid direct exposure to laser beam.

DANGER Class 2(II) laser radiation present when open and interlock defeated. Do not stare directly into the beam

DANGER Class 2(II) laser radiation present when open. Do not stare directly into the beam.

DANGER Class 2(II) LED when open and interlock defeated. Do not stare directly into the beam.

DANGER Class 2(II) LED when open. Do not stare directly into the beam.

CAUTION Moving parts.

Francais

AVERTISSEMENT Pour éviter les risques d'électrocution, ne pas retirer les capots dont l'ouverture nécessite l'utilisation d'outils.

L’instrument ne contient aucune pièce réparable par l’utilisateur. Toute intervention doit être effectuée par le personnel de service qualifié de Applied Biosystems.

DANGER Class 3B rayonnement laser en cas d’ouverture et d’une neutralisation des dispositifs de sécurité. Eviter toute exposition directe avec le faisceau.

DANGER Class 3B rayonnement laser en cas d’ouverture. Eviter toute exposition directe avec le faisceau.

DANGER de Class 2(II) rayonnement laser en cas d'ouverture et d'une neutralisation des dispositifs de securite. Eviter toute exposition directe avec le faisceau.

DANGER de Class 2(II) rayonnement laser en cas d'ouverture. Eviter toute exposition directe avec le faisceau.

DANGER de Class 2(II) LED en cas d'ouverture et d'une neutralisation des dispositifs de securite. Eviter toute exposition directe avec le faisceau.

DANGER de Class 2(II) LED en cas d'ouverture. Eviter toute exposition directe avec le faisceau.

ATTENTION Parties mobiles.

General Instrument Safety

PHYSICAL INJURY HAZARD. Use this product only as specified in this document. Using this instrument in a manner not specified by

Applied Biosystems may result in personal injury or damage to the instrument.

Moving and

Lifting the

Instrument

PHYSICAL INJURY HAZARD. The instrument is to be moved and positioned only by the personnel or vendor specified in the applicable site preparation guide. If you decide to lift or move the instrument after it has been installed, do not attempt to lift or move the instrument without the assistance of others, the use of appropriate moving equipment, and proper lifting techniques. Improper lifting can cause painful and permanent back injury. Depending on the weight, moving or lifting an instrument may require two or more persons.

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

xvii

Safety

Chemical Safety

Moving and

Lifting Stand-

Alone Computers and Monitors

Do not attempt to lift or move the computer or the monitor without the assistance of others. Depending on the weight of the computer and/or the monitor, moving them may require two or more people.

Things to consider before lifting the computer and/or the monitor:

• Make sure that you have a secure, comfortable grip on the computer or the monitor when lifting.

• Make sure that the path from where the object is to where it is being moved is clear of obstructions.

• Do not lift an object and twist your torso at the same time.

• Keep your spine in a good neutral position while lifting with your legs.

• Participants should coordinate lift and move intentions with each other before lifting and carrying.

• Instead of lifting the object from the packing box, carefully tilt the box on its side and hold it stationary while someone slides the contents out of the box.

Operating the

Instrument

Ensure that anyone who operates the instrument has:

• Received instructions in both general safety practices for laboratories and specific safety practices for the instrument.

• Read and understood all applicable Material Safety Data Sheets (MSDSs). See

“About MSDSs” on page xviii .

Chemical Safety

Chemical Hazard

Warning

CHEMICAL HAZARD. Before handling any chemicals, refer to the Material Safety Data Sheet (MSDS) provided by the manufacturer, and observe all relevant precautions.

CHEMICAL HAZARD. All chemicals in the instrument, including liquid in the lines, are potentially hazardous. Always determine what chemicals have been used in the instrument before changing reagents or instrument components. Wear appropriate eyewear, protective clothing, and gloves when working on the instrument.

About MSDSs

Chemical manufacturers supply current Material Safety Data Sheets (MSDSs) with shipments of hazardous chemicals to new customers. They also provide MSDSs with the first shipment of a hazardous chemical to a customer after an MSDS has been updated.

MSDSs provide the safety information you need to store, handle, transport, and dispose of the chemicals safely.

Each time you receive a new MSDS packaged with a hazardous chemical, be sure to replace the appropriate MSDS in your files.

xviii

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Safety

Chemical Safety

Obtaining MSDSs

You can obtain from Applied Biosystems the MSDS for any chemical supplied by

Applied Biosystems. This service is free and available 24 hours a day.

To obtain MSDSs:

1.

Go to https://docs.appliedbiosystems.com/msdssearch.html

2.

In the Search field, type in the chemical name, part number, or other information that appears in the MSDS of interest. Select the language of your choice, then click

Search.

3.

Find the document of interest, right-click the document title, then select any of the following:

• Open – To view the document

• Print Target – To print the document

• Save Target As – To download a PDF version of the document to a destination that you choose

4.

To have a copy of a document sent by fax or e-mail, select Fax or Email to the left of the document title in the Search Results page, then click RETRIEVE

DOCUMENTS at the end of the document list.

5.

After you enter the required information, click View/Deliver Selected Documents

Now.

Chemical Safety

Guidelines

To minimize the hazards of chemicals:

• Read and understand the Material Safety Data Sheets (MSDS) provided by the chemical manufacturer before you store, handle, or work with any chemicals or hazardous materials. (See

“About MSDSs” on page xviii .)

• Minimize contact with chemicals. Wear appropriate personal protective equipment when handling chemicals (for example, safety glasses, gloves, or protective clothing). For additional safety guidelines, consult the MSDS.

• Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only with adequate ventilation (for example, fume hood). For additional safety guidelines, consult the MSDS.

• Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturer’s cleanup procedures as recommended on the MSDS.

• Comply with all local, state/provincial, or national laws and regulations related to chemical storage, handling, and disposal.

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

xix

Safety

Chemical Waste Safety

Chemical Waste Safety

Chemical Waste

Hazard

HAZARDOUS WASTE. Refer to Material Safety Data Sheets and local regulations for handling and disposal.

CHEMICAL WASTE HAZARD. Wastes produced by Applied

Biosystems instruments are potentially hazardous and can cause injury, illness, or death.

Chemical Waste

Safety Guidelines

To minimize the hazards of chemical waste:

• Read and understand the Material Safety Data Sheets (MSDSs) provided by the manufacturers of the chemicals in the waste container before you store, handle, or dispose of chemical waste.

• Provide primary and secondary waste containers. (A primary waste container holds the immediate waste. A secondary container contains spills or leaks from the primary container. Both containers must be compatible with the waste material and meet federal, state, and local requirements for container storage.)

• Minimize contact with chemicals. Wear appropriate personal protective equipment when handling chemicals (for example, safety glasses, gloves, or protective clothing). For additional safety guidelines, consult the MSDS.

• Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only with adequate ventilation (for example, fume hood). For additional safety guidelines, consult the MSDS.

• Handle chemical wastes in a fume hood.

• After emptying the waste container, seal it with the cap provided.

• Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local, state/provincial, or national environmental and health regulations.

Waste Disposal

If potentially hazardous waste is generated when you operate the instrument, you must:

• Characterize (by analysis if necessary) the waste generated by the particular applications, reagents, and substrates used in your laboratory.

• Ensure the health and safety of all personnel in your laboratory.

• Ensure that the instrument waste is stored, transferred, transported, and disposed of according to all local, state/provincial, and/or national regulations.

IMPORTANT!

Radioactive or biohazardous materials may require special handling, and disposal limitations may apply.

xx

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Safety

Electrical Safety

Electrical Safety

ELECTRICAL SHOCK HAZARD. Severe electrical shock can result from operating the Applied Biosystems 3130/3130xl Genetic Analyzers without its instrument panels in place. Do not remove instrument panels. High-voltage contacts are exposed when instrument panels are removed from the instrument.

Power

ELECTRICAL HAZARD. Grounding circuit continuity is vital for the safe operation of equipment. Never operate equipment with the grounding conductor disconnected.

ELECTRICAL HAZARD. Use properly configured and approved line cords for the voltage supply in your facility.

ELECTRICAL HAZARD. Plug the system into a properly grounded receptacle with adequate current capacity.

Overvoltage

Rating

The Applied Biosystems 3130/3130xl Genetic Analyzers have an installation

(overvoltage) category of II, and is classified as portable equipment.

Physical Hazard Safety

Moving Parts

PHYSICAL INJURY HAZARD. Moving parts can crush and cut.

Keep hands clear of moving parts while operating the instrument. Disconnect power before servicing the instrument.

Laser Safety

Laser

Classification

The Applied Biosystems 3130/3130xl Genetic Analyzers use an Argon laser. Under normal operating conditions, the instrument laser is categorized as a Class I laser. When safety interlocks are disabled during certain servicing procedures, the laser can cause permanent eye damage, and, therefore, is classified under those conditions as a Class 3B laser.

The Applied Biosystems 3130/3130xl Genetic Analyzers has been tested to and complies with 21 CFR, 1040.10 and 1040.11, as applicable.”

The Applied Biosystems 3130/3130xl Genetic Analyzers have been tested to and complies with standard EN60825-1, “Radiation Safety of Laser Products, Equipment

Classification, Requirements, and User’s Guide.”

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

xxi

Safety

Workstation Safety

Laser Safety

Requirements

To ensure safe laser operation:

• The system must be installed and maintained by an Applied Biosystems Technical

Representative.

• All instrument panels must be in place on the instrument while the instrument is operating. When all panels are installed, there is no detectable radiation present. If any panel is removed when the laser is operating (during service with safety interlocks disabled), you may be exposed to laser emissions in excess of the Class

3B rating.

• Do not remove safety labels or disable safety interlocks.

Additional Laser

Safety

Information

Refer to the user documentation provided with the laser for additional information on government and industry safety regulations.

LASER HAZARD. Lasers can burn the retina causing permanent blind spots. Never look directly into the laser beam. Remove jewelry and other items that can reflect the beam into your eyes. Do not remove the instrument top or front panels.

Wear proper eye protection and post a laser warning sign at the entrance to the laboratory if the top or front panels are removed for service.

LASER BURN HAZARD. An overheated laser can cause severe burns if it comes in contact with the skin. DO NOT operate the laser when it cannot be cooled by its cooling fan. Always wear appropriate laser safety goggles.

Workstation Safety

Correct ergonomic configuration of your workstation can reduce or prevent effects such as fatigue, pain, and strain. Minimize or eliminate these effects by configuring your workstation to promote neutral or relaxed working positions.

MUSCULOSKELETAL AND REPETITIVE MOTION

HAZARD. These hazards are caused by potential risk factors that include but are not limited to repetitive motion, awkward posture, forceful exertion, holding static unhealthy positions, contact pressure, and other workstation environmental factors.

To minimize musculoskeletal and repetitive motion risks:

• Use equipment that comfortably supports you in neutral working positions and allows adequate accessibility to the keyboard, monitor, and mouse.

• Position the keyboard, mouse, and monitor to promote relaxed body and head postures.

xxii

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Safety

Safety and Electromagnetic Compatibility (EMC) Standards

Safety and Electromagnetic Compatibility (EMC) Standards

This section provides information on:

•

U.S. and Canadian Safety Standards

•

Canadian EMC Standard

•

European Safety and EMC Standards

•

Australian EMC Standards

U.S. and

Canadian Safety

Standards

This instrument has been tested to and complies with standard UL 3101-1, “Safety

Requirements for Electrical Equipment for Laboratory Use, Part 1: General

Requirements.”

This instrument has been tested to and complies with standard CSA 1010.1, “Safety

Requirements for Electrical Equipment for Measurement, Control, and Laboratory Use,

Part 1: General Requirements.”

Canadian EMC

Standard

This instrument has been tested to and complies with ICES-001, Issue 3: Industrial,

Scientific, and Medical Radio Frequency Generators.

European Safety and EMC

Standards

Safety

This instrument meets European requirements for safety (Low Voltage Directive

73/23/EEC). This instrument has been tested to and complies with standards EN 61010-

1:2001, “Safety Requirements for Electrical Equipment for Measurement, Control and

Laboratory Use, Part 1: General Requirements” and EN 61010-2-010, “Particular

Requirements for Laboratory Equipment for the Heating of Materials.”

EMC

This instrument meets European requirements for emission and immunity (EMC

Directive 89/336/EEC). This instrument has been tested to and complies with standard

EN 61326 (Class B), “Electrical Equipment for Measurement, Control and Laboratory

Use – EMC Requirements.”

Australian EMC

Standards

This instrument has been tested to and complies with standard AS/NZS 2064, “Limits and Methods Measurement of Electromagnetic Disturbance Characteristics of Industrial,

Scientific, and Medical (ISM) Radio-frequency Equipment.”

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

xxiii

Safety

Safety and Electromagnetic Compatibility (EMC) Standards

xxiv

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Chapter 1

Preparing the Instrument

1

Preparing the

Instrument

Performing a

Spatial Calibration

Performing a

Spectral Calibration

Autoanalysis and Sequencing

Analysis

Software

Autoanalysis and

SeqScape Software

Autoanalysis and

Fragment Analysis

Running the

Instrument

Notes

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Start the system

See page 7

Start Data Collection software

See page 9

Check polymer block, select capillary array, and polymer

See page 12

Prepare buffer and fill reservoirs

See page 16

See page 2

See page 2

1

Chapter 1 Preparing the Instrument

Instrument Description

Instrument Description

Instrument Layout

A typical laboratory layout is shown below.

Monitor

CPU

Keyboard

Mouse and pad

3130/3130xl

Genetic Analyzer esc esc

F9

F10

F11

F1

F2 F3

F4

F5

F6

1

2 tab caps

Q shift

3

W

A control

E

4

R

5

S

Z option

D

X

6

T

F

C

7

Y

F7

G

V

F8

8

U

H

B

I

9

J

N

0

O

K

M

_

-

P

<

L

+

=

[

F12

> delete

]

"

? /

F13 F14

F15 help x home end page down clear

7

=

4 return shift option control

8

1

5

9

*

2

6

0

3

+ enter

Notes

2

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Chapter 1 Preparing the Instrument

Application Summary Tables

Application Summary Tables

Sequencing

Resolution

Performance and

Specifications

Decide what combination of capillary array and polymer from the table below matches your resolution and performance specifications.

Capillary

Length

(cm)

Polymer

Type

24 hr Throughput

(number of samples)

Type of Run Module

Run

Time

(min)

3130

Genetic

Analyzer

3130xl

Genetic

Analyzer

QV

20

KB

Basecaller

LOR a b

Ultra rapid

Rapid 36

Fast

Long read

36

50

80

POP-4

™

UltraSeq36_POP4

POP-7

™

UltraSeq36_POP7

POP-6

™

RapidSeq36_POP6

POP-7

POP-7

RapidSeq36_POP7

FastSeq50_POP7

POP-6

POP-7

POP-4

POP-7

StdSeq50_POP4

StdSeq50_POP6

StdSeq50_POP7

LongSeq80_POP4

LongSeq80_POP7

40 144

35 164

60 96

96

60 96

150 36

120 48

210 24

170 32

576

656

384

384

384

224

144

192

96

128 a Length of Read (LOR) is the usable range of high-quality or high-accuracy bases determined by Quality Values (QV) generated by KB

™

Basecaller v1.2. The LOR is determined by using a sliding window of 20 bases, which has an average QV > 20.

b 98.5% basecalling accuracy, less than 2% Ns.

400

500

500

600

700

600

600

850

700

950

1

Notes

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

3

Chapter 1 Preparing the Instrument

Application Summary Tables

Fragment

Analysis

Resolution

Performance and

Specifications

Decide what combination of capillary array and polymer from the table below matches

your resolution and performance specifications. See “Fragment Analysis Kits and Run

Modules” on page 5 for information on capillary, polymer, and run module.

Run Modules

Capillary

Length

(cm)

Polymer

Type

Run

Time

(min)

High Throughput, Small Size Fragment Analysis

FragmentAnalysis22

_POP4

22 POP-4

SNP22_POP4

Standard Fragment Analysis

22 20

36 POP-4 45 FragmentAnalysis36

_POP4

HIDFragmentAnalysis

36_POP4

45

SNP36_POP4

FragmentAnalysis36

_POP7

FragmentAnalysis50

_POP4

FragmentAnalysis50

_POP6

FragmentAnalysis50

_POP7

50

POP-7

POP-4

POP-6

POP-7

20

30

35

65

90

50 a 20 GT (Genotypes)/capillary/injection.

b 1bp resolution at 99.99% accuracy.

24 hr Throughput (GT a

)

3130

Analyzer

3130xl

Analyzer

Resolution

(bp)

5,760

2,560

3,840

3,290

1,760

1,200

2,300

23,040

2,560 10,240

10,240

15,360

13,170

7,040 500

4,800 500

9,220

250

500

(SD)

500 0.15

120

500

500

Specification

0.15

0.15

0.50

0.15

0.15

0.15

0.15

b

Notes

4

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Chapter 1 Preparing the Instrument

Application Summary Tables

Fragment

Analysis Kits and

Run Modules

The table below lists the Applied Biosystem kit types, with the available run module(s) and dye sets.

Module

1

Application/Kit

SNaPshot

®

Multiplex System

Custom oligos

E5 E5

Linkage Mapping Set (human) v2.5

ABI P

RISM

®

Mouse Mapping Set v1.0

4-Dye Stockmarks

®

kits (bovine and canine)

5-Dye Stockmarks

®

kit (equine)

AFLP

®

kits

4-Dye AmpFlSTR

®

kits

5-Dye AmpFlSTR kits

AmpFl

STR

Kit Table

Kits

AmpFlSTR COfiler

®

Kit

AmpFlSTR Profiler Plus

®

Kit

AmpFlSTR Profiler Plus ID Kit

AmpFlSTR SGM Plus

®

Kit

Other 4-Dye AmpFlSTR Kits

AmpFlSTR SEfiler

™

Kit

AmpFlSTR Identifiler

®

Kit

AmpFlSTR Yfiler

™

Kit

Other 5-Dye AmpFlSTR Kits

Notes

D, F,

G5

D, F,

G5

D, F,

G5

D, F,

G5

D, F,

G5

D, F,

G5

D, G5 D, G5 D, G5 D, G5 D, G5 D, G5

D, G5 D, G5 D, G5 D, G5 D, G5 D, G5

F

G5

F

HIDFragmentAnalysis

36_POP4

F

G5

F

G5

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

5

Chapter 1 Preparing the Instrument

Barcode Readers

Barcode Readers

ELECTRICAL HAZARD. Power off the instrument and the computer before connecting an external barcode reader to the instrument.

External Barcode

Readers

KEYENCE BL-80VE

An external barcode reader can be used with the 3130/3130xl instrument. With the

KEYENCE BL-80VE (see photo above), which connects to the instrument computer keyboard, you can scan barcodes into any text box in the Data Collection software. The

KEYENCE BL-80VE barcode reader uses an LED as a light source.

Notes

6

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Chapter 1 Preparing the Instrument

Starting the 3130/3130xl Genetic Analyzer System

Starting the 3130/3130xl Genetic Analyzer System

Starting the Computer Workstation

IMPORTANT!

You must start the computer workstation before starting the instrument.

1.

Power on the monitor.

R2491

130 ower button 1

2.

Power on the computer. esc

F1

F2 esc

1 tab caps

2

Q

F3

3

W

A shift control

F4

4

E

F5

F6

F7 F8

F9 F10

5

R

S

Z option

D

X

6

T

F

C

Y

7

G

V

8

U

H

B

9

I

J

N

0

O

K

M

_

-

P

F11

<

L

+

=

[

F12

F13

> delete

]

"

? /

F14 help

F15 return shift option x home end control clear

7

=

4

8

1

5

9

*

2

6

0

+

3 enter tton 1

F6

F7

F8

F5

5

R

D

X

T

6

F

C

7

Y

G

V

8

U

H

B

9

I tion

F9 F10

F11

F12

J

N

0

O

K

M

_

-

P

<

L

+

=

[

> delete

]

"

? /

F13 F14

F15 return shift help x option home end control clear

7

=

4

8

1

5

9

*

2

6

0

+

3 enter

3.

In the Log On to Windows dialog box:

a.

Enter the user name.

b.

If applicable, enter a password.

Note:

If the computer is connected to a network, you do not need to log on to the network before starting the instrument.

c.

Click .

Notes

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

7

1

Chapter 1 Preparing the Instrument

Starting the 3130/3130xl Genetic Analyzer System

Starting the 3130/3130xl Genetic Analyzer

1.

Ensure that the:

• Oven door is closed and locked

• Instrument doors are closed

Note:

If the doors are open during power on, the yellow light will continue to flash until you close the doors.

2.

Ensure that the computer is powered on.

Note:

The computer must be on and running to allow the instrument to copy the firmware from the computer.

3.

Power on the instrument by pressing the on/off button on the front of the instrument.

Note:

While the instrument is booting up and performing self-checks, the yellow status light blinks.

4.

Ensure the green status light is on and not flashing before proceeding.

Note:

If the green status light does not come on, start the Data Collection software and view the log. The pathway to the log is:

E:\AppliedBiosystems\UDC\DataCollection\

Log\Instrument Name

Status lights

Press the on/off button to start the instrument

Notes

8

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Data Collection Software

IMPORTANT!

Do not rename the computer. The instrument computer was assigned a unique name before the 3130/3130xl genetic analyzer Data

Collection software was installed. Do not rename the computer once the Data Collection software has been installed. Doing so may cause the Data Collection software to malfunction.

Starting the 3130/3130xl Data Collection

Software

1.

Select Start > All Programs > Applied

Biosystems > Data Collection > Run 3130

Data Collection v3.0 or Run 3130xl Data

Collection v3.0 to display the Service Console.

By default, all applications are off, indicated by the red circles. They launch automatically with the 3130/3130xl Data Collection software.

As each application activates, the red circles (off) change to yellow triangles (activating), and then to green squares (on) when they are fully functional.

When all the applications are running (displaying all green squares–this could take several minutes), the Foundation Data Collection window displays.

Chapter 1 Preparing the Instrument

Data Collection Software

On

Activating

1

Notes

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

9

Chapter 1 Preparing the Instrument

Data Collection Software

2.

Click + to expand subfolders in the left tree pane.

All application folders are now visible.

Select items from this tree pane to open manager, viewer, and other program windows.

3.

Click + to expand subfolders in the left tree pane.

All application folders are now visible.

Select items from this tree pane to open manager, viewer, and other program windows.

Notes

10

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Chapter 1 Preparing the Instrument

Preparing the Instrument

Preparing the Instrument

Instrument Doors and Interior

Pump block

Polymer delivery pump (PDP)

Oven

Detection cell block

Capillary array

Buffer and water reservoirs

Autosampler

Lower polymer block

Anode buffer reservoir

Part

Anode buffer reservoir

Buffer and water reservoirs (four)

Autosampler

Capillary array

Detection cell block and heater

Lower polymer block

Oven

Polymer delivery pump

(PDP)

Pump block

Function

Contains 16 mL of 1X running buffer.

Each contain 16 mL of 1X running buffer or water.

Holds the sample plates and reservoirs and moves to align the samples, water, or buffer with the capillaries.

Enables the separation of the fluorescent-labeled DNA fragments by electrophoresis. It is a replaceable unit composed of 4 or 16 silica capillaries.

Holds the capillaries in place for laser detection.

Contains the buffer valve, anode electrode, and anode buffer reservoir.

Maintains uniform capillary array temperature.

Pumps polymer into the array and performs maintenance procedures.

Includes the displacement pump chamber, piston water seal, array attachment point (array port), and connection to the lower polymer block through the interconnect tube.

Notes

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

11

1

Chapter 1 Preparing the Instrument

Preparing the Instrument

Inspecting the Instrument

1.

Open the instrument doors.

2.

Perform the daily maintenance tasks -see

Chapter 1 in the Applied Biosystems 3130/3130xl

Genetic Analyzers Maintenance,

Troubleshooting and Reference Guide.

3.

Install clean drip trays.

Polymer Delivery Pump

Mounting pin

PDP motor

Syringe fitting

Water seal

Waste fitting

Water trap

Mounting pin

Piston

Pump chamber

Pump block

Check valve

Polymer supply tube

Polymer supply bottle cap with hole

Polymer supply bottle

Electrode

GR2459

Mounting pin

O-ring

PDP motor cover

Capillary array tip

Capillary array

Capillary array knob

Double-tapered ferrule

Array port

Interconnect tube

Buffer valve pin

Lower polymer block

Mounting pin

Overflow hole

Buffer fill-line

Anode buffer reservoir

(16 mL anode reservoir)

Notes

12

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Inspecting the Instrument

1.

Open the instrument doors.

2.

Perform the daily maintenance tasks -see the

Applied Biosystems 3130/3130xl Genetic

Analyzers Maintenance, Troubleshooting and

Reference Guide for more detail on maintenance.

3.

Install clean drip trays.

Installing or Replacing the Capillary Array

IMPORTANT!

The capillary array length defined in the wizard must match the array length you are using for correct autoanalysis results.

IMPORTANT!

Wear gloves while handling polymer, the capillary array, septa, or buffer reservoirs.

CHEMICAL HAZARD. POP

polymer may cause eye, skin, and respiratory tract irritation. Please read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. Use for research and development purposes only.

1.

Install a capillary array by clicking <instrument

name

>, selecting Install Array Wizard and following the prompts.

IMPORTANT!

To install or replace an array that is a different length than the one you were using, reset the active spectral calibration (see

page 47

) or create a new spectral calibration for your dye set and array length combination (see

page 37

).

2.

Optional: Select Update Cap Array Info wizard to correct any errors or update capillary array and serial number information.

Notes

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Chapter 1 Preparing the Instrument

Preparing the Instrument

13

1

Chapter 1 Preparing the Instrument

Preparing the Instrument

When to Replenish or Change Polymer

IMPORTANT!

Always replace polymer that has been on the instrument longer than one week.

Su M T W Th F S

If polymer on the instrument...

Then ...

has been on less than one week and is in sufficient quantity to complete your runs a has been on less than one week, and insufficient in quantity to complete your runs has been on longer than one week

Remove all bubbles, and then proceed with instrument preparation.

Add fresh polymer to the polymer supply by following the

Replenish Polymer Wizard.

is the wrong type (a change between POP-4, POP-6, and/or

POP-7 polymers is required)

Replace the installed polymer type with a different type by following the Change Polymer Type Wizard.

a A 3130xl genetic analyzer run uses 50 to 80 µL of polymer and a 3130 genetic analyzer run uses ~25 to 40µL of polymer.

Before Using the Polymer

1.

Remove the polymer from 4

°C storage.

2.

Loosen the cap and bring the polymer to room temperature.

3.

To dissolve deposits, tighten the cap and gently swirl the polymer.

Replenishing the Polymer

IMPORTANT!

Wear gloves while handling polymer, the capillary array, septa, or buffer reservoirs.

CHEMICAL HAZARD. POP

polymer may cause eye, skin, and respiratory tract irritation. Please read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. Use for research and development purposes only.

1.

Click < Instrument Name> in the tree pane.

Notes

14

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

2.

Select Wizards > Replenish Polymer Wizard to replenish polymer.

IMPORTANT!

The polymer type defined in the wizard must match the polymer type that you are using.

Changing Polymer Type

IMPORTANT!

Wear gloves while handling polymer, the capillary array, septa, or buffer reservoirs.

CHEMICAL HAZARD. POP

polymer may cause eye, skin, and respiratory tract irritation. Please read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. Use for research and development purposes only.

1.

Click < Instrument Name> in the tree pane.

2.

Select Wizards > Change Polymer Type

Wizard to change to a different polymer.

Chapter 1 Preparing the Instrument

Preparing the Instrument

1

Notes

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

15

Chapter 1 Preparing the Instrument

Preparing Buffer and Filling Reservoirs

Preparing Buffer and Filling Reservoirs

Required Materials

CHEMICAL HAZARD. 10X

Genetic Analyzer Buffer with EDTA may cause eye, skin, and respiratory tract irritation. Read the

MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves.

Required materials to prepare 1X running buffer:

• 10X Genetic Analyzer Buffer (P/N 402824)

• Purified (distilled or deionized) water

• 50 mL graduated cylinder

Preparing Buffer for a Single Run

1.

Add 5 mL of 10X Genetic Analyzer buffer into a graduated cylinder.

2.

Add purified water to bring the total volume up to 50 mL.

3.

Mix well.

50 mL total

Storing Buffer

Store 1X running buffer at:

• 2 to 8 °C for up to 1 month

• Room temperature for 1 week

45 mL DI H

2

O

50 mL graduated cylinder

Buffer Storage Conditions

Option A Option B

2

°C to

8

°C

20

°C to

25

°C

5 mL 10X Genetic

Analyzer buffer

Su M T W Th F S

Su M T W Th F S

7 days

1 month

Notes

16

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Chapter 1 Preparing the Instrument

Preparing Buffer and Filling Reservoirs

Replacing Buffer and Water

Replace the 1X running buffer in the anode and cathode buffer reservoirs daily, or before each batch of runs.

IMPORTANT!

Failing to replace buffer may lead to loss of resolution and data quality.

IMPORTANT!

To clean and refill the reservoirs, the autosampler is brought to the forward position, thereby leaving the capillary tips exposed to the air.

Do not leave the autosampler in this position for an extended time because the capillaries can dry out and the array may be damaged.

Filling the Water and Cathode Buffer

Reservoirs

IMPORTANT!

Wear gloves while performing the following procedure, and any other time you handle the capillary array, septa, or buffer reservoirs.

CHEMICAL HAZARD. 1

✕

Genetic Analyzer Buffer with EDTA may cause eye, skin, and respiratory tract irritation. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves.

1.

Verify the oven and instrument doors are closed.

2.

Press the Tray button on the outside of the instrument to bring the autosampler to the forward position

3.

Wait until the autosampler stops at the forward position, then open the instrument door.

Tray button

Notes

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

17

1

Chapter 1 Preparing the Instrument

Preparing Buffer and Filling Reservoirs

4.

Remove the cathode buffer and water reservoirs from the instrument.

5.

Dispose of remaining fluids and rinse out the reservoirs with deionized water.

Note:

Follow your company’s waste disposal practices for appropriate disposal procedures.

6.

Rinse the cathode reservoir with 1X running buffer, and then fill to the line with 1X running buffer (about 16 mL).

7.

Fill the two water reservoirs to the line with quality deionized water (about 16 mL).

Be sure that the septa fit securely and flush on the tops of the reservoirs to prevent damaging the capillary tips.

8.

Place a clean reservoir septa on each reservoir, and dry the outside of the reservoirs using a lint-free tissue wipe.

9.

Place the reservoirs into position on the autosampler as shown.

Fill line

Remove

Water reservoir

(waste)

Cathode reservoir

(1X running buffer)

Home position

Water reservoir

(rinse)

Water reservoir

(unused)

10.

Close the instrument doors.

Note:

Closing the doors returns the autosampler to the last known position, placing the tips of the capillaries in water or buffer.

Notes

18

Applied Biosystems 3130/3130 Genetic Analyzers Getting Started Guide

Chapter 1 Preparing the Instrument

Preparing Buffer and Filling Reservoirs

Filling the Anode Buffer Reservoir

Change the anode buffer:

• Every 24 hours

• Before each run or batch of runs

• Every time you replenish the polymer or change polymer type

CHEMICAL HAZARD. 1X

Genetic Analyzer Buffer with EDTA may cause eye, skin, and respiratory tract irritation. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves.

1.

Remove the anode buffer reservoir by firmly pulling down and twisting slowly.

2.

Discard the used buffer appropriately.

3.

Clean and rinse the reservoir with deionized water, and then rinse with buffer.

Change the Anode Buffer

Every 24 hours Before each run or batch of runs

4.

Fill the anode buffer reservoir to the fill line with fresh 1X running buffer (about 16 mL).

Note:

The meniscus should line up with the fill line.

5.

Put the anode buffer reservoir on the instrument.

6.

If the reservoir fills with fluid, repeat this procedure to discard and replace the running buffer.

Note:

The reservoir could fill during bubble removal.

Fill line

1

Notes

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

19

Chapter 1 Preparing the Instrument

Preparing Buffer and Filling Reservoirs

Notes

20

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Chapter 2

Performing a Spatial Calibration

Preparing the

Instrument

Performing a

Spatial Calibration

Start a spatial calibration run

See page 22

Performing a

Spectral Calibration

Autoanalysis and Sequencing

Analysis

Software

Autoanalysis and

SeqScape Software

Autoanalysis and

Fragment Analysis

Evaluate spatial calibration profile

See page 23

Accept or reject spatial calibration

See page 24

Running the

Instrument

Notes

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

21

2

Chapter 2 Performing a Spatial Calibration

Spatial Calibration

Spatial Calibration

The 3130/3130xl genetic analyzer Data Collection software uses images collected during the spatial calibration to establish a relationship between the signal emitted by each capillary and the position where that signal falls on and is detected by the CCD camera.

When to Perform the Calibration

You are required to perform a spatial calibration when you:

• Install or replace a capillary array

• Temporarily remove the capillary array from the detection block

• Move the instrument

Creating a Spatial Calibration File

1.

In the tree pane of the Data Collection software, click

GA Instruments >

ga3130

or ga3130xl >

instrument

name >

Spatial Run Scheduler.

Spatial profile

Start, Accept or Reject

Spatial profile

Positions (pixel) of each capillary

Notes

22

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Chapter 2 Performing a Spatial Calibration

Evaluating a Spatial Calibration File

2.

In the Spatial Protocols section, select one of the following:

• If the capillaries contain fresh polymer, select Protocol > 3130SpatialNoFill_1

• Otherwise, select Protocol

> 3130SpatialFill_1

Note:

You do not need to fill the capillaries each time you perform a spatial calibration.

Select one

3.

Click .

The calibration run lasts approximately:

• 2 min. without filling the capillaries

• 6 min. when filling the capillaries

Note:

The spatial profile window turns black when you start a spatial calibration.

Evaluating a Spatial Calibration File

1.

Evaluate the spatial calibration profile using the following criteria:

Spatial calibration profile for 3130xl system

Peak

Attribute

Height

Orange crosses

Shape

Spacing

Acceptable Criteria

Similar heights for all peaks.

One orange cross marking the top of every peak. No misplaced crosses.

Single sharp peak for each capillary.

Small shoulders are acceptable.

The difference between adjacent positions is 13 to 16 pixels.

Theoretical spacing between capillaries is 15.

Spatial calibration profile for 3130 system

Start spatial calibration

2

Notes

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

23

Chapter 2 Performing a Spatial Calibration

Evaluating a Spatial Calibration File

2.

Examine each row in the 16 or 4 Capillary

Positions table and verify that the values in both the Left spacing and Right spacing columns range between 13 to 16 pixels.

To move the cross:

a.

Type a new value in the Positions (pixels) box for the capillary of interest.

b.

Click outside of that box or press Enter.

Accepting or Rejecting a Spatial

Calibration

If the calibration:

• Passed, click to write the calibration data to the database and .ini file.

• Failed, click , then see the Applied

Biosystems 3130/3130xl Genetic Analyzers

Maintenance, Troubleshooting and

Reference Guide.

Left spacing and

Right spacing columns

Accept and

Reject buttons

Notes

24

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Examples of Spatial Profiles

Passing Profiles

3130 Genetic Analyzer

Chapter 2 Performing a Spatial Calibration

Examples of Spatial Profiles

2

3130xl Genetic Analyzer

Failing Profile

3130xl Genetic Analyzer

Notes

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

25

Chapter 2 Performing a Spatial Calibration

Examples of Spatial Profiles

Notes

26

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Chapter 3

Performing a Spectral Calibration

Preparing the

Instrument

Performing a

Spatial Calibration

Prepare spectral calibration chemistry

See page 31

Performing a

Spectral Calibration

Create a spectral instrument protocol

See page 35

Autoanalysis and Sequencing

Analysis

Software

Perform a spectral calibration

See page 37

Autoanalysis and

SeqScape Software

Evaluate the spectral calibration data

See page 43

Autoanalysis and

Fragment Analysis

Activate a spectral calibration

See page 47

Running the

Instrument

Notes

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

27

3

Chapter 3 Performing a Spectral Calibration

Spectral Calibration

Spectral Calibration

A spectral calibration creates a matrix that is used during a run to reduce raw data from the instrument to the 4-dye or 5-dye data stored in sample files. Performing a spectral calibration is similar to performing a sample run except that calibration standards are run in place of samples and a spectral calibration module is used in place of a run module.

When to Perform the Calibration

Perform a spectral calibration:

• When you use a new dye set on the instrument

• When you change capillary array length (or polymer type for fragment analysis)

• After the laser or CCD camera has been realigned/replaced by a service engineer

• If you begin to see a decrease in spectral separation (pull-up and/or pull-down peaks) in the raw or analyzed data

What Happens?

Run the spectral standards in all 16 or 4 capillaries. The Data Collection software then:

• Collects the data and stores it in 16 or 4 separate temporary files

• Analyzes the data and generates a matrix for each capillary

• Stores the spectral calibration data for the dye set run

Changing

Capillary Array

Lengths and

Polymer Type

For each dye set, a unique spectral calibration is used for each different capillary array length.

• For every sequencing dye set, you must create a separate spectral calibration for each capillary array length you use.

• For every fragment analysis dye set, you must create a separate spectral calibration for each capillary array length and polymer type combination you use.

See “Activating a Spectral Calibration” on page 47 , on how to switch calibrations once

calibrations are performed for each dye set on each capillary length.

Supported

Sequencing

Chemistries

New basecaller, mobility files, and run modules will be developed for the 3130/3130xl series instruments solely in support of the ABI P

RISM

®

BigDye

®

Terminator v1.1 and v3.1chemistry kits. For more information, please contact Applied Biosystems Technical

Support.

ABI P

RISM

®

BigDye

®

Terminator v1.0, v2.0 and v3.0 chemistry kits have been discontinued.

Notes

28

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Chapter 3 Performing a Spectral Calibration

Spectral Calibration

Types of

Calibration

Standards

There are two types of spectral calibration standards:

• Matrix standards for fragment analysis or sequencing - A tube that contains four or five fragments each labeled with a different single dye.

•

BigDye® v3.1 or BigDye® v1.1 Terminator Sequencing Standard

- A tube of a standard chemistry reaction that contains multiple labeled fragments in each of the four dyes

Dye Set Tables

Determine the correct dye set and matrix standard set for the application you are using by using the tables below.

Sequencing Chemistry Dye Sets, Calibration Standards, and Chemistry File

Sequencing Chemistry Dye Set Spectral Calibration Standard Chemistry File

• BigDye ® Terminator v3.1 Cycle

Sequencing Kit

• ABI P

RISM

®

dGTP BigDye

®

Terminator v 3.0 Cycle Sequencing

Ready Reaction Kit a

• BigDye ® Terminator v1.1 Cycle

Sequencing Kit

• ABI P

RISM

®

BigDye ® Primer Cycle

Sequencing Kits

• ABI P

RISM

®

dGTP BigDye

®

Terminator Cycle Sequencing Ready

Reaction Kit a

ABI P

RISM

®

dRhodamine Terminator

Cycle Sequencing Kit

Z_BigDyeV3

E_BigDyeV1

BigDye ® v3.1 Matrix Standards

BigDye ® v3.1 Terminator

Sequencing Standard

DS-01 Matrix Standards

BigDye ® v1.1 Terminator

Sequencing Standard dRhodamine Matrix Standards Kit

Matrix Standard

Sequence Standard

Matrix Standard

Sequence Standard

Matrix Standard a dGTP kits are not supported on capillary electrophoresis instruments due to compressions on certain sequence context regions; you can run the kits if compression is not an issue.

3

Notes

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

29

Chapter 3 Performing a Spectral Calibration

Spectral Calibration

Fragment Analysis Dye Sets, Calibration Standards, and Chemistry File

Fragment Analysis Chemistry

Custom oligos

• ABI P

RISM

®

Mouse Mapping Set v1.0

• Custom oligos

• AFLP

®

kits

• Stockmarks

®

Kits 4-dye (bovine and canine)

• AmpFlSTR

®

COfiler

®

Kit

• AmpFlSTR ® Profiler Plus ® Kit

• AmpFlSTR ® Profiler Plus ® ID Kit

• AmpFlSTR

®

SGM Plus

®

Kit

• Other 4-Dye AmpFlSTR Kits

ABI P

RISM

®

SNaPshot ® Multiplex System

• ABI P

RISM®

Linkage Mapping Set v2.5

• Stockmarks

®

Kit 5-dye (equine)

• Custom Oligos

• AmpFlSTR ® Identifiler ® Kit

• AmpFlSTR ® SEfiler

™

Kit

• AmpFlSTR ® Yfiler

™

Kit

• Other 5-Dye AmpFlSTR Kits

Dye Set

D

D

F

E5

G5

Spectral Calibration Standard

DS-30 Matrix Standards

DS-31 Matrix Standards

DS-32 Matrix Standards

DS-02 Matrix Standards

DS-33 Matrix Standards

Chemistry File

Matrix Standard

Notes

30

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Chapter 3 Performing a Spectral Calibration

Preparing the Spectral Calibration Chemistry

Preparing the Spectral Calibration Chemistry

Preparing the Calibration Standard

1.

Prepare one of the following:

CHEMICAL HAZARD.

Formamide causes eye, skin, and respiratory tract irritation. It is a possible reproductive and birth defect hazard. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves.

• ABI P

RISM

BigDye Terminator v1.1 or v3.1

Sequencing Standard:

– Remove a tube of the Sequencing

Standard from the freezer.

– Add 170 µL of Hi-Di

™

formamide to resuspend the BigDye Terminator v1.1 or v3.1 Sequencing Standard.

• Sequencing or fragment analysis matrix standards:

– Remove a tube of the matrix standard from the refrigerator.

– Mix thoroughly, then spin briefly in a microcentrifuge.

– Follow the matrix standard insert for matrix standard and Hi-Di formamide ratios.

2.

Vortex thoroughly.

Briefly centrifuge the mixture.

Vortex

00:00:05

<1500

×g

00:00:05

95

°C

3.

Heat the standard tube at 95 °C for 5 minutes to denature the DNA.

00:05:00

4.

Cool the tubes on ice for 2 minutes.

0

°C

00:02:00

Notes

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

31

3

Chapter 3 Performing a Spectral Calibration

Loading Samples

Loading Samples

CHEMICAL HAZARD. All chemicals on the instrument, including liquid in the lines, are potentially hazardous. Please read the

MSDS, and follow the handling instructions. Wear appropriate eyewear, protective clothing, and gloves when working on the instrument.

or damaged plates.

Do not use warped

Note:

The efficient way to lay out samples in a plate is illustrated here. See

Appendix A, “Plate Mapping,”

for the relationship between sample position and priority in scheduled injections.

To load samples:

1.

Add the denatured samples to the wells of a 96or 384-well reaction plate:

• If using a 3130xl genetic analyzer:

– 96-well plate – Add 10 µL of denatured standard to wells A1 through H2.

– 384-well plate – Add 5 µL of denatured standard into alternating wells of the plate:

Row 1: A1, C1, E1, …K1, M1, O1

Row 2: Empty

Row 3: A3, C3, E3, …K3, M3, O3

• If using 3130 genetic analyzer:

– 96-well plate – Add 10 µL of denatured standard to wells A1, B1, C1 and D1.

– 384-well plate – Add 5 µL of denatured standard into alternating wells of the plate:

Row 1: A1, C1, E1 and G1

Prepared standard

(from

step 4 on page 31 )

96-Well

Plate

3130xl instrument layout

3130 instrument layout

D

E

F

G

H

A

B

C

1 2 3 4 5 6 7 8 9 10 11 12

GR1315c

Add 10 µL prepared standard

384-Well

Plate

3130xl instrument layout

3130 instrument layout

I

J

K

F

G

H

C

D

E

A

B

O

P

L

M

N

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

GR1316c

Add 5 µL prepared standard into alternating wells

Notes

32

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

2.

Seal the plate:

a.

Place the plate on a clean, level surface.

b.

Lay the septa flat on the plate.

c.

Align the holes in the septa strip with the wells of the plate, then firmly press downward onto the plate.

IMPORTANT!:

Do not heat plates that are sealed with septa.

3.

To prevent damage to the capillary array, inspect the plate and septa to verify the septa fits snugly and flush on the plate.

Chapter 3 Performing a Spectral Calibration

Loading Samples

Plate septa

Sample plate

Septa and well not aligned

Septa and well not aligned

3

4.

Briefly centrifuge the plate.

5.

Remove the plate from the centrifuge and verify that each sample is positioned correctly in the bottom of its well.

If the reagents of any well contain bubbles or are not located at the bottom of the well, repeat

steps 4 and 5

.

6.

Leave the plate on ice until you are ready to prepare the plate assembly and place the assembly on the autosampler.

<1500

× g

0:05

Prepared standard

Sample is at the bottom of the well

Notes

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

33

7.

Chapter 3 Performing a Spectral Calibration

Loading Samples

Assemble the plate assembly:

a.

Place the sample plate into the plate base.

b.

Snap the plate retainer onto the plate and plate base.

8.

Verify that the holes of the plate retainer and the septa strip are aligned. If not, re-assemble the

plate assembly (see step 7

).

IMPORTANT!

Damage to the array tips will occur if the plate retainer and septa strip holes do not align correctly.

Plate retainer

6a

6b

Plate base

Assembled components

Plate retainer holes and septa holes are not aligned

Notes

34

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Chapter 3 Performing a Spectral Calibration

Creating a Spectral Instrument Protocol

Creating a Spectral Instrument Protocol

1.

In the tree pane of the Data Collection software, click

GA Instruments > ga3130xl or

ga3130 > Protocol Manager to open the

Protocol Manager window.

Create instrument protocols here

Create analysis protocols here

2.

In the Instrument Protocols pane, click

The Protocol Editor dialog box opens.

.

3

3.

Complete the Protocol Editor dialog box.

a.

Type a name for the protocol.

b.

Type a description for the protocol

(optional).

c.

Select Spectral in the Type drop-down list.

d.

Select the correct dye set for your run. See

“Dye Set Tables” on page 29 .

Notes

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

3a

3b

3c

3d

3e

3f

3g

3h

35

Chapter 3 Performing a Spectral Calibration

Creating a Spectral Instrument Protocol

e.

f.

Select the Polymer and Array Length from the appropriate drop-down list. See table,

“Polymer, Array Length and Spectral Run

Modules.”

Select your chemistry file in the Chemistry drop-down list. Failure to select the correct chemistry file for your spectral calibration samples results in a failing spectral run. See

“Dye Set Tables” on page 29 for matching

chemistry files.

Note:

The chemistry file for fragment analysis dye sets defaults to the Matrix

Standard.

g.

In the Run Module drop-down list, select the run module. See the table

“Polymer,

Array Length and Spectral Run Modules.”

Note:

The modules list is filtered based on the polymer type, then the array length you

selected in step e

. You may have only one run module option available.

Polymer, Array Length and Spectral Run Modules

Polymer

Type

POP-4

POP-6

POP-7

Array

Length (cm)

22

36

50

80

36

50

36

50

80

Run Module

Spect22_POP4

Spect36_POP4

SpectSQ36_POP4

Spect50_POP4

Spect80_POP4

Spect36_POP6

Spect50_POP6

Spect36_POP7

Spect50_POP7

Spect80_POP7

h.

Click .

Notes

36

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Chapter 3 Performing a Spectral Calibration

Performing a Spectral Calibration

Performing a Spectral Calibration

Creating the Plate Record

1.

In the tree pane of the Data Collection software, click

GA Instruments > ga3130xl or

3130 >

instrument name >

Plate

Manager.

2.

Click box.

to open the New Plate dialog

3.

Complete the New Plate dialog box:

a.

Enter a name for the plate.

b.

Optional: Enter a description for the plate record.

c.

In the Application drop-down list, select

Spectral Calibration.

d.

In the Plate Type drop-down list, select 96-

Well or 384-Well

e.

Enter a name for the owner.

f.

Enter a name for the operator.

g.

Click .

4.

In the Spectral Calibration Plate Editor dialog box:

a.

In the Sample Name column, enter a sample name, then click the next cell. The value

100 automatically displays in the Priority column.

b.

Optional: In the Comments column, enter any additional comments or notations for the sample at the corresponding position of the plate.

c.

In the Instrument Protocol 1 column, select a protocol from the list or create a new

protocol (see step 2 on page 35

)

5.

Highlight the entire row.

4a

4b

Plate Manager

4c

3a

3b

3c

3d

3e

3f

3g

Notes

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

37

3

Chapter 3 Performing a Spectral Calibration

Performing a Spectral Calibration

6.

Select Edit > Fill Down Special.

Based on your plate type (96- or 384-well) and capillary array (16 or 4 capillaries), the software automatically fills in the appropriate well numbers for a single run.

7.

Click .

You have successfully created the plate record for the spectral calibration plate.

Notes

38

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Chapter 3 Performing a Spectral Calibration

Performing a Spectral Calibration

Placing the Plate Assembly into the

Instrument

1.

Verify the oven and front doors are closed.

2.

Press the Tray button and wait for the autosampler to stop at the forward position.

3.

Open the front doors.

4.

Place the plate assembly on the autosampler in position A or B for the 3130xl genetic analyzer and position B for the 3130 genetic analyzer.

Note:

There is only one orientation for the plate, with the notched end of the plate base away from you.

A

B

3130xl genetic analyzer

B

5.

Ensure the plate assembly fits flat in the autosampler. Failure to do so may allow the capillary tips to lift the plate assembly off of the autosampler.

6.

Close the instrument doors.

Note:

Closing the doors returns the autosampler to the home position, placing the tips of the capillaries in buffer.

3130 genetic analyzer

Notes

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

39

3

Chapter 3 Performing a Spectral Calibration

Performing a Spectral Calibration

Running the Spectral Calibration Plate

1.

In the tree pane of the Data Collection software, click

GA Instruments >

ga3130xl

or

3130 >

instrument name >

Run

Scheduler >

Plate View.

2.

Search for your plate record. There are two options:

• Select Barcode in the Type of Search dropdown list.

– In the Scan or Type Plate ID text box, type in the plate name and click Search to find the plate.

– Or if you have a limited number of plates in the database, click .

All plates in the database display in plate record section.

• Perform an advanced search by selecting

Advanced in the Type of Search drop-down list.

– Use the drop-down list to define search conditions for a category or multiple categories (Run Name, Results Group

Name, Plate Name, etc.)

Note:

Your entry for Plate Name will also be set as the Plate ID.

– For each category with a condition selected, type a value (primary search string) in the Value 1 column.

– Click . All plates in the database that match the search criteria display in the plate record section.

Notes

40

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Chapter 3 Performing a Spectral Calibration

Performing a Spectral Calibration

3.

Link the plate.

a.

Select the plate record you want to run.

b.

Click the plate position indicator that matches the plate you want linked.

The plate map color will change from yellow to green when it is successfully linked.

Note:

The 3130 genetic analyzer has only one plate position (Bay B) to link a plate record.

4.

In the toolbar of the Data Collection software window, click to begin the run.

5.

The Processing Plates dialog box opens. Click

.

Note:

The instrument may pause before running the plate to raise the oven temperature.

.

Spectral Calibration Approximate Run

Times

Capillary Length

(cm)

22

36

50

80

Run Type

Spec22_POP4

Spec36_POP4

SpecSQ36_POP4

Spec36_POP6

Spec36_POP7

Spec50_POP4

Spec50_POP6

Spec50_POP7

Spec80_POP4

Spec80_POP7

Ramp Time

(min)

3

10

10

10

5

7.5

10

7.5

10

10

Approximate Total Run

Time (min)

21

35

48

54

30

110

95

52

140

120

3a

3b

3

Notes

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

41

Chapter 3 Performing a Spectral Calibration

Performing a Spectral Calibration

Viewing the Pass/Fail Status After the Run

After the spectral calibration run, the pass or fail status of each capillary is recorded in the Event Log section of the Instrument Status window.

1.

In the tree pane of the Data Collection software, click

GA Instruments >

ga3130xl

or

ga3130 >

instrument name >

Instrument Status > Event Log.

2.

In the Event Messages section of the window, view the status of each capillary.

Note:

The window below shows the dye set G5 status results.

Cap #

Pass/fail status

Q-value

Condition Number

Notes

42

Each capillary should have a Q-value above 0.95 and a Condition number range shown in the table.

Note:

If the entire spectral calibration failed, see the Applied Biosystems 3130/3130xl Genetic

Analyzers Maintenance, Troubleshooting and

Reference Guide for troubleshooting information.

Dye Set

Acceptable

Condition

Number Range

Sequencing Analysis

Z_BigDyeV3 3 to 5

E_BigDyeV1

Fragment Analysis

D

F

E5

G5

Any 4- or 5-Dye set

4 to 8.5

6 to 12

2.5 to 4

8.5 to 14.5

1 to 20

Q-Value

0.95

0.80

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Chapter 3 Performing a Spectral Calibration

Evaluating the Spectral Calibration Data

Evaluating the Spectral Calibration Data

IMPORTANT!

Evaluate the spectral calibration profile for each capillary, even if the Spectral Calibration

Results box indicates that they passed.

Note:

Pages 49 to

50 for examples of passing

sequencing spectral calibration profiles. See

pages 51

to 52 for examples of passing fragment analysis

spectral calibration profiles.

Evaluating the Spectral Profile and Raw

Data

1.

In the tree pane of the Data Collection software, click

GA Instruments >

ga3130xl

or

ga3130 >

instrument name >

Spectral

Viewer.

3

Plate diagram

Click each cell to verify the spectral profile and raw data

Notes

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Spectral profile

Raw data

(matrix standards)

View, rename, or set the active spectral calibration for the selected dye set

43

Chapter 3 Performing a Spectral Calibration

Evaluating the Spectral Calibration Data

2.

In the Dye Set drop-down list, select the dye set you just created.

Note:

If the spectral calibration failed (no spectral profiles are created), see the Applied

Biosystems 3130/3130xl Genetic Analyzers

Maintenance, Troubleshooting and Reference

Guide for more information.

3.

In the plate diagram, select a well on the plate diagram to view the capillary spectral results.

Note:

A failing capillary is automatically assigned the spectral profile of its nearest passing capillary.

Well A1

Capillary status:

Passed (dark green)

Passed well elected (light green)

Failed (tan)

Failed well selected (light tan)

4.

Evaluate the spectral profile and raw data for the selected capillary:

a.

Verify that the order of the peaks in the spectral profile from left to right are:

– 4-dye: blue-green-yellow-red

– 5-dye: blue-green-yellow-red-orange

Do the peaks in the profile appear in the correct order?

Yes

No

Then

Go to step b .

The calibration run has failed. See the

Applied

Biosystems

3130/3130xl

Genetic Analyzers

Maintenance,

Troubleshooting, and Reference

Guide.

Blue Green Yellow Red

Example of a 4-dye spectral profile

Blue Green Yellow Red

Example of a 5-dye spectral calibration profile

Orange

Notes

44

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Chapter 3 Performing a Spectral Calibration

Evaluating the Spectral Calibration Data

b.

Verify that the order of the peaks in the raw data profile from left to right are:

Sequencing

– 4-dye: red-yellow-blue-green

Fragment Analysis

– 4-dye: red-yellow-green-blue

– 5-dye: orange-red-yellow-green-blue

Are peaks in the wrong order or extraneous peaks adversely affecting the spectral profile?

Yes

No

Then

The calibration run has failed. See the

Applied

Biosystems

3130/3130xl

Genetic Analyzers

Maintenance,

Troubleshooting, and Reference

Guide.

Go to step c

.

c.

Verify that the peaks in the spectral profile do not contain gross overlaps, dips, or other irregularities (see

“Magnifying the Spectral

Profile or Raw Data” on page 46 ).

Are the peaks separate and distinct?

Yes

Then

No

The capillary has passed. Go to step 5.

The calibration run has failed. See the

Applied

Biosystems

3130/3130xl

Genetic Analyzers

Maintenance,

Troubleshooting, and Reference

Guide.

Red

Yellow Blue Green

Example of a 4-dye sequencing raw data profile

Orange Red Yellow Green Blue

Example of a 5-dye fragment analysis raw data profile

Peaks are distinct, regular and in the proper order – pass

Red peak is not distinct, regular or in the proper order – fail

Notes

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

45

3

Chapter 3 Performing a Spectral Calibration

Evaluating the Spectral Calibration Data

5.

Repeat steps 3 and 4 for each capillary in the array.

6.

Optional: Rename the spectral run. The spectral file default name is the day, date, and time of the run.

a.

Click .

b.

In the Rename Calibration dialog box, enter a descriptive name for the spectral calibration including the dye set, array length and polymer type (optional).

c.

Click .

Magnifying the Spectral Profile or Raw Data

1. In the tree pane of the Data Collection software, click

GA Instruments >

ga3130 >

instrument

ga3130xl or

name > Spectral Viewer.

2. In the spectral profile or raw data display, click-drag the cursor to create a box around the area of interest.

Selecting an area to magnify in a spectral profile

3. Release the mouse button.

The Data Collection software displays the selected region.

4. Press r to reset the view.

Magnified area of that spectral profile

Notes

46

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Chapter 3 Performing a Spectral Calibration

Activating a Spectral Calibration

Activating a Spectral Calibration

IMPORTANT!

A run cannot start unless a calibration file that matches the dye set and capillary array length combination to be used for the run, is active.

IMPORTANT!

While the software does not force you to recalibrate spectrally after changing polymer types, it is strongly recommended that you perform a spectral calibration after a polymer type change.

IMPORTANT!

Whenever you install or replace an array or switch dye sets, you must either set the active spectral calibration for that dye set and array length combination or perform a new spectral calibration and then activate it.

IMPORTANT!

set.

Every new spectral calibration is automatically the active one for that dye

Setting an Active

Spectral

Calibration

You may activate a previously created spectral calibration for a run as long as the calibration matches the dye set and array length combination (and, for fragment analysis, the polymer type) that you wish to use. Use an active spectral calibration for:

• Sequencing analysis applications that require a separate spectral calibration (for the same dye set) for different capillary array lengths and polymer type

• Fragment analysis applications that require a separate spectral calibration (for the same dye set) for different capillary array lengths and polymer type

• Repeat spectral calibrations where the original calibration is better than the second one

1.

In the tree pane of the Data Collection software, click

GA Instruments >

ga3130xl

or ga3130 >

instrument

name >

Spectral Viewer.

IMPORTANT!

If the Spectral Viewer window is blank and deactivated, then either:

–The spectral calibration for that dye set is not in the database or,

–You changed the array length and you do not have a spectral calibration file activated for that dye set and array length combination.

2.

In the Dye Set drop-down list, select a dye set.

3

Notes

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

47

Chapter 3 Performing a Spectral Calibration

Activating a Spectral Calibration

3.

In the List of Calibrations for Dye Set drop-down list, select the spectral calibration you want to use. The spectral profile and raw data is displayed.

4.

If the spectral calibration is acceptable, then click

. Otherwise, run a new spectral calibration.

Notes

48

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Chapter 3 Performing a Spectral Calibration

Examples of Passing Sequencing Spectral Calibrations

Examples of Passing Sequencing Spectral Calibrations

Dye Set Z Created from Matrix

Standard

Dye Set Z Created from a

Sequencing

Standard

3

Notes

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

49

Chapter 3 Performing a Spectral Calibration

Examples of Passing Sequencing Spectral Calibrations

Dye Set E Created from Matrix

Standard Set

DS-01

Dye Set E Created from a

Sequencing

Standard

Notes

50

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Chapter 3 Performing a Spectral Calibration

Examples of Passing Fragment Analysis Spectral Calibrations

Examples of Passing Fragment Analysis Spectral

Calibrations

Dye Set G5

Created from

Matrix Standard

Set DS-33

Dye Set F Created from Matrix

Standard Set

DS-32

3

Notes

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

51

Chapter 3 Performing a Spectral Calibration

Examples of Passing Fragment Analysis Spectral Calibrations

Dye Set D

Created from

Matrix Standard

Set DS-30

Dye Set E5

Created from

Matrix Standard

Set DS-02

Notes

52

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Chapter 4

Autoanalysis and

Sequencing Analysis Software

Preparing the

Instrument

Performing a

Spatial Calibration

Performing a

Spectral Calibration

Autoanalysis and Sequencing

Analysis

Software

Autoanalysis and

SeqScape Software

Autoanalysis and

Fragment Analysis

Running the

Instrument

Notes

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Create protocols for automated sequencing analysis

See page 59

Create a

Sequencing Analysis

Plate Record

See page 77

53

4

Chapter 4 Autoanalysis and Sequencing Analysis Software

Sequencing Analysis Software

Sequencing Analysis Software

See the Applied Biosystems DNA Sequencing Analysis Software v5.1 User Guide

(P/N 4346366) for details on sequencing analysis or the Sequencing Analysis

Software v5.2 User Bulletin titled New Features (P/N 4358355) for an update on

Sequencing Analysis Software v5.2.

Analyze your sequencing samples automatically using autoanalysis or manually.

Autoanalysis

Perform autoanalysis of sequencing samples using features of the 3130/3130xl genetic analyzer Data Collection software and Sequencing Analysis Software v5.2 or higher.

The Sequencing Analysis Software must be installed and registered with the

3130/3130xl genetic analyzer Data Collection software before you can create files required for autoanalysis.

Autoanalysis can only be performed on the same computer that collected the sample files. If you perform autoanalysis on samples, but wish to edit/review results on another computer, transfer the analysis protocol to the Sequencing Analysis Software database.

To analyze samples on another computer, transfer the files to that location.

Manual Analysis

If the run is not set up for autoanalysis, see the Applied Biosystems DNA Sequencing

Analysis Software v5.1 User Guide and the Sequencing Analysis Software v5.2 User

Bulletin - New Features.

File-Naming

Convention

Some alphanumeric characters are not valid for user names or file names. Do not use the characters below: spaces

\ / : * ? " < > |

IMPORTANT!

An error message is displayed if you use any of these characters. You must remove the invalid character to continue.

Notes

54

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Chapter 4 Autoanalysis and Sequencing Analysis Software

About Plate Records and Sequencing Analysis

About Plate Records and Sequencing Analysis

Overview

Plate records are data tables in the instrument database that contain the following information:

• Plate name, type, and owner

• Position of the sample on the plate (well number)

• Sample Name

• Mobility file (in Analysis Protocol)

• Comments about the plate and about individual samples

• Name of the run module and Dye set information (run modules specify the conditions to run the samples)

• Name of the Analysis Protocol

Note:

A plate record is similar to a sample sheet or an injection list that you may have used with other ABI P

RISM

®

instruments.

When to Create a

Plate Record

Create a plate record for each plate of samples for the following types of runs:

• Spectral calibrations

• Sequencing Analysis Software

• SeqScape Software analysis

• GeneMapper

®

software

• Mixed (sequencing and fragment analysis samples - see the Applied Biosystems

3130/3130xl Genetic Analyzers Maintenance, Troubleshooting, and Reference

Guide for creating a mixed plate record)

For runs to begin, you must create a plate record for and link it to a plate loaded on the instrument. However, you can create plate records for new plates while a run is in progress.

About a

Sequencing

Analysis Plate

Record

The Plate Editor displays an empty plate record for the application selected in the New

Plate dialog box. The data fields within a given plate record vary depending on the selected application. This section describes the sequencing analysis plate record’s data fields.

The table below describes required plate editor fields:

Parameters Description

Instrument Protocol Contains the run module and dye set needed to run the instrument.

Analysis Protocol Contains everything needed to analyze sequencing data.

Results Group Defines the file type, the file name, file save locations, analysis software and autoanalysis.

See Page

59

62

70

Notes

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

55

4

Chapter 4 Autoanalysis and Sequencing Analysis Software

About Plate Records and Sequencing Analysis

Elements of a Sequencing Analysis Plate Record

Plate Manager

Plate Record

Results Group Instrument Protocol Analysis Protocol

Results Group

Name

Analysis software and autoanalysis

File storage location

File and run folder name preferences

Instrument

Protocol Name

Run type

(Regular)

Run module

Dye set

Analysis Protocol

Name

Basecaller and

DyeSet/Primer file

Bases called

(pure or mixed)

Assigning Quality

Values (QVs)

Post processing

(clear range

trimming)

IMPORTANT!

For data collection and autoanalysis to succeed, each run of samples must have an Instrument Protocol, an Analysis Protocol, and a Results Group assigned within a plate record.

Notes

56

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

A Blank

Sequencing

Analysis Plate

Record

Chapter 4 Autoanalysis and Sequencing Analysis Software

About Plate Records and Sequencing Analysis

1

2

3

4

5

6

Default is one sample run, to add additional runs see

page 79

Columns inserted in a plate record for a sequencing analysis run

Number and

Column

1. Sample Name

2. Comment

3. Priority

Description

Name of the sample

Comments about the sample (optional)

A default value of 100 to each sample. Changing the value to a smaller number increases the priority of that set of 16 or 4 samples to run before the others in the injection list.

• New: Opens the Results Group Editor dialog box

• Edit: Opens the Results Group Editor dialog box for the Results

Group listed in the cell

• None: Sets the cell to have no selected Results Group

• Select one of the available Results groups from the list

Note:

You must have a Results Group selected for each sample entered in the Sample Name column.

See

“Results Group for Sequencing Analysis” on page 70 .

4

Notes

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

57

Chapter 4 Autoanalysis and Sequencing Analysis Software

About Plate Records and Sequencing Analysis

Number and

Column

5. Instrument

Protocol

6. Analysis Protocol

Description

• New: Opens the Protocol Editor dialog box.

• Edit: Opens the Protocol Editor dialog box for the Instrument

Protocol listed in the cell.

• None: Sets the cell to have no selected protocol.

• List of Instrument Protocols: In alpha-numeric order.

Note:

You must have an Instrument Protocol selected for each sample entered in the Sample Name column.

See

“Instrument Protocol for Sequencing Analysis” on page 59 .

• New: Opens the Analysis Protocol Editor dialog box.

• Edit: Opens the Analysis Protocol Editor dialog box for the

Instrument Protocol listed in the cell.

• None: Sets the cell to have no selected protocol.

• List of Analysis Protocols: In alpha-numeric order

Note:

You must have an Analysis Protocol selected for each sample entered in the Sample Name column.

See

“Analysis Protocol for Sequencing Analysis” on page 62 .

Notes

58

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Chapter 4 Autoanalysis and Sequencing Analysis Software

Creating Protocols for Automated Sequencing Analysis

Creating Protocols for Automated Sequencing Analysis

If the appropriate instrument protocol, analysis protocol, and results group have been created, proceed

to “Filling Out a Sequencing Analysis Plate Record” on page 77 .

Instrument Protocol for Sequencing Analysis

An instrument protocol contains all the settings necessary to run the instrument: the protocol name, type of run, run module, and dye set.

Do not edit the instrument protocol currently being used, while the instrument is running.

Creating an Instrument Protocol

1.

In the tree pane of the Data Collection software, click

GA Instruments >

ga3130xl

or

ga3130 > Protocol Manager.

Create instrument protocols here

Create analysis protocols here

4

Notes

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

59

Chapter 4 Autoanalysis and Sequencing Analysis Software

Creating Protocols for Automated Sequencing Analysis

2.

In the Instrument Protocol section, click

.

The Protocol Editor opens.

3.

Complete the Protocol Editor:

a.

Type a name for the protocol.

b.

Type a description for the protocol

(optional).

c.

Select Regular in the Type drop-down list.

d.

Select the correct run module for your run.

See “Sequencing Resolution Performance and Specifications” on page 3 .

Note:

To customize a run module, see the

Applied Biosystems 3130/3130xl Genetic

Analyzers Maintenance, Troubleshooting,

and Reference Guide.

3a

3b

3c

3d

3e

3f

Notes

60

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Chapter 4 Autoanalysis and Sequencing Analysis Software

Creating Protocols for Automated Sequencing Analysis

e.

Select the correct Dye Set for your run by using the table below.

POP-4

Polymer

POP-6

Polymer

POP-7 Polymer

Chemistry Dye Set

BigDye

®

Terminator v3.1 Cycle Sequencing Kit Z_BigDye

V3

ABI P

RISM

®

dGTP BigDye

®

Terminator v3.0

Cycle Sequencing Ready Reaction Kit

BigDye

®

Terminator v1.1 Cycle Sequencing Kit E_BigDye

V1

ABI P

RISM

®

dGTP BigDye

®

Terminator Cycle

Sequencing Kit*

ABI P

RISM

®

dRhodamine Dye Terminator

Cycle Sequencing Ready Reaction Kit

ABI P

RISM

®

BigDye ® Primer Cycle Sequencing

Kits

✓

—

✓

—

✓

—

✓

—

✓

—

✓

—

✓

—

✓

—

✓

—

✓

—

✓

—

✓

✓

✓

—

✓

—

✓

✓

✓

—

✓

—

—

—

✓

—

✓

—

—

—

✓

—

✓

—

—

—

✓

—

✓

—

—

—

✓

—

✓

—

—

—

*dGTP kits are not supported on capillary electrophoresis instruments due to compressions on certain sequence context regions; you can run the kits if you do not care about the compression issues.

f.

Click .

Importing an Instrument Protocol

1.

Click in the Instrument Protocols pane of the Protocol Editor window to display the standard File Import dialog box.

2.

Navigate to the location of the .xml file you want to import.

3.

Select the .xml file and click Open.

Exporting an Instrument Protocol

1.

In the Instrument Protocols pane, highlight the protocol you want to export.

2.

Click to display the standard File

Export dialog box.

3.

Browse to the desired folder location.

4.

Click .

Notes

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

61

4

Chapter 4 Autoanalysis and Sequencing Analysis Software

Analysis Protocol for Sequencing Analysis

Analysis Protocol for Sequencing Analysis

An analysis protocol contains all the settings necessary for analysis and post processing:

• Protocol name – The name, description of the analysis protocol, and the sequence file formats to be used

• Basecalling settings – The basecaller,

DyeSet/Primer file, and analysis stop point to be used

• Mixed Bases (Optional) – When two bases are found at the same position. Define the percent value of the second highest to the highest peak

• Clear Range – The high quality sequence remaining after trimming low quality sequences typically found at the beginning and ends of the sequence. The clear range can be based on base positions, sample quality values, and/or number of ambiguities (Ns) present

Note:

If you created an appropriate analysis protocol in the Sequencing Analysis software, you can use it in

Data Collection software.

IMPORTANT!

Do not delete an analysis protocol during a run while it is being used for that run.

Autoanalysis will not be performed if you do so.

Creating an Analysis Protocol

See the Applied Biosystems DNA Sequencing Analysis

Software v5.2 User Guide (P/N 4346366) for more information on analysis protocols.

1.

In the Analysis Protocol section of the Protocol

Manager, click .

If more than one analysis application is installed on the data collection computer, the Analysis

Applications dialog box opens.

Note:

When you name your analysis protocol, assign a version name to help you distinguish your analysis protocols.

Notes

62

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Chapter 4 Autoanalysis and Sequencing Analysis Software

Analysis Protocol for Sequencing Analysis

2.

Select Sequencing Analysis, then click to open the Analysis Protocol Editor dialog box.

3.

In the General tab:

a.

Enter a unique name and description for the new protocol.

b.

Select the appropriate Sequence File formats settings.

Option

Write .Seq File check box

Write Standard

Chromatogram

Format file

(.scf)

Write Phred

(.phd.1) File

Creates…

A .seq file for printing the sequence as text file or for use in other software.

• Use ABI format with

Applied Biosystems software.

• Use FASTA format with other software.

A .scf file to use with other software. When created, the .scf extension is not appended to the file name.

A .phd.1 file to use with other software if you used the KB

™

basecaller.

3a

3b

4

Notes

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

63

Chapter 4 Autoanalysis and Sequencing Analysis Software

Analysis Protocol for Sequencing Analysis

4.

Select the Basecalling tab.

a.

See “3130/3130xl Genetic Analyzer

Basecaller and DyeSet/Primer Files” on page 66 to select your basecaller and

DyeSet primer file.

Note:

Sequencing Analysis Software and

3130/3130xl genetic analyzer Data

Collection software filter .mob file choices to match the chosen .bcp file.

Notes

64

b.

In the Processed data pane, select True or

Flat Profile.

Option Used to display data as processed traces scaled...

Uniformly - the average height of peaks in the region of strongest signal is about equal to a fixed value. The profile of the processed traces will be very similar to that of the raw traces.

Semi-locally - the average height of peaks in any region is about equal to a fixed value. The profile of the processed traces will be flat on an intermediate scale (> about 40 bases).

Note:

This option is applied to data that is analyzed with the KB

™

basecaller only. If you use the ABI basecaller, the profile option reverts to True Profile.

c.

If desired, select one or more stop points for data analysis.

d.

Select your Threshold Quality option.

Option Function

With the KB

™

basecaller, use this setting to assign a base to every position, as well as the QV.

With the KB basecaller, use this setting to assign Ns to base with

QVs less than the set point. The QV is still displayed.

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

4d

4b

4a

4c

Chapter 4 Autoanalysis and Sequencing Analysis Software

Analysis Protocol for Sequencing Analysis

5.

Select the Mixed Bases tab.

Note:

This function is active with the KB

Basecaller only.

a.

For mixed bases only, select Use Mixed

Base Identification.

b.

Use the default setting of 25% or change the detection level by entering a new value or dragging the % line up or down.

Note:

Do not use less than 15% as your detection limit.

5a

5b

6.

Select the Clear Range tab.

Note:

The clear range is the region of sequence that remains after excluding the low-quality or error prone sequence at both the 5´ and 3´ ends.

Select one or more Clear Range methods. If you apply multiple methods, the smallest clear range results.

7.

Click to save the protocol and close the

Sequence Analysis Protocol Editor dialog box.

Use with ABI and

KB Basecallers

Use with

KB Basecaller

Use with ABI and

KB Basecallers

4

Notes

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

65

Chapter 4 Autoanalysis and Sequencing Analysis Software

Analysis Protocol for Sequencing Analysis

3130/3130xl Genetic Analyzer Basecaller and DyeSet/Primer Files

Basecaller and DyeSet/Primer Files Used with BigDye

®

Terminator Chemistry and KB Basecalling

DNA Sequencing

Chemistry

BigDye

®

Terminator v1.1

Cycle Sequencing Kit

BigDye

®

Terminator v3.1

Cycle Sequencing Kit

Polymer

POP-4

™

POP-6

™

POP-7

™

POP-4

POP-6

POP-7

KB Basecalling Run

Module

UltraSeq36_POP4

StdSeq50_POP4

LongSeq80_POP4

RapidSeq36_POP6

StdSeq50_POP6

UltraSeq36_POP7

RapidSeq36_POP7

FastSeq50_POP7

StdSeq50_POP7

LongSeq80_POP7

UltraSeq36_POP4

StdSeq50_POP4

LongSeq80_POP4

RapidSeq36_POP6

StdSeq50_POP6

UltraSeq36_POP7

RapidSeq36_POP7

FastSeq50_POP7

StdSeq50_POP7

LongSeq80_POP7

DyeSet/Primer

KB_3130_POP4_BDTv1.mob

KB_3130_POP6_BDTv1.mob

KB_3130_POP7_BDTv1.mob

KB_3130_POP4_BDTv3_.mob

KB_3130_POP6_BDTv3.mob

KB_3130_POP7_BDTv3.mob

Basecaller

KB.bcp

Basecaller and DyeSet/Primer Files Used with BigDye

®

Terminator Chemistry and ABI Basecalling

DNA

Sequencing

Chemistry

BigDye

®

Terminator v1.1

Kit

Polymer

POP-4

™

POP-6

™

ABI Basecalling Run

Module

UltraSeq36_POP4

LongSeq80_POP4

RapidSeq36_POP6

StdSeq50_POP6

Basecaller

Basecaller-

3130POP4UR.bcp

Basecaller-

3130POP4_80cmv3.bcp

Basecaller-

3130POP6RRv2.bcp

Basecaller-

3130POP6SR.bcp

DyeSet/Primer

DT3130POP4LR{BD}v1.

mob

DT3130POP6{BD}v2.mob

Notes

66

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Chapter 4 Autoanalysis and Sequencing Analysis Software

Analysis Protocol for Sequencing Analysis

Basecaller and DyeSet/Primer Files Used with BigDye

®

Terminator Chemistry and ABI Basecalling (continued)

BigDye

Terminator v3.1

Cycle

Sequencing Kit

ABI P

RISM

® dRhodamine

Dye Terminator

Cycle

Sequencing

Ready Reaction

Kit

POP-4

POP-6

POP-4

POP-6

UltraSeq36_POP4

LongSeq80_POP4

RapidSeq36_POP6

StdSeq50_POP6

UltraSeq36_POP4

LongSeq80_POP4

RapidSeq36_POP6

StdSeq50_POP6

Basecaller-

3130POP4UR.bcp

Basecaller-

3130POP4_80cmv3.bcp

Basecaller-

3130POP6RRv2.bcp

Basecaller-

3130POP6SRv2.bcp

Basecaller-

3130APOP4UR.bcp

Basecaller-

3130POP4_80cmv3.bcp

Basecaller-

3130POP6RRv2.bcp

Basecaller-

3130POP6SR.bcp

DT3130POP4{BDv3}v1.m

ob

DT3130POP6{BDv3}v1.m

ob

DT3130POP4{dRhod}v2.

mob

DT3130POP6{dRhod}v2.

mob

Dye Primer Chemistry

Basecaller and DyeSet/Primer Files Used for Dye Primer Chemistry

DNA

Sequencing

Chemistry

ABI P

RISM

®

BigDye

®

Primer

Cycle

Sequencing Kit

Polymer

POP-6

ABI Basecalling Run

Module

RapidSeq36_POP6

StdSeq50_POP6

Basecaller

Basecaller-

3130POP6RRv2.bcp

Basecaller-

3130POP6SR.bcp

DyeSet/Primer

DP3130POP6{BD

-21M13}v1.mob

DP3130POP6{BD

-21M13Rev}v1.mob

4

Notes

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Chapter 4 Autoanalysis and Sequencing Analysis Software

Analysis Protocol for Sequencing Analysis

Editing and Deleting Analysis Protocols

Editing an Analysis Protocol

1.

In the Analysis Protocols pane in the Analysis

Protocol Manager, highlight the protocol you want to edit.

2.

Click .

3.

Make changes in the General, Basecalling,

Mixed Bases and Clear Range tabs, if appropriate.

4.

Click to save the protocol and close the

Analysis Protocol Editor dialog box.

Deleting an Analysis Protocol

IMPORTANT!

Do not delete an analysis protocol during a run while it is being used for that run.

Autoanalysis will not be performed if you do so. You must first delete any plate records using the analysis protocol before you can delete or modify the analysis protocol for these plate records.

1.

In the Analysis Protocols pane in the Analysis

Protocol Manager, highlight the protocol you want to delete.

2.

Click to display the Deletion

Confirmation dialog box.

3.

Click .

Notes

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Chapter 4 Autoanalysis and Sequencing Analysis Software

Analysis Protocol for Sequencing Analysis

Exporting an Analysis Protocol

1.

In the Analysis Protocols pane in the Analysis

Protocol Manager, highlight the protocol you want to export.

2.

Click to display the standard File

Export dialog box.

3.

Browse to the desired folder location.

4.

Click Save.

Importing an Analysis Protocol

1.

Click to display the standard File

Import dialog box.

2.

Browse for the .xml file to import and click

Open.

Note:

For multiple applications, select the appropriate application to associate with the analysis protocol.

4

Notes

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69

Chapter 4 Autoanalysis and Sequencing Analysis Software

Results Group for Sequencing Analysis

Results Group for Sequencing Analysis

A Results Group is a component within Data

Collection that organizes samples and certain user settings under a single name. It is called a Results

Group because it is used to name, sort, and deliver samples that result from a run.

Creating a Results Group

1.

In the tree pane of the Data Collection software, click

GA Instruments > Results Group.

2.

Click window.

to display the Results Group Editor

3.

Complete the General tab:

a.

Type a unique Results Group Name. The name can be used in grouping sample files.

b.

Type a Results Group Owner (optional).

The owner name can be used in naming and sorting sample files.

c.

Type a Results Group Comment (optional).

3a

3b

3c

Notes

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Chapter 4 Autoanalysis and Sequencing Analysis Software

Results Group for Sequencing Analysis

4.

Select the Analysis tab, then:

a.

Select Sequencing Analysis from the

Analysis Type drop-down list.

b.

In the Analysis Actions section, select Do

Autoanalysis, if you want your data automatically analyzed after a run.

Note:

Login ID and password are not required for Sequencing Analysis software.

4a

4b

5.

Select the Destination tab, then use the default destination or define a new location for data storage.

To use …

default location* custom location

Then …

skip to step 6.

complete steps a-b.

a.

Click Use Custom Location, then click

to navigate to a different save location.

b.

Click

to test the Location path name connection:

If it…

Then a message box displays …

Fails

<“path”>.

Test failed:<“path”>.

*Sample File Destinations

Locations where sample files are placed during extraction:

• Default Destination, default folder naming: E:\AppliedBiosystemsUDC\DataCollection\data\<instrument type>\

<instrument name>\run folder

• Default Destination, custom folder naming: E:\AppliedBiosystemsUDC\DataCollection\data\top custom folder\ subfolders, etc.

• Custom Destination, default folder naming: Destination\<instrument type>\<instrument name>\run folder

• Custom Destination, custom folder naming: Destination\top customer folder\subfolders, etc.

5a

5b

5c

4

Notes

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Chapter 4 Autoanalysis and Sequencing Analysis Software

Results Group for Sequencing Analysis

6.

Select the Naming tab.

Use the Naming tab to customize sample file and run folder names. You have two options:

• Use the default names for the folder and sample file.

• Use custom names for the folder and sample

file. See “Optional: Completing Sample File

Name Format Pane” on page 73 for

elements of the Naming tab.

Note:

Sample name, run folder name, and path name, combined, can total no more than 250 characters.

Sample

File Name

Format pane

Run Folder

Name

Format pane

Notes

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Chapter 4 Autoanalysis and Sequencing Analysis Software

Results Group for Sequencing Analysis

Optional: Completing Sample File Name

Format Pane

Note:

The default sample file naming for sequencing analysis is: seq_<capillary number>_<wellposition>.ab1

1.

Select the Naming tab.

2.

Type a prefix for the file name in the Prefix box.

Your entry is shown in the Example line.

3.

Select the symbol that separates the Format elements in the file name from the Name

Delimiter drop-down list. Only one delimiter symbol may be chosen.

4.

Click the Format list and then select the components that you want in the sample name.

Note:

All the samples from a single run can be placed in the same run or results folder, so the name of every sample from a single run should be different. Most of the Format options are not different between samples, so select at least one of the options that make the sample names unique within a run.

Notes

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4

Chapter 4 Autoanalysis and Sequencing Analysis Software

Results Group for Sequencing Analysis

If a unique identifier is not included in the name, a warning message displays. Select from the elements to make a unique sample file name.

Examples are:

– Well position

– Capillary number

– Run sequence number

– Plate Quadrant

Note:

some identifiers, such as well position, capillary number and plate quadrant are not unique in multiple sample instances. Plate

Quadrant is not a unique identifier for a 96-well plate.

As you select the elements for the file name, they are placed in the Example line. An additional element drop-down menu displays allowing you the option of selecting an additional element.

Warning message

Notes

74

The names of the Format elements eventually truncate, but the Example field remains visible

(up to 72 characters).

Note:

Resize the Results

Group window horizontally to view the element options.

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Chapter 4 Autoanalysis and Sequencing Analysis Software

Results Group for Sequencing Analysis

5.

Type the suffix for the file name in the Suffix box.

Note:

The File Extension field displays the file extension generated from the Analysis Type specified on the Analysis tab (see

page 71

). For example, Sequencing Analysis produces sample files with an .ab1 extension.

Optional: Run Folder/Sub-Folder Name

Format Pane

Note:

The default run folder naming for sequencing analysis is Run_<Instrumentname>_<Dateand

TimeofRun>_<RunSeq#>.

Follow the same steps described for the Sample File

Name Format pane (see page 73

) to change the subfolder name within the run folder. Make the subfolder name unique, such as using the Run Folder name.

Saving a Results Group

Click from any tab once all the elements within the Results Group have been chosen.

Note:

Even if you create a custom run folder location, the system generates a separate default run folder that contains the log file.

Importing and Exporting a Results Group

Results Groups can be imported from, or exported to,

XML files allowing sharing of identical Results

Groups between instruments.

Importing a Results Group

1.

In the tree pane of the Data Collection software, click

GA Instruments > Results Group.

2.

Click to display the standard File

Import dialog box.

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4

Chapter 4 Autoanalysis and Sequencing Analysis Software

Results Group for Sequencing Analysis

3.

Navigate to the file you want to import.

Note:

Import file type is .xml (XML file).

4.

Click .

Exporting a Results Group

1.

In the tree pane of the Data Collection software, click

GA Instruments > Results Group.

2.

Click the Results Group name to select it.

3.

Click .

A standard file export dialog box displays with the chosen Results Group name.

4.

Navigate to the location where you want to save the exported file.

5.

Click .

Note:

If there is a name conflict with a Results

Group that already exists at the save location, then duplicate the Results group. Duplication copies the settings into a similar Results Group without the risk of user error when copying it manually (see procedure below).

Duplicating a Results Group

1.

Click the Results Group name.

2.

Click .

Note:

When you duplicate a Results Group, you are asked to type a name for the new Results

Group and for the analysis application type.

Notes

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Chapter 4 Autoanalysis and Sequencing Analysis Software

Filling Out a Sequencing Analysis Plate Record

Filling Out a Sequencing Analysis Plate Record

Creating a Sequencing Analysis Plate

Record

1.

In the tree pane of the Data Collection software, click

GA Instruments >

ga3130xl

or

ga3130 > Plate Manager.

2.

Click dialog box.

to display the New Plate Dialog

3.

Complete the information in the New Plate

Dialog:

a.

Type a name for the plate.

b.

Type a description for the plate (optional).

c.

Select your sequencing application in the

Application drop-down list.

d.

Select 96-well or 384-well in the Plate Type drop-down list.

e.

Type a name for the owner and operator.

f.

Click to open the Sequencing

Analysis Plate Editor.

3a

3b

3c

3d

3e

3f

3f

4

Notes

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Chapter 4 Autoanalysis and Sequencing Analysis Software

Filling Out a Sequencing Analysis Plate Record

Completing a Sequencing Analysis Plate

Record

1.

In the Sample Name column of a row, enter a sample name, then click the next cell. The value

100 automatically displays in the Priority column.

2.

In the Comments column, enter any comments or notations for the sample.

3.

In the Priority column, change the priority value, if desired. A lower number for each 4 or 16 set, receives a higher run priority.

4.

In the Results Group 1 column, select a group

from the drop-down list (see page 70

) or create a new Results Group.

5.

In the Instrument Protocol 1 column, select a

protocol from the drop-down list (see page 59 ) or

create a new instrument protocol.

6.

In the Analysis Protocol 1 column, select a

protocol from the drop-down list (see page 62 ) or

create a new analysis protocol.

7.

Complete the plate record based on the samples loaded in your plate:

• For the single runs that use the same samples and protocols – Highlight the entire row, then select Edit > Fill Down Special.

Based on your plate type (96- or 384-well) and capillary array (16 or 4 capillaries), the software automatically fills in the appropriate well numbers for a single run or

• For entire plates that use the same samples and protocols – Highlight the entire row, then select Edit > Fill Down to fill down the entire plate editor.

• For plates with different samples and protocols – complete the entries manually.

5

1 2 3 4

6

Notes

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Chapter 4 Autoanalysis and Sequencing Analysis Software

Filling Out a Sequencing Analysis Plate Record

8.

To do more than one injection of the same sample, select Edit > Add Sample Run.

Results Group, Instrument Protocol and Analysis

Protocol columns are added to the right end of the plate record.

9.

Complete the columns for the additional runs.

10.

Click .

Note:

After clicking OK in the Plate Editor, the completed plate record is stored in the Plate

Manager database.The plate record can be searched for, edited, duplicated, exported, or deleted in the Plate Manager.

4

Notes

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Chapter 4 Autoanalysis and Sequencing Analysis Software

Filling Out a Sequencing Analysis Plate Record

Notes

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Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Chapter 5

Autoanalysis and

SeqScape Software

Preparing the

Instrument

Performing a

Spatial Calibration

Performing a

Spectral Calibration

Autoanalysis and Sequencing

Analysis

Software

Autoanalysis and

SeqScape Software

Create protocols for automated

SeqScape Software Analysis

See page 88

Autoanalysis and

Fragment Analysis

Create a

SeqScape Software

Plate Record

See page 101

Running the

Instrument

Notes

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5

Chapter 5 Autoanalysis and SeqScape Software

SeqScape Software

SeqScape Software

Analyzing your sequencing samples may be done by autoanalysis or manual analysis.

Autoanalysis

Use the ABI P

RISM

®

SeqScape

®

software to automatically analyze Applied Biosystems

3130/3130xl Genetic Analyzers sequencing data. Perform autoanalysis only on the same instrument computer that collected the sample files. You can configure the software packages to perform data collection and then data analysis without requiring user interaction.

Install and register the SeqScape software with the 3130/3130xl genetic analyzer Data

Collection software before creating required files for autoanalysis.

See Appendix B in the SeqScape Software User Guide v2.5 (P/N 4359442), for more details on setting up autoanalysis.

Required

Software

Packages

Autoanalysis requires three software packages:

• 3130/3130xl genetic analyzer Data Collection software

The Data Collection software runs the instrument and collects fluorescent data from samples. For autoanalysis to occur, the software must communicate with downstream software.

Analysis parameters used for data collection as well as that created in SeqScape software are accessible in Data Collection software.

• Autoanalysis Manager

The Autoanalysis Manager software is part of the integration between the data collection, SeqScape, and GeneMapper

®

software. It can queue messages and track their processing status. Each message is considered a batch job, whether it contains a single sample, samples from a result group, or an entire run of samples.

Autoanalysis Manager is installed by Seqscape or GeneMapper software when loaded on a system with Data Collection software.

• A version of SeqScape software without a user interface

This SeqScape software version is identical to the regular software version except that there is no user interface. The Autoanalysis Manager opens and uses this version of software to analyze the data in the projects.

The automated processing version and the standard version of SeqScape software are installed from the SeqScape Software installation CD.

IMPORTANT!

When installing SeqScape software on a computer that is connected to a

3130/3130xl genetic analyzer, the Data Collection software must be running. Otherwise, the SeqScape software does not register with the Data Service. See Chapter 2 in the

SeqScape Software User Guide v2.5, for information on installing the software.

Notes

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Chapter 5 Autoanalysis and SeqScape Software

About Plate Records and Seqscape Software

Importing Reports and Projects After

Autoanalysis

In the SeqScape Software v2.5, select Tools > Options tab.You can set the options to automatically import:

• Reports after autoanalysis

• Projects after autoanalysis

See the SeqScape Software User Guide v2.5 (P/N 4359442) for more details.

File-Naming

Convention

Some alphanumeric characters are not valid for user names or file names. Do not use the invalid characters below: spaces

\ / : * ? " < > |

IMPORTANT!

An error message is displayed if you use any of these characters. You must remove the invalid character to continue.

Manual Analysis

See the SeqScape Software User Guide v2.5 (P/N 4359442) for information on performing manual analysis.

About Plate Records and Seqscape Software

Requirements

Successful automatic analysis requires that:

• SeqScape software is installed properly

• SeqScape software is registered and the appropriate user IDs have been created

• Autoanalysis Manager software is running

• The 3130/3130xl genetic analyzer is set up to run, and samples are prepared

• Files for a Data Collection software plate record are available:

– An instrument protocol

– An analysis protocol

– A results group assigned within a plate record

When to Create a

Plate Record

Create a plate record for each plate of samples for the following types of runs:

• Spectral calibrations

• Sequencing Analysis Software

• SeqScape Software analysis

• GeneMapper

®

software

• Mixed (sequencing and fragment analysis samples - see the Applied Biosystems

3130/3130xl Genetic Analyzers Maintenance, Troubleshooting and Reference

Guide for creating a mixed plate record)

Notes

5

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83

Chapter 5 Autoanalysis and SeqScape Software

About Plate Records and Seqscape Software

For runs to begin, you must create a plate record for and link it to a plate loaded on the instrument. However, you can create plate records for new plates while a run is in progress.

SeqScape

Software Plate

Record Overview

The Plate Editor displays an empty plate record for the application selected in the New

Plate dialog box. The data fields within a given plate record vary depending on the application. This section describes the SeqScape Plate Record’s data fields.

IMPORTANT!

For data collection and SeqScape software autoanalysis to succeed, each run of samples must have an instrument protocol, an analysis protocol, and a results group assigned within a plate record. You are also required to have a project, project template, and specimen information.

The table below describes required plate editor fields.

Parameters Description

Instrument Protocol Contains the run module and dye set needed to run the instrument.

Created in Data Collection software.

Analysis Protocol Contains everything needed to analyze sequencing data.

Created in Data Collection software or SeqScape software. If you prefer to use analysis protocols created in SeqScape software, then select “Always use this protocol” - in the SeqScape software analysis defaults settings.

Results Group

Project

Project Template

Specimen

Defines the file type, the file name, file save locations, default analysis protocols linked to sample injections, and user name and password.

Created in Data Collection software.

A group of related sequences that share the same reference or for which there is no explicit reference.

Contains a reference data group (RDG), analysis defaults, and display settings.

The container that holds all the sample data as assembled contigs from a biological source or PCR product

See Page

88

92

98

See the

SeqScape

Software

User Guide v2.5

for more information

Notes

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Chapter 5 Autoanalysis and SeqScape Software

About Plate Records and Seqscape Software

Elements of a

SeqScape Plate

Record

Data Collection software files:

Results Group

Results Group

Name

Analysis software and autoanalysis

File storage location

File and run folder name preferences

Instrument Protocol

Instrument

Protocol Name

Run type

(Regular)

Run module

Dye set

Plate Manager

Plate Record

Analysis Protocol *

Analysis Protocol

Name

Basecaller and

DyeSet/Primer file

Bases called

(pure or mixed)

Assigning Quality

Values (QVs)

Post processing

(clear range

trimming)

Filter settings

SeqScape software files:

Project Template

- Analysis Protocol *

- Analysis Defaults

- RDG

- Display Settings

Project

Specimen

*You can create Analysis Protocols in either

SeqScape or Data Colletion software

5

Notes

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Chapter 5 Autoanalysis and SeqScape Software

About Plate Records and Seqscape Software

SeqScape

Software Plate

Record

A Seqscape software plate record displaying the columns and descriptions is shown below.

1 2 3 4 5

6

7 8

9

1.

2.

3.

Number and Column

Sample Name

Comment

Priority

4.

Project

5.

6.

Project Template

Specimen

Default is one sample run, to add runs

see page 103

Description

Name of the sample.

Comments about the sample (optional).

A default value of 100 to each sample. Changing the value to a smaller number causes that set of 16 or 4 samples to run to before the others in the injection list.

Select one of the available Project from the list that was created in Data Collection or

SeqScape software.

Completed automatically based on the Project selected.

Select one of the available Specimen from the list that was created in Data

Collection or SeqScape software.

Notes

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Chapter 5 Autoanalysis and SeqScape Software

About Plate Records and Seqscape Software

7.

Number and Column

8.

Instrument Protocol

9.

Analysis Protocol

Description

• New: Opens the Results Group Editor dialog box

• Edit: Opens the Results Group Editor dialog box for the Results Group listed in the cell

• None: Sets the cell to have no selected Results Group

• Select one of the available Results groups from the list

Note:

You must have a Results Group selected for each sample entered in the

Sample Name column.

See “Creating a Results Group” on page 98

.

• New: Opens the Protocol Editor dialog box.

• Edit: Opens the Protocol Editor dialog box for the Instrument Protocol listed in the cell.

• None: Sets the cell to have no selected protocol.

• List of Instrument Protocols: In alpha-numeric order.

Note:

You must have an Instrument Protocol selected for each sample entered in the Sample Name column.

See “Creating an Instrument Protocol” on page 88 .

• New: Opens the Analysis Protocol Editor dialog box.

• Edit: Opens the Analysis Protocol Editor dialog box for the Instrument Protocol listed in the cell.

• None: Sets the cell to have no selected protocol.

• List of Analysis Protocols: In alpha-numeric order

Note:

You must have an Analysis Protocol selected for each sample entered in the

Sample Name column.

See “Creating an Analysis Protocol” on page 93 .

5

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Chapter 5 Autoanalysis and SeqScape Software

Creating Protocols for Automated SeqScape Software Analysis

Creating Protocols for Automated SeqScape Software

Analysis

If the appropriate SeqScape software protocols and

results group have been created, proceed to “Filling

Out a SeqScape Software Plate Record” on page 101 .

Instrument Protocol for SeqScape Software

An instrument protocol contains all the necessary settings to run the instrument:

• Protocol name

• Type of run

• Run module

• Dye set

Creating an Instrument Protocol

1.

In the tree pane of the Data Collection software, click

GA Instruments >

ga3130

or

ga3130xl > Protocol Manager.

Create instrument

Create analysis protocols here

2.

In the Instrument Protocols section, click

to open the Protocol Editor.

Notes

88

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

3.

Complete the Protocol Editor:

a.

Type a name for the protocol.

b.

Type a description for the protocol

(optional).

c.

Select Regular in the Type drop-down list.

Chapter 5 Autoanalysis and SeqScape Software

Creating Protocols for Automated SeqScape Software Analysis

3a

3b

3c

3d

3e

3f d.

Select the correct run module for your run.

See “Sequencing Resolution Performance and Specifications” on page 3

to see a list of modules or the Applied Biosystems

3130/3130xl Genetic Analyzers

Maintenance, Troubleshooting and

Reference Guide to modify a default module.

5

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Chapter 5 Autoanalysis and SeqScape Software

Creating Protocols for Automated SeqScape Software Analysis

e.

Select the correct Dye Set for your run using the table below.

POP-4

Polymer

POP-6

Polymer

POP-7 Polymer

Chemistry Dye Set

BigDye

®

Terminator v3.1 Cycle Sequencing Kit Z_BigDye

V3

ABI P

RISM

®

dGTP BigDye

®

Terminator v3.0

Cycle Sequencing Ready Reaction Kit

BigDye

®

Terminator v1.1 Cycle Sequencing Kit E_BigDye

V1

ABI P

RISM

®

dGTP BigDye

®

Terminator Cycle

Sequencing Kit*

ABI P

RISM

®

dRhodamine Dye Terminator Cycle

Sequencing Ready Reaction Kit

ABI P

RISM

®

BigDye ® Primer Cycle Sequencing

Kits

✓

—

✓

—

✓

—

✓

—

✓

—

✓

—

✓

—

✓

—

✓

—

✓

—

✓

—

✓

✓

✓

—

✓

—

✓

✓

✓

—

✓

—

—

—

✓

—

✓

—

—

—

✓

—

✓

—

—

—

✓

—

✓

—

—

—

*dGTP kits are not supported on capillary electrophoresis instruments due to compressions on certain sequence context regions; you can run the kits if you do not care about the compression issues.

✓

—

✓

—

—

—

f.

Click .

Notes

90

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Importing an Instrument Protocol

1.

Click in the Instrument Protocols pane of the Protocol Editor window to display the standard File Import dialog box.

Chapter 5 Autoanalysis and SeqScape Software

Creating Protocols for Automated SeqScape Software Analysis

2.

Navigate to the location of the .xml file you want to import.

3.

Select the .xml file and click Open.

Exporting an Instrument Protocol

1.

In the Instrument Protocols pane, highlight the protocol you want to export.

2.

Click to display the standard File

Export dialog box.

3.

Browse to the desired folder location.

4.

Click .

5

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Chapter 5 Autoanalysis and SeqScape Software

Creating Protocols for Automated SeqScape Software Analysis

Analysis Protocol for SeqScape Software

About Analysis

Protocols

An analysis protocol contains all the settings necessary for analysis and post processing:

• Protocol name – The name, description of the analysis protocol, and the sequence file formats to be used

• Basecalling settings – The basecaller, DyeSet/Primer file, and analysis stop point to be used

• Mixed Bases – (Optional) – When two bases are found at the same position. Define the percent value of the second highest to the highest peak

• Clear Range – The high quality sequence remaining after trimming low quality sequences typically found at the beginning and ends of the sequence. The clear range can be based on base positions, sample quality values, and/or number of ambiguities (Ns) present

• Filter – The settings that define which samples will be used in the assembly

Note:

If you created an appropriate analysis protocol in the SeqScape software, you can use it in the Data Collection software. See Appendix B in the SeqScape Software User

Guide v2.5 (P/N 4359442) for more information on autoanalysis and analysis protocols.

IMPORTANT!

Do not delete the current analysis protocol being used, during a run.

Autoanalysis will not be performed if you do so.

IMPORTANT!

You are required to select the Always use this Analysis Protocol in

Analysis Defaults > Analysis Protocol in the Seqscape software if you want to use it for

SeqScape analysis. See the SeqScape Software User Guide v2.5 (P/N 4359442) for more information.

Notes

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Creating an Analysis Protocol

IMPORTANT!

If you created an appropriate analysis protocol in SeqScape software, you can use it in the

Data Collection software. You can also create an analysis protocol in the SeqScape software.

1.

In the Analysis Protocol section of the Protocol

Manager, click .

If more than one analysis application is installed on the data collection computer, the Analysis

Applications dialog box opens.

Chapter 5 Autoanalysis and SeqScape Software

Creating Protocols for Automated SeqScape Software Analysis

2.

Select SeqScape, then click

Analysis Protocol Editor dialog box.

to open the

3.

In the General tab, enter a unique name and description (optional) for the new protocol.

5

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Chapter 5 Autoanalysis and SeqScape Software

Creating Protocols for Automated SeqScape Software Analysis

4.

Select the Basecalling tab, then:

a.

Select the appropriate basecaller and

DyeSet primer based on the chemistry, capillary array length and polymer type you are using. The dye set primer files displayed are based on the basecaller selected.

See “3130/3130xl Genetic Analyzer

Basecaller and DyeSet/Primer Files” on page 66 for a table on basecallers and

dyeset/primer files.

Note:

SeqScape Software and the Data

Collection software filter .mob file choices to match the chosen .bcp file.

b.

In the Processed data pane, select True or

Flat Profile.

Option Function

Displays data as processed traces scaled uniformly. The average peak height in the region of strongest signal is about equal to a fixed value. The profile of the processed traces is similar to that of the raw traces.

Displays data as processed traces scaled semi-locally. The average peak height in any region is about equal to a fixed value. The profile of the processed traces is flat on an intermediate scale (> about 40 bases).

Note:

This option is applied to data analyzed with the KB

™

basecaller only. If you use the ABI basecaller, the profile option reverts to True Profile.

c.

If desired, select one or more stop points for data analysis.

d.

Select your Threshold Quality option.

Option setting Function using KB basecaller

Assigns a base to every position, as well as the QV.

Assigns Ns to bases with QVs less than the set point. The QV will still be displayed.

Notes

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Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

5.

Select and fill in the Mixed Bases tab:

Note:

This function is active with the KB basecaller only.

a.

For mixed bases only, select Use Mixed

Base Identification.

b.

Use the default setting of 25% or change the detection level by entering a new value or dragging the % line up or down.

Note:

Do not use less than 15% as your detection limit.

Chapter 5 Autoanalysis and SeqScape Software

Creating Protocols for Automated SeqScape Software Analysis

6.

Select the Clear Range tab, then, if desired, select one or more stop points for data analysis.

Select the recommended settings, which are the default settings:

a.

Use Quality values, fewer than 4 bases out of 20 have QVs less than 20.

b.

Use reference trimming.

Note:

The clear range is the region of the sequence that remains after excluding the lowquality or error-prone sequence at both the 5´ and

3´ ends.

5

Notes

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Chapter 5 Autoanalysis and SeqScape Software

Creating Protocols for Automated SeqScape Software Analysis

7.

Select the Filter tab, then, if desired, change one or more of the settings.Select the recommended settings, which are the default settings:

a.

Maximum Mixed Bases (%): 20

b.

Maximum Ns (%): 10.0

c.

Minimum Clear Length (bp): 50

d.

Minimum Sample Score: 20

8.

Click to save the protocol and close the

Analysis Protocol Editor dialog box.

Editing an Analysis Protocol

1.

In the Analysis Protocols pane in the Analysis

Protocol Manager, highlight the protocol you want to edit.

2.

Click .

3.

Make changes in the General, Basecalling,

Mixed Bases and Clear Range tabs, as appropriate.

4.

Click to save the protocol and close the

Analysis Protocol Editor dialog box.

Notes

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Deleting an Analysis Protocol

IMPORTANT!

Do not delete an analysis protocol during a run while it is being used for that run or

Autoanalysis will not be performed. Also, you must first delete any plate records using the analysis protocol before you can delete or modify the analysis protocol for these plate records.

1.

In the Analysis Protocols pane in the Analysis

Protocol Manager, highlight the protocol you want to delete.

2.

Click .

The Deletion Confirmation dialog box displays.

3.

Click .

Exporting an Analysis Protocol

1.

In the Analysis Protocols pane in the Analysis

Protocol Manager, highlight the protocol you want to export.

2.

Click to display the standard File

Export dialog box.

3.

Browse to the desired folder location.

4.

Click Save.

Importing an Analysis Protocol

1.

Click to display the standard File

Import dialog box.

2.

Browse to the .xml file to import and click Open.

Note:

For multiple applications, select the appropriate application to associate with the analysis protocol.

Chapter 5 Autoanalysis and SeqScape Software

Creating Protocols for Automated SeqScape Software Analysis

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5

Chapter 5 Autoanalysis and SeqScape Software

Creating Protocols for Automated SeqScape Software Analysis

Results Group for SeqScape Software

A Results Group is a component within Data

Collection that organizes samples and certain user settings under a single name. It is called a Results

Group because it is used to name, sort, and deliver samples that result from a run.

Creating a Results Group

1.

In the tree pane of the Data Collection software, click

GA Instruments > Results Group.

2.

Click .

The Results Group Editor window displays.

3.

Complete the General tab:

a.

Type a Results Group Name. The name can be used in naming and sorting sample files.

It must be unique (see page for a list of accepted characters).

b.

Optional: Type a Results Group Owner. The owner name can be used in naming and sorting sample files.

c.

Optional: Type a Results Group Comment.

4.

Select the Analysis tab, then:

a.

Select SeqScape_ computer name in the

Analysis Type drop-down list.

b.

Select Do Autoanalysis in the Analysis

Actions section.

c.

Type a valid SeqScape Software Login ID and Password in the text boxes.

Note:

Failure to use the proper login and password causes your samples not to be analyzed automatically.

Notes

98

3a

3b

3c

4a

4b

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

5.

Select the Destination tab, then use the default destination or define a new location for data storage.

To use …

default location* custom location

Then …

skip to step 6 complete steps a-c

a.

Click Use Custom Location, then click

to navigate to a different save location.

b.

Click

to test the Location path name connection:

If it…

Then a message box displays …

Fails

<“path”>.

Test failed:<“path”>.

6.

Select the Naming tab, then define custom names for the sample file and the run folder name. See

“Optional: Completing Sample File

Name Format Pane” on page 73

for more detailed information.

7.

Click

Group Editor.

to save and close the Results

Chapter 5 Autoanalysis and SeqScape Software

Creating Protocols for Automated SeqScape Software Analysis

5a

5a

5b

5

*Sample File Destinations

Locations where sample files are placed during extraction:

• Default Destination, default folder naming: E:\AppliedBiosystemsUDC\DataCollection\data\<instrument type>\

<instrument name>\run folder

• Default Destination, custom folder naming: E:\AppliedBiosystemsUDC\DataCollection\data\top custom folder\ subfolders, etc.

• Custom Destination, default folder naming: Destination\<instrument type>\<instrument name>\run folder

• Custom Destination, custom folder naming: Destination\top customer folder\subfolders, etc.

Notes

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Chapter 5 Autoanalysis and SeqScape Software

Creating Protocols for Automated SeqScape Software Analysis

Importing and Exporting a Results Group

Note:

Importing or exporting of SeqScape software

Results Groups are not supported for transfer between separate computers.

1.

In the tree pane of the Data Collection software, click

GA Instruments > Results Group.

2.

Click to open a File Import dialog box.

3.

Navigate to the file you want to import.

Note:

Import file type is .xml (XML file).

4.

Click .

Note:

When you import or duplicate a Results

Group, you are asked to type a name for the new

Results Group and for the analysis application type.

Exporting a Results Group

1.

In the tree pane of the Data Collection software, click

GA Instruments > Results Group.

2.

Click the Results Group name to select it.

3.

Click .

A standard File Export dialog box displays with the chosen Results Group name.

4.

Navigate to the location where you want to save the exported file.

5.

Click .

Note:

If there is a name conflict with a Results

Group that already exists at the save location, the

Results groups can be duplicated to copy settings into a similar Results Group without the risk of user error when copying it manually (see

“Duplicating a Results Group” on page 100

).

Duplicating a Results Group

1.

Click the Results Group to select it.

Notes

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Chapter 5 Autoanalysis and SeqScape Software

Filling Out a SeqScape Software Plate Record

2.

Click .

Note:

When you import or duplicate a Results

Group, you are asked to type a name for the new

Results Group and for the analysis application type.

Filling Out a SeqScape Software Plate Record

Creating a SeqScape Software Plate

Record

1.

Click the Plate Manager icon in the navigation pane.

2.

Click .

The New Plate Dialog dialog box opens.

3.

Complete the New Plate dialog box:

a.

Type a name for the plate.

b.

Type a description for the plate (optional).

c.

Select SeqScape_computer name in the

Application drop-down list.

d.

Select 96-well or 384-well in the Plate Type drop-down list.

e.

Type a name for the owner.

f.

Type a name for the operator.

g.

Click .

The SeqScape Plate Editor opens.

3a

3b

3c

3d

3e

3f

3g

5

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Chapter 5 Autoanalysis and SeqScape Software

Filling Out a SeqScape Software Plate Record

Completing a SeqScape Software Plate

Record

1.

In the Sample Name column of a row, enter a sample name, then click the next cell. The value

100 automatically display in the Priority column.

2.

In the Comments column, enter any additional comments or notations for the sample.

3.

In the Priority column, change the priority value, if desired. A lower number for each 4 or 16 set, receives a higher run priority. (See the Applied

Biosystems 3130/3130xl Maintenance,

Troubleshooting, and Reference Guide for changing priority values).

4.

In the Project column, select a project from the drop-down list or create a new project with the corresponding project template.

Based on the project you select, the project template is filled in automatically.

5.

In the Specimen column, select or create a specimen.

6.

In the Results Group 1 column, select a group

from the drop-down list (see page 98 ).

7.

In the Instrument Protocol 1 column of the row, select a protocol from the drop-down list (see

page 88 ).

8.

In the Analysis Protocol 1 column of the row, select a protocol from the drop-down list (see

page 93 ).

9.

To complete the rest of the plate record based on the samples loaded in your plate, do one of the following:

• For the same samples and protocols –

Highlight the entire row, then select Edit >

Fill Down Special.

Based your plate type (96- or 384-well) and capillary array (16 or 4 capillaries), the software automatically fills in the appropriate well numbers for a single run.

Notes

6

1 2 3 4

7 8

102

5

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

• For the same samples and protocols –

Highlight the entire row, then select Edit >

Fill Down.

• For the different samples and protocols –

Complete the entries manually.

10.

If you want to do more than one run, then select

Edit > Add Sample Run.

Additional Results Group, Instrument Protocol and Analysis Protocol columns are added to the right end of the plate record.

Add additional runs by selecting Edit > Add

Sample Run again.

11.

Complete the columns for the additional runs.

12.

Click to save, then close the plate record.

Note:

After clicking OK within the Plate Editor, the completed plate record is stored in the Plate

Manager database.The plate record can be searched for, edited, exported, or deleted in the

Plate Manager.

For sample preparation and running the instrument, see

Chapter 7, “Running the Instrument.”

Chapter 5 Autoanalysis and SeqScape Software

Filling Out a SeqScape Software Plate Record

5

Notes

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Chapter 5 Autoanalysis and SeqScape Software

Filling Out a SeqScape Software Plate Record

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Chapter 6

Autoanalysis and Fragment Analysis

Preparing the

Instrument

Performing a

Spatial Calibration

Performing a

Spectral Calibration

Autoanalysis and Sequencing

Analysis

Software

Autoanalysis and

SeqScape Software

Create protocols for automation

See page 112

Autoanalysis and

Fragment Analysis

Create a

GeneMapper Software

Plate Record

See page 123

Running the

Instrument

Notes

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6

Chapter 6 Autoanalysis and Fragment Analysis

GeneMapper Software

GeneMapper Software

Analyze your fragment analysis samples automatically using autoanalysis or manually.

Note:

This chapter is written for both GeneMapper

®

Software v3.7 or higher and

GeneMapper

®

ID Software v3.2. Graphic examples are from GeneMapper software v3.7.

GeneMapper

Software v3.7 and

GeneMapper ID

Software v3.2

Perform autoanalysis of fragment analysis samples with features of the 3130/3130xl

Data Collection and GeneMapper software using the same instrument computer that collected the sample files or on a remote computer.

• Autoanalysis can be performed on the same instrument computer that collected the sample files or on another computer using the remote analysis feature of

GeneMapper software.

• After autoanalysis, if you wish to edit/review results on another computer, then transfer the GeneMapper software project, analysis methods, size standards, panel, and bin set information to the other GeneMapper software database. All components need to be exported and imported individually.

• When completing the plate record, fill in the instrument protocol and other information for Data Collection software to complete the run.

• When creating a new Results Group for a set of samples to be autoanalyzed, check the Do Autoanalysis check box and enter the appropriate GeneMapper Software user name and password. For remote analysis, define a default location for sample file storage.

File-Naming

Convention

Some alphanumeric characters are not valid for user names or file names. Do not use the invalid characters below: spaces

\ / : * ? " < > |

IMPORTANT!

An error message is displayed if you use any of these characters. You must remove the invalid character to continue.

Manual Analysis

For information on manual analysis, see the GeneMapper Software v3.7 User Guide

(PN 4359413) or GeneMapper ID Software V3.1 User Guide (PN 4338775), or the User

Bulletin titled GeneMapper ID Software v3.2, Subject: New Features and Installation

Procedures for GeneMapper ID Software v3.2 (PN 4352543).

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Chapter 6 Autoanalysis and Fragment Analysis

GeneMapper Software

About Fragment

Analysis and Data

Collection

When GeneMapper software is installed on a computer that has 3130/3130xl genetic analyzer Data Collection software, two applications are available through the Results

Group Editor (see

page 115

):

• GeneMapper-Generic

• GeneMapper-<Computer Name>

GeneMapper-

Generic

Use GeneMapper-Generic to generate .fsa files, without performing autoanalysis. When completing the Sample Sheet, fill in basic information for the Data Collection software to complete the run; all other GeneMapper software related fields are text entries. Text entries are useful if you are using other software analysis applications or choose to analyze your samples in GeneMapper software on another computer, but do not have the same entries in the GeneMapper software database stored on the Data Collection computer. For example, if you have a customized size standard definition on another

GeneMapper software computer, you can type in that size standard name in the size standard text field and it will populate that column in your GeneMapper software project.

GeneMapper-

<Computer

Name>

Use GeneMapper-<Computer Name> for autoanalysis. The Size Standard, Analysis

Method, and Panel columns in the Sample Sheet window read directly from the

GeneMapper software database. Create these components in GeneMapper software prior to setting up the plate record for a run. New entries cannot be created for these columns once you are in the plate editor dialog box. If you create a new GeneMapper software component while the plate record dialog box is open, the columns will not update. The plate record must be closed and reopened to update the GeneMapper software components. For more information see,

“Creating Protocols for Automated

Fragment Analysis” on page 112

.

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6

Chapter 6 Autoanalysis and Fragment Analysis

About GeneMapper Software Plate Records

About GeneMapper Software Plate Records

A plate record is similar to a sample sheet or an injection list that you may have used with other ABI P

RISM

®

instruments.

Plate records are data tables in the instrument database that store information about the plates and the samples such as:

• Plate name, type, and owner

• Position of the sample on the plate (well number)

• Comments about the plate and about individual samples

• Dye set information (in Instrument protocol)

• Name of the run module where run modules specify information about how samples are run (in Instrument protocol)

When to Create a

Plate Record

Create a plate record for each plate of samples for the following types of runs:

• Spectral calibrations

• Sequencing Analysis Software

• SeqScape Software analysis

• GeneMapper

®

software

• Mixed (sequencing and fragment analysis samples - see the Applied Biosystems

3130/3130xl Genetic Analyzers Maintenance, Troubleshooting, and Reference

Guide for creating a mixed plate record)

For runs to begin, you must create a plate record for and link it to a plate loaded on the instrument. However, you can create plate records for new plates while a run is in progress.

Files needed to create a GeneMapper software plate record:

Parameters

Instrument

Protocol

Results Group

Description

Contains everything needed to run the instrument.

Defines the file type, the file name, autoanalysis, and file save locations that are linked to sample injections.

See

Page

112

115

IMPORTANT!

For data collection and auto-analysis to be successful, each run of samples must have an Instrument Protocol, a Results Group and files created in

GeneMapper software assigned within a plate record.

Notes

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Chapter 6 Autoanalysis and Fragment Analysis

About GeneMapper Software Plate Records

Elements of a GeneMapper software plate record

Plate Manager

Plate Record

Data Collection software files:

Results Group

Results Group

Name

Analysis software and autoanalysis

File storage location

File and run folder name preferences

Instrument Protocol

Instrument

Protocol Name

Run type

(Regular)

Run module

Dye set

GeneMapper software definitions:

Size standard

Analysis method

Bin set

Panel

SNP Set

Notes

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6

Chapter 6 Autoanalysis and Fragment Analysis

About GeneMapper Software Plate Records

1

2 3

4

5

6 7

8

9

10

11

Defaults to one sample run, to add additional runs see page 125

Blank GeneMapper software plate record

Columns inserted in a plate record for a fragment analysis run

Number and Column

1.

Sample Name

2.

Comment

3.

Priority

4.

Sample Type

5.

Size Standard

IMPORTANT!

For GeneMapper-<Computer

Name> ONLY:

Create the Size Standard, Panel, and Analysis

Method in GeneMapper software before creating a new plate

6.

Panel

IMPORTANT!

For GeneMapper-<Computer

Name> ONLY:

Create the Size Standard, Panel, and Analysis

Method in GeneMapper software before creating a new plate

Description

Name of the sample

Comments about the sample (optional)

A default value of 100 to each sample. Changing the value to a smaller number causes that set of 16 or 4 samples to run to before the others in the injection list.

Use to identify the sample as Sample, Positive Control, Allelic

Ladder or Negative Control.

• GeneMapper-Generic (optional):

Manually enter size standards in the text field

• GeneMapper-<Computer Name>:

Select a saved size standard from the drop-down list

• GeneMapper-Generic (optional):

Manually enter panels in the text field

• GeneMapper-<Computer Name>:

Select a saved panel from the drop-down list

Notes

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Chapter 6 Autoanalysis and Fragment Analysis

About GeneMapper Software Plate Records

Number and Column Description

7.

Analysis Method

IMPORTANT!

For GeneMapper <Computer

Name> ONLY:

Create the Size Standard, Panel, and Analysis

Method in GeneMapper software before creating a new plate

8.

SNP Set

IMPORTANT!

For GeneMapper <Computer

Name> ONLY:

The SNP set, created in the GeneMapper software, links a SNP name to a marker name.

9.

Three User-defined columns

• GeneMapper-Generic (optional):

Manually enter analysis methods in the text field

• GeneMapper-<Computer Name>:

Select a saved analysis method from the drop-down list

• GeneMapper-Generic (optional):

Manually enter SNP set in the text field

• GeneMapper-<Computer Name>

Optional text entries

10.

Results Group Options:

• New: Opens the Results Group Editor dialog box

• Edit: Opens the Results Group Editor dialog box for the

Results Group listed in the cell

• None: Sets the cell to have no selected Results Group

• Select one of the available Results groups from the list

Note:

You must select a Results Group for each sample entered in the Sample Name column.

See

“Results Group for Fragment Analysis” on page 115 .

11.

Instrument Protocol Options:

• New: Opens the Protocol Editor dialog box.

• Edit: Opens the Protocol Editor dialog box for the

Instrument Protocol listed in the cell.

• None: Sets the cell to have no selected protocol.

• List of Instrument Protocols: In alpha-numeric order.

Note:

You must select an Instrument Protocol for each sample entered in the Sample Name column.

See

“Results Group for Fragment Analysis” on page 115 .

6

Notes

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Chapter 6 Autoanalysis and Fragment Analysis

Creating Protocols for Automated Fragment Analysis

Creating Protocols for Automated Fragment Analysis

If the appropriate fragment analysis protocols and results group have been created, proceed to

“Creating a GeneMapper Software Plate Record” on page 123

.

Instrument Protocol for Fragment Analysis

An instrument protocol contains all the settings necessary to run the instrument:

• Protocol name

• Type of run

• Run module

• Dye set

Creating an Instrument Protocol

1.

In the tree pane of the Data Collection software, click

GA Instruments >

ga3130xl

or

ga3130 > Protocol Manager.

Create instrument

Create analysis protocols here

2.

In the Instrument Protocols section, click

to open the Protocol Editor.

Notes

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Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

3.

Complete the Protocol Editor:

a.

Type a name for the protocol.

b.

Type a description for the protocol

(optional).

c.

Select Regular in the Type drop-down list.

Chapter 6 Autoanalysis and Fragment Analysis

Creating Protocols for Automated Fragment Analysis

3a

3b

3c

3d

3e

3f d.

Using the table below, select the run module for your run. To customize a run module, see the Applied Biosystems 3130/3130xl

Genetic Analyzers Maintenance,

Troubleshooting, and Reference Guide on modifying a module.

Application or Kit

SNaPshot

®

Multiplex System

• LMS v2.5

• ABI P

RISM

®

Mouse Mapping

Set v1.0

• Custom oligos

• Stockmarks

• AFLP

®

• AmpFlSTR

®

COfiler

®

• AmpFlSTR Profiler Plus

®

• AmpFlSTR SGM Plus

®

• AmpFlSTR Profiler Plus ID

• AmpFlSTR SEfiler

™

• AmpFlSTR Yfiler

™

• Other 4-Dye AmpFlSTR

• AmpFlSTR Identifiler

• Other 5-Dye AmpFlSTR

Capillary Array

Length

(cm)

Run Module

22 SNP22_POP4_1

36 SNP36_POP4_1

22 FragmentAnalysis22_POP4_1

36 FragmentAnalysis36_POP4_1

FragmentAnalysis36_POP7_1

50 FragmentAnalysis50_POP4_1

FragmentAnalysis50_POP6_1

FragmentAnalysis50_POP7_1

36 HIDFragmentAnalysis36_POP4_1

Notes

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Chapter 6 Autoanalysis and Fragment Analysis

Creating Protocols for Automated Fragment Analysis

e.

Using the table below, select the correct Dye

Set for your run.

Application or Kit

Custom oligos

• ABI P

RISM

Mouse Mapping Set v1.0

• Custom oligos

• AmpFlSTR COfiler

• AmpFlSTR Profiler Plus

• AmpFlSTR SGM Plus

• AmpFlSTR Profiler Plus ID

• Other 4-Dye AmpFlSTR

• AFLP

• Stockmarks -Canine and Bovine

ABI P

RISM

SNaPshot Multiplex System

• ABI P

RISM

Linkage Mapping Set v2.5

• Custom Oligos

• AmpFlSTR Identifiler™

• AmpFlSTR SEfiler

• AmpFlSTR Yfiler

• Other 5-Dye AmpFlSTR

• Stockmarks-Equine

f.

Click .

Importing an Instrument Protocol

1.

Click in the Instrument Protocols pane of the Protocol Editor window to display the standard File Import dialog box.

Dye Set

D

D

F

E5

G5

Matrix Standard

Set

DS-30

DS-31

DS-32

DS-02

DS-33

2.

Navigate to the location of the .xml file you want to import.

3.

Select the .xml file and click Open.

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Exporting an Instrument Protocol

1.

In the Instrument Protocols pane, highlight the protocol you want to export.

2.

Click to display the standard File

Export dialog box.

3.

Browse to the desired folder location.

4.

Click .

Results Group for Fragment Analysis

A Results Group is a component within Data

Collection that organizes samples and certain user settings under a single name. It is called a Results

Group because it is used to name, sort, and deliver samples that result from a run.

Creating a Results Group for Autoanalysis

1.

In the tree pane of the Data Collection software, click

GA Instruments > Results Group.

2.

Click window.

to open the Results Group Editor

Chapter 6 Autoanalysis and Fragment Analysis

Creating Protocols for Automated Fragment Analysis

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Chapter 6 Autoanalysis and Fragment Analysis

Creating Protocols for Automated Fragment Analysis

3.

Complete the General tab:

a.

Type a unique Results Group Name. The name can be used in naming and sorting sample files.

b.

Optional: Type a Results Group Owner. The owner name can be used in naming and sorting sample files.

c.

Optional: Type a Results Group Comment.

4.

Select the Analysis tab, then:

a.

Click Analysis Type and then select one of the following:

If You Select ...

None

GeneMapper-

Generic

GeneMapper-

<Computer

Name>

Then ...

Only raw data files are generated.

Autoanalysis is not enabled and only .fsa files are generated.

Autoanalysis of completed runs is enabled.

Steps b, c, and d below apply only to

GeneMapper-<Computer

Name> (not GeneMapper-

Generic).

Notes

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3a

3b

3c

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4a

4c

4d

4b

Chapter 6 Autoanalysis and Fragment Analysis

Creating Protocols for Automated Fragment Analysis

b.

Select an option in the Analysis Actions section using the table below.

If You Select … Then …

Do Autoanalysis

Do Autoanalysis and

Results Entry Group

Complete

Samples are analyzed after each run of

16 or 4 samples.

Samples are analyzed after all pending samples using the same results group have been run.

Use with Setting from Automated

Processing Tab ( page 118 )

When every run completes

Only when the result group is complete

c.

Type the Login ID.

d.

Type the login password.

Note:

The login ID and password relate to the

GeneMapper software UserName and Password.

These items can only be created through the

GeneMapper software Options Users tab.

Autoanalysis will fail if the incorrect username and password are entered.

5.

Select the Destination tab, then use the default destination or define a new location for data storage.

To use a …

default location* custom location

Use for remote analysis using GeneMapper v3.7

Then …

skip to step 6.

complete steps a-b.

5a

5b

5c

*Sample File Destinations

Locations where sample files are placed during extraction:

• Default Destination, default folder naming: E:\AppliedBiosystemsUDC\DataCollection\data\<instrument type>\

<instrument name>\run folder

• Default Destination, custom folder naming: E:\AppliedBiosystemsUDC\DataCollection\data\top custom folder\ subfolders, etc.

• Custom Destination, default folder naming: Destination\<instrument type>\<instrument name>\run folder

• Custom Destination, custom folder naming: Destination\top customer folder\subfolders, etc.

Notes

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Chapter 6 Autoanalysis and Fragment Analysis

Creating Protocols for Automated Fragment Analysis

a.

Click Use Custom Location, then click

to navigate to a different save location.

b.

Click

to test the Location path name connection:

If it…

Then a message box displays …

Fails

<“path”>.

Test failed:<“path”>.

6.

Select the Naming tab to customize sample file and run folder names.

Note:

The default sample file naming for fragment analysis is: frag_<capillary number>_<wellposition>.fsa

Note:

Sample name, run folder name, and path name, combined, can total no more than 250 characters.

For defining the elements of the Naming tab, see

page 119 .

Sample File Name Format

Run Folder Name Format pane

7.

Select the Automated Processing tab.

In “Autoanalysis is performed” select when you want your samples autoanalyzed using the table below.

Select an autoanalysis option

If You Select … Then …

Only when the result group is complete

Samples are analyzed after all pending samples using the same results group have been run.

When every run completes

Samples are analyzed after each run of

16 or 4 samples.

Use with Settings from Analysis Tab

(

page 117

)

Do Autoanalysis and Results Entry

Group Complete

Do Autoanalysis

8.

Click to save the Results Group.

Notes

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Completing the Sample File Name Format Pane

1.

Type a prefix for the file name in the Prefix box.

Anything that you type here is shown in the

Example line.

2.

Click the Name Delimiter list choose the symbol that will separate the Format elements in the file name (see step 3 below). Only one delimiter symbol may be chosen.

Chapter 6 Autoanalysis and Fragment Analysis

Creating Protocols for Automated Fragment Analysis

3.

Click the Format list and then select the components that you want in the sample name.

Note:

All the samples from a single run can be placed in the same run or results folder; the name of every sample from a single run should be different. Most of the Format options will not be different between samples; select at least one of the options to make the sample names unique within a run.

If a unique identifier is not included in the name, a warning message displays.Select from the elements to make a unique sample file name.

Examples are:

– Well position

– Capillary number

– Run sequence number

– Plate Quadrant

Note:

Some identifiers, such as well position, capillary number and plate quadrant are not unique in multiple sample instances. Plate

Quadrant is not a unique identifier for a 96-well plate.

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Creating Protocols for Automated Fragment Analysis

As you select the elements for the file name, they are placed in the Example line. An additional element drop-down menu displays allowing you the option of selecting an additional element.

The names of the Format elements eventually truncate, but the Example field remains visible

(up to 72 characters).

4.

Click the Suffix box (optional) and type the suffix for the file name.

The File Extension field displays the file extension generated from the Analysis Type specified on the Analysis tab (see

page 116 ). For

example, Sequencing Analysis produces sample files with an .ab1 extension.

Run Folder/Sub-Folder Name Format Pane

Follow the same steps described above for the Sample

File Name Format pane (see

page 119

) to change the sub-folder name within the run folder.

Notes

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Chapter 6 Autoanalysis and Fragment Analysis

Creating Protocols for Automated Fragment Analysis

Importing and Exporting a Results Group

Importing or exporting of GeneMapper software

Results Groups are not supported for transfer between separate computers.

Importing a Results Group

1.

In the tree pane of the Data Collection software, click

GA Instruments > Results Group.

2.

Click .

A standard File Import dialog box displays.

3.

Navigate to the file you want to import.

Note:

Import file type is .xml (XML file).

4.

Click .

Note:

When you import or duplicate a Results

Group, you are asked to type a name for the new

Results Group and for the analysis application type.

Exporting a Results Group

1.

In the tree pane of the Data Collection software, click

GA Instruments > Results Group.

2.

Click the Results Group name to select it.

3.

Click .

A standard file export dialog box displays with the chosen Results Group name.

4.

Navigate to the location where you want to save the exported file.

5.

Click .

Note:

If there is a name conflict with a Results

Group that already exists at the save location, the

Results groups can be duplicated in order to copy settings into a similar Results Group without the risk of user error when copying it manually (see

“Duplicating a Results Group” on page 122

).

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Duplicating a Results Group

1.

Click the Results Group.

2.

Click .

Note:

When you import or duplicate a Results

Group, you are asked to type a name for the new

Results Group and for the analysis application type.

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Chapter 6 Autoanalysis and Fragment Analysis

Creating a GeneMapper Software Plate Record

Creating a GeneMapper Software Plate Record

Creating the Plate Record for Autoanalysis

1.

In the tree pane of the Data Collection software, click

GA Instruments >

ga3130xl

or

ga3130 > Plate Manager.

2.

Click to open New Plate Dialog.

3.

Complete the information in the New Plate

Dialog:

a.

Type a name for the plate.

b.

Type a description for the plate (optional).

c.

Select your GeneMapper application in the

Application drop-down list.

d.

Select 96-well or 384-well in the Plate Type drop-down list.

e.

Type a name for the owner.

f.

Type a name for the operator.

g.

Click .

The GeneMapper software Plate Editor opens.

3a

3b

3c

3d

3e

3f

3g

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Chapter 6 Autoanalysis and Fragment Analysis

Creating a GeneMapper Software Plate Record

Completing a GeneMapper Software Plate

Record for Autoanalysis

1.

In the Sample Name column of a row, enter a sample name, then click the next cell. The value

100 automatically display in the Priority column.

2.

In the Comment column, enter any additional comments or notations for the sample.

3.

In the Priority column, change the priority value, if desired.

4.

In the Sample Type column, select a sample type from the drop-down list.

5.

In the Size Standard column, select a size standard from the drop-down list.

6.

In the Panel column, select a panel from the drop-down list.

7.

In the Analysis Method column, select a method from the drop-down list.

8.

In the Snp Set column, select a SNP set from the drop-down lis if applicable, otherwise select

None.

9.

Enter text for User-Defined columns 1 to 3.

10.

In the Results Group 1 column, create a new

Results Group or select a group from the drop-

down list (see page 115

).

11.

In the Instrument Protocol 1 column, create a new instrument protocol or select a protocol from

the drop-down list (see page 112

).

6

9

1 2 3 4

7

10

8

11

9

5

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12.

To complete the plate record:

• For the same samples and protocols –

Highlight the entire row, then select Edit >

Fill Down Special.

Based on your plate type (96- or 384-well) and capillary array (16 or 4 capillaries), the software automatically fills in the appropriate well numbers for a single run.

• For the same samples and protocols –

Highlight the entire row, then select Edit >

Fill Down.

• For the different samples and protocols –

Complete the entries manually.

13.

To do more than one run, select Edit > Add

Sample Run.

Additional Results Group, Instrument Protocol and Analysis Protocol columns are added to the right end of the plate record.

Add additional runs by selecting Edit > Add

Sample Run again.

14.

Complete the columns for the additional runs.

15.

Click to save, then close the plate record.

IMPORTANT!

After clicking OK in the Plate

Editor, the plate record is stored in the Plate

Manager database and can now be located, edited, exported, or deleted.

Chapter 6 Autoanalysis and Fragment Analysis

Creating a GeneMapper Software Plate Record

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Chapter 6 Autoanalysis and Fragment Analysis

Creating a GeneMapper Software Plate Record

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Chapter 7

Running the Instrument

Preparing the

Instrument

Prepare samples and

Heading 1text plate assemblies

See page 128

Performing a

Spatial Calibration

Place Plate onto

Autosampler

See page 128

Performing a

Spectral Calibration

See page 128

Autoanalysis and Sequencing

Analysis

Software

See page 129

Autoanalysis and

SeqScape Software

See page 130

Autoanalysis and

Fragment Analysis

Set up for continuous operation

See page 134

Running the

Instrument

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Chapter 7 Running the Instrument

Preparing Samples and Plate Assemblies

Preparing Samples and Plate Assemblies

Review Table

Generalized sample preparation and plate assembly

procedures are discussed in Chapter 3, “Performing a

Spectral Calibration,” on page 27.

Topic...

Sample Preparation

Sealing plates and plate assembly

Placing plate assemblies in the instrument

Searching for plate records to link to a plate

See

“Preparing the Calibration Standard” on page 31 and “Loading

Samples” on page 32 to review handling samples.

Follow your individual kit protocol to prepare sample load volume:

10 to 30 µL for a 96-well plate

5 to 15 µL for a 384-well plate

“Seal the plate:” on page 33.

“Placing the Plate Assembly into the Instrument” on page 39.

“Running the Spectral Calibration Plate” on page 40.

Linking and Unlinking a Plate

Link a plate on the autosampler to your newly created plate record before running the plate.

Select the desired plate record, then click the plate position indicator corresponding to the plate position in the instrument. The plate position (A or B) displays in the link column.

Note:

The 3130 genetic analyzer has only one plate position (B) to link a plate record.

The plate position indicator changes from yellow to green when linked and the green run button becomes active.

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Unlinking a Plate Record

1.

Click the plate record that you want to unlink.

2.

Click .

Chapter 7 Running the Instrument

Linking and Unlinking a Plate

1

2

Viewing the Run Schedule

For more information on run scheduling and sample

injection order, see “Plate Mapping” on page 161.

To verify that runs are scheduled correctly:

1.

In the tree pane of the Data Collection software, click

GA Instruments > ga3130xl or

ga3130 >

instrument name >

Run

Scheduler > Run View.

2.

Select a row for any run. The corresponding wells to be injected for that run are highlighted in the plate diagram.

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Chapter 7 Running the Instrument

Running the Instrument

Running the Instrument

Starting the Run

1.

Verify that the active spectral calibration matches your dye set and capillary array length for all scheduled runs.

To change the active spectral calibration, see

“Activating a Spectral Calibration” on page 47.

To create a new spectral calibration, see

“Creating a Spectral Instrument Protocol” on page 35.

2.

Click the green button in the toolbar.

3.

The Processing Plates dialog box opens, then click .

The software automatically performs a run validation:

• If the validation passes, the run starts

• If any of the validation test fails, the run does not start. Check the event log for information.

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Starting the Autoanalysis Manager for

SeqScape and/or GeneMapper Software

Use the Autoanalysis Manager (AAM) software with

SeqScape

®

and/or GeneMapper

®

software to automatically analyze the data.

1.

Select Start > Programs > Applied Biosystems

> Autoanalysis Manager > Autoanalysis

Manager 3.0.

Note:

Autoanalysis Manager does not start automatically. Open Autoanalysis Manager to receive messages from the data collection software for autoanalysis in SeqScape and/or

GeneMapper software.

The Autoanalysis Manager window opens.

Chapter 7 Running the Instrument

Running the Instrument

2.

Quit the SeqScape and/or GeneMapper software.

No other interaction with the AAM software is

needed until the runs finish. See “Using

Autoanalysis Manager Software” on page 155 on

how to use the Autoanalysis Manager.

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Chapter 7 Running the Instrument

Running the Instrument

Basic Run Module

Steps

Automated module steps when the run starts

Module Steps

Turn Oven On

Wait for oven to equilibrate

Initialize autosampler

Fill Array

PreRun

Inject samples

Start separation

Ramp voltage

Collect Data

Run ends:

Leave oven on

Laser to idle

Total time prior to separation:

• Oven unheated: ~25 min

• Oven at set temperature: ~6.5 min

Approximate Time

N/A

1 min 40 sec

3-4 min

3 min

10 to 30 sec

10 min

Variable

Until next run starts

To customize a run module, see the Applied Biosystems 3130/3130xl Genetic Analyzers

Maintenance, Troubleshooting and Reference Guide.

Note:

The PostBatch Utility, which runs automatically, turns off both the oven and the laser at end of a batch of runs.

DNA Sequencing

Run Times

Approximate run times of common DNA sequencing analysis runs

Type of Run Run Module

Ultra rapid UltraSeq36_POP4

UltraSeq36_POP7

Rapid RapidSeq36_POP6

RapidSeq36_POP7

Fast FastSeq50_POP7

Standard StdSeq50_POP4

StdSeq50_POP6

StdSeq50_POP7

Long read LongSeq80_POP4

LongSeq80_POP7

Run Time (min)

40

35

60

60

100

150

120

210

170

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Chapter 7 Running the Instrument

Controlling the Run

Fragment

Analysis Run

Times

Approximate run times of common fragment analysis runs

Type of Run

High Throughput, Small Size

Fragment Analysis

Run Modules

FragmentAnalysis22_POP4

SNP22_POP4

Run Time

(min)

20

20

Standard Fragment Analysis FragmentAnalysis36_POP4

HIDFragmentAnalysis36_POP4

SNP36_POP4

FragmentAnalysis36_POP7

FragmentAnalysis50_POP4

FragmentAnalysis50_POP6

FragmentAnalysis50_POP7

35

65

90

50

45

45

30

For the latest size standard modules (for example, run modules for the LIZ 1200 system), go to http://www.appliedbiosystems.com, then click the link for Support, then Software

Downloads, then GeneScan

™

Size Standards, then Run Modules.

Controlling the Run

Using the Toolbar

Use the toolbar at the top of the data collection software window to control the run.

Click ...

Start Run

Stop

Stop After Current Run

Starts the run

Description

Stops the current run, and all other scheduled runs

Completes the current run, then stops all other scheduled runs

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Chapter 7 Running the Instrument

Set Up for Continuous Operation

Click ...

Description

Stops the current run, then starts the other scheduled runs

Skip to Next Run

Pauses the current run a

Pause Run a Pausing the instrument for too long, especially after sample injection, will adversely affect data quality.

Set Up for Continuous Operation

Overview

Use the continuous run feature to create and link a plate during a run. You can:

• Run one or more plates

• Remove the plate(s) once samples have run

• Link and run additional plates

When using the continuous run feature:

• Only mount or unmount plates when the instrument is paused.

• Create the plate record before pausing the instrument to make linking more efficient, reducing long pauses.

• Link new plates after a run has resumed.

Adding or

Replacing a Plate

During a Run

During a run, you can mount and unmount plates while the instrument is paused. The

plate record can be created, then linked after the run has been resumed.

If the plate is in use, see “Adding, Replacing, or Removing a Plate During a Run” on page 135.

IMPORTANT!

Pausing the instrument for too long, especially after sample injection, will affect data quality.

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Adding, Replacing, or Removing a Plate

During a Run

Replace a completed plate or add a new plate to an unused plate bay (3130xl genetic analyzer only).

1.

Prepare your plate and create the plate record.

2.

Click

In the Pause dialog box, read the pause warning, then click to pause the run.

The Resume run dialog box opens when the run is paused.

IMPORTANT!

Do not click OK to resume the run. Temporarily ignore the dialog box.

3.

Remove the old plate, if applicable.

a.

Press the Tray button to bring the autosampler forward.

b.

Open the instrument door.

c.

Remove the old plate.

4.

Mount the new plate.

5.

Close the door.

6.

Click OK in the Resume Run dialog box.

7.

In the Completed Run dialog box, click continue if the samples have been injected, or click to abort the run and return the instrument to an idle state.

to

IMPORTANT!

If you click OK, the instrument will continue running the current run regardless of whether the samples have actually been injected or not. If the samples have not been injected, the samples will be injected from the new plate.

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Set Up for Continuous Operation

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Chapter 7 Running the Instrument

Set Up for Continuous Operation

8.

Search for the plate record, then link the new plate.

The new plate runs after the current plate completes all scheduled injections.

Note:

If you unmount the currently running plate prior to the first frame of data being collected but after sample injection (clicked OK to continue), the plate status changes to processed even though the run is actually continuing.

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Chapter 7 Running the Instrument

Set Up for Continuous Operation

Viewing Data During a Run

Run Scheduler >

Plate View

In the tree pane of the Data Collection software, click

GA Instruments > ga3130xl or ga3130 >

instrument name >

Run

Scheduler > Plate View.

Note:

The

Run Scheduler and Plate View windows display the same information.

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Chapter 7 Running the Instrument

Set Up for Continuous Operation

Run Scheduler >

Run View

In the tree pane of the Data Collection software, click

GA Instruments > ga3130xl or ga3130 >

instrument name >

Run

Scheduler > Run View to monitor the status of the scheduled runs.

Note:

For default load maps, see Appendix A, “Plate Mapping.”

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Chapter 7 Running the Instrument

Set Up for Continuous Operation

Instrument Status

In the tree pane of the Data Collection software, click

GA Instruments > ga3130xl or ga3130 >

instrument name >

Instrument

Status to monitor the status of the instrument or the current run.

Open the Event Log to monitor system messages

System Status must be

‘Ready’ before a run starts

Array and polymer information

System Status changes from green to flashing red when errors occur, see Event Log.

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Chapter 7 Running the Instrument

Set Up for Continuous Operation

Instrument Condition Sensor States Pane

The color of the indicator provides a quick way to check the status of the item to the left.

See the table below for a definition of each color.

For...

Laser

EP

Oven

Front Doors

Oven Door

Autosampler

A green box indicates...

Laser is off

Electrophoresis is off

Oven is off

Doors are closed

Door is closed

Autosampler is homed

A red box indicates...

Laser is on

Electrophoresis is on

Oven is on

Doors are open

Door is open

Autosampler is forward

A yellow box indicates...

Laser is idle

—

—

—

—

—

Events Box

The Events box lists the:

• Instrument’s recent actions

• Status of each capillary as passed or failed at the end of a spectral calibration

• Calibration data at the end of a spatial calibration

Some of the events listed in the Events box provide information for service engineers.

Errors Box

The Errors box lists errors that have occurred during the current run.

Some of the error messages provide information for service engineers. A “fatal” error usually requires that you restart the data collection software.

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Chapter 7 Running the Instrument

Set Up for Continuous Operation

Instrument Status

> EPT Chart

In the tree pane of the Data Collection software, click

GA Instruments >

ga3130xl or ga3130 >

instrument name >

Instrument Status > EPT

Chart. The EPT chart displays real-time electrophoresis (EP) data during a run.

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Chapter 7 Running the Instrument

Set Up for Continuous Operation

Instrument Status

> Event Log

In the tree pane of the Data Collection software, click

GA Instruments >

ga3130xl or ga3130 >

instrument name >

Instrument Status >

Event

Log. The Event log itemizes events such as errors and general information for all data collection steps.

Clear error messages by clicking Clear Errors. The System Status light flashes red until all errors are cleared. Take corrective action based on error message.

Note:

This view can also be used to monitor spectral calibration results in real time to verify the capillary-by-capillary processing status.

Clear Errors changes the System status from red to green (ready state).

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Chapter 7 Running the Instrument

Set Up for Continuous Operation

Capillaries Viewer

In the tree pane of the Data Collection software, click

GA Instruments >

ga3130

or ga3130xl > instrument name >

Capillaries Viewer. Use the Capillary Viewer to examine the quality of the raw data during a run for several capillaries at once.

Click individual colors to view or hide them

Select check boxes of capillaries to display

Raw, multicomponented data

Notes

Check Boxes

Select the check boxes of the capillaries to view their electropherograms. The capillaries are displayed in the order in which the boxes are checked. The more boxes that are selected, the slower the refresh window rate.

Raw Data

An electropherogram is a graph of relative dye concentration against time, plotted for each dye. The displayed raw data has been corrected for spectral overlap

(multicomponented).

How to Zoom

To zoom in and out:

1.

Select a rectangular area over the area of interest by holding down the mouse button. Release the mouse button to zoom in.

2.

Click to return to full view.

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Set Up for Continuous Operation

Cap/Array Viewer

In the tree pane of the Data Collection software, click

GA Instruments >

ga3130

or ga3130xl > instrument name >

Cap/Array Viewer. Use the window during a run to examine the quality of your data, which is displayed as color data for the entire capillary array. You can view all the capillaries (vertical axis) as a function of time/scan numbers (horizontal axis).

Click individual colors to view or hide them

Raw data display for selected capillary (4)

Capillary order

1

4

Electropherogram displays

0

End

How to Zoom

To zoom in and out:

1.

Select a rectangular area over the area of interest by holding down the mouse button. Release the mouse button to zoom in.

2.

Click to return to full view.

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Chapter 7 Running the Instrument

Working with Data in The Run History View

Working with Data in The Run History View

Run History Components

Elements of the

Run History Utility

Use the Run History utility only with completed runs stored in the local 3130/3130xl genetic analyzer Data Collection database. It does not provide real-time viewing of collecting runs.

In the left tree pane, click the icon next to the function to launch it.

Elements Within the Run History Utility

Icon Element

EPT Viewer

Displays the...

Electrophoresis data of the run, such as voltage, current, and oven temperature profiles.

Event Log Event messages associated with the run.

Instrument

Protocol

Spatial

Calibration

Viewer

Capillaries

Viewer

Cap/Array

Viewer

Spectral

Viewer

Reextraction

Instrument protocol and run module settings used for the run.

Spatial calibration associated with the run.

Individual electropherogram for each sample in the run.

Array view of the run.

Spectral calibration used for the run.

Extraction and analysis status for all samples in the run.

See “Viewing Autoextraction Results” on page 147 for

more details.

Note:

If Cleanup Database has been used, you cannot view processed data in Run History.

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Chapter 7 Running the Instrument

Viewing Data from a Completed Run in the Data Collection Software

Viewing Data from a Completed Run in the Data Collection

Software

View data using two formats within the 3130/3130xl genetic analyzer Data Collection software under the

Run History icon:

• In the Cap/Array Viewer window (in much the same way that you might view the gel file output from an ABI P

RISM

®

slab gel instrument).

• In the Capillary Viewer window, capillary-bycapillary.

Viewing Data from a Completed Run

1.

In the tree pane of the Data Collection software, click

GA Instruments > ga3130xl or

ga3130 > Run History to select the run you want to view.

2.

Search for your run by either Barcode or

Advanced search.

3.

After choosing the run, click the run history elements, such as Cap/Array Viewer or the

Capillary Viewer from the left tree pane to look at the run data.

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Chapter 7 Running the Instrument

Viewing Autoextraction Results

Viewing Autoextraction Results

Overview

After a run is completed, extraction and analysis is performed automatically, using the settings in the Plate Editor and the Results Group. View the results of extraction and analysis in the Reextraction Panel. Reextract samples with the same settings, or with different Analysis Protocols or Results Groups. This process can be useful for many reasons:

• The destination location may not have been available during extraction.

• Some samples may have failed analysis and a different Analysis Protocol might be more successful.

• Samples might be saved in different locations, or with no analysis at all to save space.

• Sample names may need to be edited.

Sample File

Destinations

Locate the Results Group’s Naming Tab and check the Destination Tab and Run Folder

Name Format to find the sample file destination.

Effects of

Changes Made in the Reextraction

Panel

Changes made in the Reextraction Panel to a Results Group, Analysis protocol, sample names, or Comments, also change in the original plate record. The original plate information is overwritten.

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Chapter 7 Running the Instrument

Viewing Autoextraction Results

Selecting and Queuing Samples for

Extraction

Queue individual samples for reextraction to experiment with different Analysis Protocols for samples that have failed initial extraction.

1.

Click (Run History).

2.

Enter the plate name for a plate that has been completed, or click Search. Plates that have runs still pending cannot be reextracted. All the runs from that plate appear in the window.

3.

Select a run from the list.

4.

Click (Reextraction) in the left tree pane to display the Reextraction window.

5.

Click the check boxes in the Extract column to select the samples to be reextracted.

6.

Click Extract to start the reextraction.

Note:

Reextracted sample files are saved in the original folder that data was extracted to unless this destination was changed through the Results

Group.

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Chapter 7 Running the Instrument

Viewing Autoextraction Results

Elements of the Reextraction Window

All the samples are displayed with the results of extraction and analysis.

Note:

Sort the columns of the re-extraction panel by holding the shift key and then clicking on a column header.

Reextraction Window for Sequencing Analysis

Use check boxes to select samples to be reextracted

Select a run

Results of extraction and analysis

Click here to start extraction

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Use these buttons if several samples are highlighted

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Viewing Autoextraction Results

Reextraction Window for Fragment Analysis

Use check boxes to select samples to be reextracted Select a run

Results of extraction

Click here to start extraction

Use these buttons if several samples are highlighted

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Chapter 7 Running the Instrument

Viewing Autoextraction Results

Results Column

The results of extraction and analysis are color coded in the Results column. The following table lists the colors and their values for Sequencing Analysis.

Color

Red

Yellow

Green

Value

Extraction or analysis failed

Note:

Descriptive messages can be viewed by resizing the Results column to view all text.

Warnings for extraction or analysis

Note:

Descriptive messages can be viewed by resizing the Results column to view all text.

Successful extraction (with no analysis intended), or successful extraction and analysis.

The Results column, by default, shows only the beginning of any processing message.

The entire message and the sample file location can be viewed by expanding the cell.

There is a tooltip view for each sample results message.

Tooltip view. Access by placing the cursor over the sample of interest

Drag the cell’s edge to expand the column

Notes

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151

7

Chapter 7 Running the Instrument

Viewing Autoextraction Results

Notes

152

Expanded column

Quality Column

The Quality column represents the quality values for an entire sequence. Quality Values are only assigned to analyzed samples when using the KB

™

Basecaller.

Colors displayed and their associated value range

Color Quality Value Range

Red

Orange

Yellow

< 15

≥ 15 and < 20

≥ 20 and < 30

> 30 Green

Note:

For more information on KB Basecaller and Quality Values, see the Applied

Biosystems DNA Sequencing Analysis Software v5.1 User Guide, PN 4346366.

The column is empty (white) if:

• Analysis was not performed

• Analysis failed

• ABI Basecaller was used for analysis. This basecaller does not assign

Quality Values.

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Results Group and Analysis Protocol

Columns

You can edit and make changes for reextraction to the

Results Group and the Analysis Protocol (Analysis

Method in the GeneMapper ® software).

Note:

Select an entire column in the Reextraction window by clicking on the column header. For example, clicking on the Extract column header selects all samples. Clicking the Uncheck or Check buttons at the bottom of the window, enables or disables the check boxes for each sample. The filldown command (Ctrl+D) works the same here as in the Plate Editor for easier information input.

Sorting the Samples

1.

Hold down the shift key while clicking on the column header to sort samples according to the column properties.

2.

Shift-click again to sort them in reverse order.

Sort by:

• Capillary number

• Well position

• Results

• Quality

• Extract column

Note:

For example, bring all of the failed analysis or extraction samples to the top of the column. Now they can be viewed without having to scroll down to each sample individually.

Chapter 7 Running the Instrument

Viewing Autoextraction Results

Notes

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153

7

Chapter 7 Running the Instrument

Viewing Autoextraction Results

Reextracting Selected Samples

1.

Expand the Results column cells for any yellow or red results to see a description of the warning or failure.

2.

If desired, select a new Results Group, or edit the current one. Selecting a new Results Group allows you to turn off autoanalysis, change the samples and folder naming options, the location where they are placed, and the owner of the

Results Group.

3.

If desired, change the Analysis Protocol to experiment with different ways of analyzing the sample, using a different basecaller for example.

4.

Check the check box in the Extract column for the samples you wish to extract again.

5.

Click Extract.

Note:

Reextraction creates an entirely new sample file and does not replace the previously saved sample file. The presence of a previous sample file has no effect on the creation of a new sample file. If the same naming options that are used for reextraction are identical to those used previously, a number is appended to the filename. For example, if the first sample is,

“sample 01.ab1” then the second sample would be, “sample 01 (1).ab1.”

Notes

154

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Chapter 7 Running the Instrument

Using Autoanalysis Manager Software

Using Autoanalysis Manager Software

Use Autoanalysis Manager software with SeqScape and/or GeneMapper software. The

Autoanalysis Manager software is installed by the SeqScape or GeneMapper software installation CD.

Autoanalysis occurs in the following sequence:

1.

When data collection software finishes a run, the Message Service sends the message “Run Completed.”

2.

The Autoanalysis Manager receives the message, and the job is submitted. The job appears in the General tab.

3.

The Autoanalysis Manager polls for jobs every 2 minutes and opens the automated processing version of SeqScape and/or GeneMapper software to analyze the data.

4.

At the end of analysis, the status in the Autoanalysis Manager is updated.

Files Created

The data collection software stores the sample files in the location specified in the results group. The Autoanalysis Manager copies the files into the Data Store for SeqScape or

GeneMapper processing.

To maintain sufficient storage space on your hard drive, delete or transfer unnecessary sample files to a different network or CD.

Autoanalyzed

Samples

Note:

If your GeneMapper or SeqScape software was opened during autoanalysis, then close your analysis application and relaunch the Autoanalysis Manager.

Once an internal message from the instrument is received by the Autoanalysis Manager, it opens the automated processing version of SeqScape and/or GeneMapper software to autoanalyze the samples. The standard user version of SeqScape and/or GeneMapper software must be closed in order for autoanalysis to begin.

Notes

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

155

7

Chapter 7 Running the Instrument

Using Autoanalysis Manager Software

Components

The Autoanalysis Manager has two or three tabs:

• General tab

• GeneMapper tab, if GeneMapper v3.7 or GeneMapper ID v3.2 software is installed

• SeqScape tab, if SeqScape software is installed

General Tab

The General tab shows the jobs that have been submitted and their status.

Notes

156

Command Buttons in the General Tab

Command Button Functions in the General Tab

Button Name

Delete Job

Delete Completed Jobs

Move Job Up

Move Job Down

Function

Deletes an individual job/project from the Autoanalysis Manager list.

Does not delete sample files, SeqScape or GeneMapper software project.

Deletes all completed jobs/projects from the Autoanalysis

Manager list. Only successful jobs are deleted.

Does not delete sample files, SeqScape or GeneMapper software projects.

The active job/project is always given a queue number of 1. Once job 1 is finished analyzing, job 2 becomes job 1 and all other numbers are changed accordingly. Use the Move Up/Down buttons if you want to rearrange the analysis order.

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Chapter 7 Running the Instrument

Using Autoanalysis Manager Software

GeneMapper

Software Tab

The GeneMapper software tab shows the jobs that have been submitted and their status.

Command Buttons in the GeneMapper Software Tab

Command Button Functions in the GeneMapper Software Tab

Button Name

Configure Schedule

Edit Properties

Function

• Next Analysis Time:

Enables you to set a start time for autoanalysis. Before this time arrives, autoanalysis of projects will not occur.

• Periods restricting automated analysis:

Enables you to set times during which autoanalysis will not occur. Useful if you know that you are going to be reviewing data during a certain time period and don’t want to be bothered by the “Runs ready for processing” dialog box. Runs build up in the queue until the restricting time period is over at which time the runs will be autoanalyzed.

• Automatic Deletion of Completed Jobs:

Enables you to set the software to automatically delete successfully completed jobs. Jobs that failed or have not been analyzed will not be deleted. Only the Autoanalysis Manager job is deleted, sample files and GeneMapper software projects are not.

Enables you to change the following settings:

• Job/Project Name

• UserName: GeneMapper software UserName

• Password: Matching password for GeneMapper software

UserName

• Queue position: Enter a new queue position number for the project

Notes

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157

7

Chapter 7 Running the Instrument

Using Autoanalysis Manager Software

Button Name

Requeue Job

Delete Job

Delete Completed Jobs

Function

• Samples that need to be autoanalyzed have queue numbers listed in the # column.

• Samples that are already analyzed or failed, have a blank cell in the # column.

To resubmit a job for autoanalysis, use the Requeue Job button to assign a queue number to that job.

• Deletes an individual job/project from the AutoAnalysis

Manager list.

Does not delete sample files or GeneMapper software project.

• Deletes all completed jobs/projects from the AutoAnalysis

Manager list. Only successful jobs are deleted.

Does not delete sample files or GeneMapper software projects.

Notes

158

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Chapter 7 Running the Instrument

Using Autoanalysis Manager Software

SeqScape

Software Tab

The SeqScape software tab shows the jobs, project, and status information.

Command Button Functions in the SeqScape Software Tab

Button Name

Details

Function

Displays the project in the navigation pane

Resubmit

Edit Properties

Delete

Submits a job for analysis

Edits the name and password (active only if analysis failed)

Deletes a job from the Autoanalysis Manager

Notes

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

159

7

Chapter 7 Running the Instrument

Viewing Analyzed Data

Viewing Analyzed Data

Locating Sample

Files

Analyzed sample files are extracted into a run folder defined in the Naming tab of your

Results Group, and placed in a location defined in the Destination tab.

The default location is:

E:\AppliedBiosystems\UDC\Data Collection\Data\ <instrument type>\<instrument

name>\<Run Name>

Notes

160

Locating Sample Files

Locate the reextracted data in the location defined by the Results Group or the default destination location.

Viewing Sample

Files

Use the Sequencing Analysis, SeqScape, or the GeneMapper Software to view reextracted sample electropherogram data, both raw, and analyzed.

Sample file type

Sequencing, SeqScape

Fragment analysis

File extension

.ab1

.fsa

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Appendix A

Plate Mapping

Injection Scheduling

Samples are scheduled for injection based on their position in a 96-or 384-well plate and the capillary number. Study the appropriate plate mapping patterns described in

“96-

Well Plate Mapping”

or “384-Well Plate Mapping” to arrange your samples according to

your preferred injection priority.

The injection schedule system is based on the:

• Order of the linked plates (3130xl instrument only). See

page 128 for linking plate

information.

• Sample priority value in the plate record.

If all priorities are set to 100 (default), then runs are scheduled as outlined below. See the

Applied Biosystems 3130/3130xl Genetic Analyzers Maintenance, Troubleshooting, and

Reference Guide on “Run Priority Scheduling”.

Capillary Array Map

The capillary numbers in the capillary array are shown in the layout below. The capillary array layout is the same for both 96- and 384-well plates.

Capillary Number Layout

Notes

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

161

Appendix A

96-Well Plate Mapping

96-Well Plate Mapping

3130xl Genetic

Analyzer

For a 96-well plate, injections are made from every well in two consecutive rows, starting with an odd row. A full 96-well plate requires six runs to inject all samples. See the examples below where samples (grey) and capillary number (number) positions can affect your number of runs.

Efficient Sample Placement

Inject 48 samples using three runs

Inefficient Sample Placement

Inject 32 samples using four runs

15 13 11 9

15 13 11 9

7

16 14 12 10 8

7

16 14 12 10

15 13 11 9

8

7

16 14 12 10 8

5

6

5

6

5

6

3

4

3

4

3

4

1

2

1

2

1

2

Run 1

Run 2

Run 3

15 13 11 9

15 13 11 9

7

16 14 12 10 8

7

16 14 12 10

15 13 11 9

8

7

16 14 12 10 8

15 13 11 9 7

16 14 12 10 8

5

6

5

5

6

6

5

6

3

4

3

3

4

4

3

4

1

2

1

1

2

2

1

2

Run 1

Run 2

Run 3

Run 4

3130 Genetic

Analyzer

For a 96-well plate, injections are made from four consecutive wells in a row. A full plate of 96 sample requires 24 runs to inject all samples once.

In the following example of a 96-well plate, the gray circles represent samples and the number in the well indicates capillary number. It takes four runs to inject 16 samples.

Run 2

Run 4

4

4

3

3

2

2

1

1

4

4

3

3

2

2

1

1

Run 1

Run 3

Notes

162

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Appendix A

384-Well Plate Mapping

384-Well Plate Mapping

3130xl Genetic

Analyzer

For a 384-well plate, injections are made from every other well and every other row. A full plate of 384 samples requires 24 runs to inject all samples once.

The example below is the injection pattern for the first four injections, starting with well

A01. The light gray circles represent samples and the dark gray circles indicate the injection pattern.

Run 1 Run 2

Run 3

Run 4

3130 Genetic

Analyzer

For a 384-well plate, injections are made from every other well. A full plate of 384 sample requires 96 runs to inject all samples once.

The example below is the injection pattern for the first six injections, starting with well

A01. The light gray circles represent samples and the dark gray circles indicate the injection pattern.

Run 1

Run 2

Run 3

Run 4

Run 5 Run 6

Notes

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

163

Appendix A

384-Well Plate Mapping

Notes

164

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Index

Numerics

3130 basecaller and DyeSet/Primer files

66 ,

67

A

ABI P

RISM

®

BigDye® Primer Cycle Sequencing Kits table, dye sets and standards

29

ABI P

RISM

®

dGTP BigDye® Terminator Cycle

Sequencing Ready Reaction Kit, table of dye set and standards

29

ABI P

RISM

®

dGTP BigDye® Terminator v 3.0 Cycle

Sequencing Ready Reaction Kit table of dye set and standards

29

ABI P

RISM

®

dRhodamine Terminator Cycle Sequencing

Kit table of information

29

Activating spectral calibration

47

AmpFlSTR kits matrix standard

30

table of dye sets, filter, modules

5

analysis protocol deleting

68

,

97

editing

68 ,

96

export

69

,

97

general description

55

import

69

,

97

options

58

options, SeqScape software

87

analysis, manual

54

,

83 ,

106

analyzing GeneMapper Software data

160

anode buffer reservoir filling

19

function

11

Applied Biosystems contacting

xi

customer feedback on documentation

x

Information Development department

x

Technical Support

xi

array length, polymer type, table

36

array port, illustration of

12

assembling the plate

34

assumptions for using this guide

ix

Australian EMC standards

xxiii

autoanalysis

GeneMapper Software, computer name 107

manually, fragment analysis documentation 106

manually, Seqscape software documentation

83

manually, sequencing documentation 54 sequencing 54

Autoanalysis Manager

components 155 explained

82

general tab

156

starting

131

using

155

autoextraction

overview 147

Automated Processing tab 118

automated SeqScape analysis 82

automatic analysis, before you start 83

autosampler 18 function

11

B

barcode reader 6

barcode, type of search 40

basecaller and DyeSet/Primer files 66

basecaller files 66 , 67

BigDye® Terminator chemistry, information tables 66

BigDye® Terminator v3.1 Cycle Sequencing Kit table, dye set and standard 29 biohazardous waste, handling

xx

bold text, when to use ix buffer hazards 32 making

16

buffer fill-line 12

buffer reservoir, anode, illustration of 12

buffer valve pin, illustration of 12

C

calibration standards types, fragment analysis 30 types, sequencing 29

Canadian safety standards xxiii

cap/array viewer 144 capillaries viewer

143

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

165

capillary array function 11

illustration of 12

installing or replacing 13

capillary array knob and tip, illustration of 12

capillary array length polymer type, table 36

cathode buffer reservoir, filling 17

CAUTION description xiv example xv

Change Polymer Type Wizard, using 15

characters acceptable 54 , 83 , 106 check valve diagram of 12 chemical safety

xviii

chemical safety guidelines xix

chemical waste hazards xx safety

xx safety guidelines xx chemical waste disposal, guidelines xx

computer assigning a unique name 9 starting 7

condition number, spectral calibration 42

conventions bold text ix

IMPORTANTS!

ix

in this guide ix italic text ix

menu commands ix

Notes ix

user attention words ix

conventions, safety xiv creating analysis protocol for autoanalysis 62 , 93

GeneMapper Software plate record for

autoanalysis 123

SeqScape Software plate record 101

Sequencing Analysis plate record for

autoanalysis 77

customer feedback, on Applied Biosystems

documents x

D

DANGER description xiv example xv data how to zoom 143 viewing 137

166

viewing analyzed sequencing data 160

viewing complete run 146

Data Collection software starting 9

data collection software, starting 9

destinations 147

detection cell block and heater

function 11

documentation

GeneMapper Software User Guide (PN) 106 related x

double-tapered ferrule, illustration of 12

dye primer chemistry, information table 67

dye set fragment analysis 30 , 114 table, sequencing

29

Dye Terminator chemistry, files 66

DyeSet/Primer files 66 , 67 list of 64 , 94

E

E dye set, sequencing kit table 61 electrical hazard symbol xvi electrical safety xxi

electrical shock hazards xxi electrical symbols, on instruments xv

electrode, illustration of 12

electromagnetic compatibility standards. See EMC

standards

electropherogram, definition 143

EMC standards xxiii

Australian

xxiii

Canadian

xxiii

European

xxiii

EPT chart

141

ergonomic safety xxii

errors box 140

European EMC standards xxiii

European safety standards

xxiii

event log 142

icon, displays 145

events, box 140

F

file extension 160

file naming, invalid characters 54

fill down special, function 38

fill line, anode buffer reservoir 19

filter set

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

fragment analysis 5 find all plate search

40

fragment analysis data analysis 160 filter set 5 kit types

5 passing profiles, spectral calibration 51

run modules 113

run, average time 133

function 11

G

GeneMapper Software computer name 107 elements 109 generic

107

instrument protocol 108

plate records 108 results group

108

GeneMapper Software tab 157

GeneMapper™ Software and GeneMapper™ ID

Software 106

general hazard symbol xvi

guidelines chemical safety xix

chemical waste disposal xx

chemical waste safety

xx

waste disposal xx

H

hard drive space, automatic checking 132 hazard icons accompanying safety alert words xiv components xiv described

xiv in documents xiv

on instruments

xiv , xvi

See also hazard symbols

See also safety symbols hazard symbols

electrical xvi general

xvi hot surface xvi

in documents

xv

laser hazard

xvi

moving parts

xvi

on instruments

xvi

See also hazard icons

See also safety symbols hazards

chemical waste xx electrical shock

xxi

laser xxi

moving parts xxi

moving/lifting instrument xvii

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide physical xxi repetitive motion xxii

I

illustration of 12

IMPORTANT description

xiv

example xv

IMPORTANTS!, description ix

Information Development department, contacting x

Install Array Wizard using 13 installation category xxi instrument condition, status

140

diagram of interior 11 documentation x

EPT status 141 layout 2 operation

130

setup

12

starting the system 8

instrument operational safety, instructions for xviii

instrument protocol creating 35 creating for SeqScape software 88

creating, for fragment analysis 109 , 112

general description 55 icon, displays 145

options, SeqScape software 87

options,s equencing 58

instrument status 139 interconnect tube 12 invalid characters in names 54 , 83 , 106 italic text, when to use ix

K

KB basecalling run modules 66

L

labels, instrument safety xvi laser hazard xxi symbol xvi warnings

xxii

laser safety

xxi

laser classification xxi

requirements xxii

Launching Data Collection software 9

layout, instrument 2 length of read, definition 3 linking a plate 128

167

LOR definition

3

lower polymer block 11

lower polymer block, illustration of 12

Luer fitting, illustration of 12

M

manual analysis, fragment analysis documentation 106 manual analysis, Seqscape software documentation

83

manual analysis, sequencing documentation 54 matrix standard fragment analysis 30 matrix standards fragment analysis 114

preparing for sequencing 31

sequencing 29

menu commands, conventions for describing ix

mixed plate record 55 modules fragment analysis 4 , 5

KB basecalling

66

sequencing 3 spectral 36

mounting pin, illustration of 12

moving and lifting computer xviii monitor

xviii moving and lifting instrument, safety xvii

moving parts hazard xxi

hazard symbol xvi

safety xxi

MSDSs description xviii obtaining

xix

referring to

xix , xx when to review

xviii

N

Naming tab, fragment analysis 118

Notes, description ix

O

O-ring, illustration of 12 oven function 11

overflow hole, illustration of 12

overvoltage category (rating)

xxi

overvoltage rating xxi

P

pausing a run

133

PDP motor cover, illustration of

12

physical hazard safety

xxi

physical hazards

xxi

piston, illustration of

12

plate assembly diagram

34

placing in instrument

39

plate editor, filling in

37

plate manager, access

37

plate record creating

37

creating for GeneMapper Software autoanalysis

123

creating for SeqScape Software

101

creating for Sequencing Analysis autoanalysis

77

discussed

55

,

83 ,

108

elements, sequencing analysis

56

linking and unlinking

128

mixed

55

search

40

plate view

137

plates linking and unlinking from plate records

128

sealing

33

polymer array length, table

36

changing to new type

15

replenishing or changing

14

when to add or change

14

polymer delivery pump diagram

12

polymer supply bottle cap with hole, illustration of

12

polymer supply bottle, illustration of

12

polymer supply tube illustration of

12

protocol manager, access

35

pump block

12

function

11

pump chamber, illustration of

12

Q

quality column

152

quality value range

152

Q-value, spectral calibration

42

R

radioactive waste, handling

xx

reader, barcode

6

168

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

reextraction editing Results Group, Analysis Protocol, Analysis

Method

153

results column

151

selected samples

154

selecting

148

window elements

149

reextraction panel effects of changes made in

147

repetitive motion hazard

xxii

Replenish Polymer Wizard using

15

reservoirs filling

17

positions on the autosampler

18

resolution fragment analysis

4

results column, reextraction

151

Results Group creating for sequencing analysis

70

creating, fragment analysis

115

creating, SeqScape software

98

editor, GeneMapper

107

general description

55

importing and exporting

100

options

57

options, SeqScape software

165

run fragment analysis

133

requires plate record

55

,

84 ,

108

sensor states

140

sequencing, average time

132

starting

130

starting, stopping, skipping, pausing

133

viewing data

137

run history utility

145

run modules automated steps, description

132

selecting for fragment analysis

113

selecting spectral

36

run schedule, viewing

129

run scheduler spatial

22

verifying schedule

129

run time fragment analysis

4

spectral calibration

41

running buffer, making and storing

16

S

safety before operating the instrument

xvii

chemical

xviii

chemical waste

xx

conventions

xiv

electrical

xxi

ergonomic

xxii

instrument

xvii

instrument operation

xviii

laser

xxi

moving and lifting instrument

xvii

moving parts

xxi

physical hazard

xxi

standards

xxiii

workstation

xxii

safety alert words accompanying hazard icons

xiv

CAUTIONS

xiv

DANGERS

xiv

description

xiv

IMPORTANTS

xiv

WARNINGS

xiv

safety labels, on instruments

xvi

safety requirements, laser

xxii

safety standards

Canadian

xxiii

European

xxiii

U.S.

xxiii

safety symbols on instruments

xvi

See also hazard symbols

sample file

147

default location

160

type

160

samples autoanalyzing

155

loading

32

sensor states

140

SeqScape software tab

159

SeqScape Software v2.5

autoanalysis

82

plate editor

84

plate record elements

85

user guide P/N

82

sequencing automated, creating protocols

59

automation, required files

55

data, automating the analysis

82

matrix standards

31

passing profiles, spectral calibration

49

plate editor

55

run time

132

specifications

3

spectral run modules

36

viewing analyzed data

160

sequencing chemistry supported kits

28

service console, using

9

set (activate) spectral calibration

47

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

169

software, starting

9

spatial calibration accepting or rejecting

24

creating

22

evaluating profiles

23

examples of profiles

25

viewer

145

when required

22

specifications fragment analysis

4

sequencing

3

spectral calibration creating plate record

37

evaluating results

43

pass/fail status

42

passing profiles, fragment analysis

51

passing profiles, sequencing

49

performing a

37

preparing standards for

31

run times

41

set spectral calibration

47

when to perform

28

spectral calibration standard, types

29

spectral viewer

145

spectral viewer, blank or deactivated

47

standards

EMC

xxiii

safety

xxiii

starting run

133

spectral calibration run

40

status instrument

139

service console

9

status lights on instrument startup

8

stopping a run

133

supported sequencing chemistry kits

28

symbols hazard

xvi

hot surface

xvi

symbols on instruments electrical

xv

safety

xvi

T

tables summary

3

Technical Support, contacting

xi

text conventions

ix

toolbar

133

training, information on

xi

170

U

unlinking a plate

129

Update Cap Array Info using

13

US safety standards

xxiii

user attention words, described

ix

utility, run history

145

V

viewer cap/array

144

capillaries

143

icons and display information

145

spectral

145

W

WARNING, description

xiv

waste disposal, guidelines

xx

water seal, illustration of

12

water trap illustration of

12

wizard

Replenish Polymer Wizard, using

15

Update Cap Array Info, using

13

workstation safety

xxii

Z

Z dye set, sequencing kit table

61

Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide

Headquarters

850 Lincoln Centre Drive

Foster City, CA 94404 USA

Phone: +1 650.638.5800

Toll Free (In North America): +1 800.345.5224

Fax: +1 650.638.5884

Worldwide Sales and Support

Applied Biosystems vast distribution and service network, composed of highly trained support and applications personnel, reaches 150 countries on six continents. For sales office locations and technical support, please call our local office or refer to our Web site at www.appliedbiosystems.com.

Appli ed Biosystems is committed to providing the world’s leading technology and information for life scientists.

6/2010

Part Number 4352715 Rev.

D