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Factura

™

2.1

Feature Identification Software

User’s Manual

© Copyright 2001, Applied Biosystems

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

Notice to Purchaser: License Disclaimer

Purchase of this software product alone does not imply any license under any process, instrument or other apparatus, system, composition, reagent or kit rights under patent claims owned or otherwise controlled by Applied Biosystems, either expressly, or by estoppel.

ABI P

RISM

and its Design, Applied Biosystems, BioLIMS, GeneScan, Genotyper, INHERIT, SeqEd, and Sequence Navigator are registered trademarks of Applera Corporation or its subsidiaries in the U.S. and certain other countries.

ABI, AutoAssembler, BaseSprinter, Factura, and Primer Express are trademarks of Applera Corporation or its subsidiaries in the U.S. and certain other countries.

AppleScript and Macintosh are registered trademarks of Apple Computer, Inc.

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

Contents

iii

1 About the Factura User’s Manual . . . . . . . . . . . . . . 1-1

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-1

Manual Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2

What is the Factura Process? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-4

Technical Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-5

2 Getting Started . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1

Installation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2

Inside the Factura Applications Folder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-3

Factura Input and Output Sequence Files . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-5

About Processing a Sequence File. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-6

How to Process Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-7

Factura Windows . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-9

Example of Using Factura . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-13

3 Using the Log Window . . . . . . . . . . . . . . . . . . . . . . . 3-1

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-1

Overview of the Log Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-2

Using the Log Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-6

4 Using the Factura Settings Window. . . . . . . . . . . . . 4-1

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-1

About the Factura Settings Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-2

Working with Settings Files. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-9

iv

5 Creating Vector Patterns . . . . . . . . . . . . . . . . . . . . . .5-1

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-1

About Vector Patterns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-2

The Create Vector Pattern Window. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-3

How to Create and Edit Vector Patterns . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-4

Reloading Vector Pattern Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-7

6 Using the Sequence Window . . . . . . . . . . . . . . . . . . .6-1

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-1

About the Sequence Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-2

How to Open the Sequence Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-3

The Sequence Window and Views . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-4

Changing the Appearance of the Sequence. . . . . . . . . . . . . . . . . . . . . . . . . . 6-9

Using the Electropherogram View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-11

Electropherogram View Options. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-15

7 Working with Sequences . . . . . . . . . . . . . . . . . . . . . .7-1

Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-1

Sequence Files Factura Recognizes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-2

How to Open an Existing Sequence File . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-3

How to Create a New Sequence File. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-4

How to Change Sequences in Sequence View . . . . . . . . . . . . . . . . . . . . . . . 7-7

How to Edit and Add Bases In Electropherogram View. . . . . . . . . . . . . . . . 7-9

How to Print . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-11

How to Save Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-14

How to Export Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-15

How to Close a Sequence Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-16

8 Using Sequence Features . . . . . . . . . . . . . . . . . . . . .8-1

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-1

Feature Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-2

How to Add, Remove, or Change Features. . . . . . . . . . . . . . . . . . . . . . . . . . 8-3

v

How to Assign the Status to a Feature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-7

How to Join Features in the Feature Table. . . . . . . . . . . . . . . . . . . . . . . . . . . 8-8

How to Change the Appearance of Features . . . . . . . . . . . . . . . . . . . . . . . . . 8-9

How to Change a Marking Style . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-11

How to Automatically Mark Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-14

How to Search for Patterns in a Sequence . . . . . . . . . . . . . . . . . . . . . . . . . . 8-15

A Using Factura for UNIX . . . . . . . . . . . . . . . . . . . . . A-1

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-1

About Factura for UNIX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-2

Installation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-3

About the Factura Settings File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-7

How to Change the Factura Settings File . . . . . . . . . . . . . . . . . . . . . . . . . . A-9

How to Create Vector Patterns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-11

Types of Input Files Supported . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-13

How to Process a Sequence File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-14

What is the Output? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-16

Using the BioLIMS Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-18

B Using the BioLIMS Database . . . . . . . . . . . . . . . . . B-1

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .B-1

About the BioLIMS Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .B-2

Installing the BioLIMS Client Package, Including Factura. . . . . . . . . . . . . .B-3

How to Configure for Server Connection . . . . . . . . . . . . . . . . . . . . . . . . . .B-10

How to Access the BioLIMS Database . . . . . . . . . . . . . . . . . . . . . . . . . . . .B-12

How to Set the BioLIMS Preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . .B-14

Working with Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .B-16

How to Use the Sequence Chooser Window . . . . . . . . . . . . . . . . . . . . . . . .B-17

C Technical Support. . . . . . . . . . . . . . . . . . . . . . . . . . . C-1

Technical Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C-1

vi

D License and Warranty . . . . . . . . . . . . . . . . . . . . . . . D-1

Software License and Limited Product Warranty . . . . . . . . . . . . . . . . . . . . . D-1

Index

About the Factura User’s Manual 1-1

About the Factura User’s Manual 1

Introduction

In This Chapter

This chapter provides a general introduction to the Factura™ Feature Identification Software. It provides information about the organization of this manual and instructions on how to get help from Applied Biosystems.

Topics in this chapter include the following:

Topic See page

Manual Contents 1-2

What is the Factura Process? 1-4

Technical Support 1-5

1

1-2 About the Factura User’s Manual

Manual Contents

The following table describes the contents of the Factura User’s Manual.

Table 1-1

Chapter contents

Chapter Content See page

1 “About the Factura User’s Manual” is a general introduction to this manual.

It also provides information about the organization of this manual, about special text usage in the manual, and instructions on how to get from Applied Biosystems.

1-4

2 “Getting Started” lists the files provided with the Factura program.

It also describes Factura input and output files, windows, views, and a step-by-step walk through of the using the Factura program.

2-1

3 “Using the Log Window” describes the information displayed in the Log window, and how to save and print the window’s contents.

3-1

4 “Using the Factura Settings Window” describes the information you must input to process sequence files, and how to create, save and load new settings.

4-1

5 “Creating Vector Patterns” explains how to use the Create Vector Pattern File window to create vector patterns, and how and where to save the files.

5-1

6 “Using the Sequence Window” describes the appearance of sequence windows, their four views, and how to change the appearance of the data within the sequence windows.

6-1

7 “Working with Sequences” describes how to open, create, edit, save and print sequences from a sequence window.

7-1


8 “Using Sequence Features” describes how to find, mark, format, and display specific bases or ranges of bases within a sequence.

You can create these features or the Factura program can designate them.

8-1

Appendix A

“Using Factura for UNIX” is an explanation of the variables and commands used with the Factura 2.1 for the UNIX operating system.

There is also a description of how to access the BioLIMS database

®

and how to process sequences using the UNIX operating system.

A-1

AppendixB

“Using the BioLIMS Database” describes how to access the database and how to process sequences and collections that are a result of a search of the database.

This database accommodates multiple users and editions while preserving the original data.

B-1

AppendixC

“Technical Support” is a listing of addresses and phone/fax numbers of Applied Biosystems and its representatives for service and technical assistance.

C-1

AppendixD

“License and Warranty” information. D-1

Index The

Factura User’s Manual

concludes with an index.

Index-1

Table 1-1

Chapter contents

(continued)

Chapter Content See page

1-4 About the Factura User’s Manual

What is the Factura Process?

ProcessingSequence Files

The Factura™ program is used to process sequence files produced by Applied Biosystems instruments and collection software. Raw DNA sequences from automated sequencers often contain vector sequence and ambiguously called bases at both ends that you should remove prior to assembly or analysis.

The Factura program automatically processes sequence files by identifying designated vectors and ambiguous regions, and flagging these features in the sequence file. Assembly and analysis applications can then disregard these ambiguous regions, using only the clear range from the insert.

SequenceIdentifying

Features

After the Factura program identifies ambiguous features, you can take advantage of the editable data in the ABI P

RISM

®

Sample Files (Applied Biosystems automated sequencer integrated data files) to identify and mark additional features in the sequence files.

You view and edit the sequence files using a Factura Sequence window and its four views. In this way, you can further identify features and ambiguous regions in the sequence, and create an unambiguous range of data for input to assembler and analysis applications.

Locating VectorPatterns

To locate vectors and remove ambiguous regions from the sequence, you designate settings information and create a folder of vector patterns appropriate to your data. You assign a vector, a primer, and a cloning site to create a vector pattern file.

Note

To enable the vector identification by the Factura program, you must create vector pattern files that are appropriate to your samples and save them to the Vector Patterns folder.


Technical Support

Release Notes

Release notes are sent to users as necessary to provide updates for information in this manual.

U.S. andWorldwide

Technical SupportCenter

Service and technical help is available to Applied Biosystems customers throughout the world. Please see Appendix C, “Technical Support” for further information.

Getting Started 2-1

Getting Started 2

Overview

In This Chapter

This chapter contains a description of the files provided with the Factura program, descriptions of Factura input and output files, and an introduction to the Factura windows and dialog boxes.


Topic See page

Installation 2-2

Inside the Factura Applications Folder 2-3

Factura Input and Output Sequence Files 2-5

About Processing a Sequence File 2-6

How to Process Files 2-7

Factura Windows 2-9

Example of Using Factura 2-13

2

2-2 Getting Started

Installation

Installing Factura

The Factura program is bundled with the Applied Biosystems analysis applications that accompany ABI P

RISM

®

instruments. For information on installing the Factura program when bundled with the BioLIMS

®

application files, see “Installing the BioLIMS Client Package, Including Factura” on page B-3.

To install the program, insert the installer disk in your Macintosh® computer and double-click the Installer icon.

The installer installs the Factura 2.1 folder that holds the Factura files.

The following is the open Factura application folder following a typical Factura installation. For information on the contents of the folder, see “Inside the Factura Applications Folder” on page 2-3.

Getting Started 2-3

Inside the Factura Applications Folder

Applications foldercontents

The following are the contents of the Factura application folder:

�

About Factura

�

Vectors, Primers, and Sites folders

�

Example AppleScripts

�

FacturaSettings file

�

Factura Log

About Factura

This is a help file that contains special or late-breaking information that may apply to your installation, setup, or processing needs.

Vectors, Primers,and Sites Folders

The three folders used to create vector patterns that enable the Factura program’s ability to distinguish between the vector and the DNA insert are:

�

Vectors

�

Primers

�

Sites

For more information, see “Creating Vector Patterns” on page 5-1.

Note

For the program to access the vector pattern files that you create, save them in the Vector Patterns folder.

continued on next page

2-4 Getting Started

ExampleAppleScripts

You can use Apple’s Macintosh AppleScript® to automate Factura file processing. This folder contains two example scripts. Refer to documentation available from Apple computer to write your own AppleScripts.

FacturaSettingsFile

The FacturaSettings file contains the parameters, displayed in the Settings window, that are the active processing settings. When you change the parameters in the Settings window the new settings become active and are saved to this file automatically.

Factura Log

The Factura Log file is a record of the Factura program’s activities and results, which is displayed in the Log window.

Note

Both the Settings and Log files are automatically accessed by the program. If the files are thrown away or moved out of the Factura program folder, then the program creates a new, empty Log file and a new settings file that contains the built-in default settings.

For each sequence processed, the Log Window displays a description of:

�

Identified features (vector range, ambiguity range, confidence range)

�

Details of the clear range of data (the number of bases in the clear range, the percentage of ambiguities in the clear data, and the original length of the sequence)

Getting Started 2-5

Factura Input and Output Sequence Files

What is aSequence File

A sequence file contains the nucleic acid characters (bases) of a sequence and any annotation associated with the sequence.

The Factura program uses sequence files as input. The sequences you process can come from external sources or you can create them using the program.

The Factura program accepts Sample Files (from ABI P

RISM

instruments), INHERIT

®

files, or text files as sequence input.

Sample Files

A Sample File is a specific data file created by ABI P

RISM

®

sequencing and analysis instruments and data collection software. These files contain base calls, peak locations, feature tables, electropherograms, and other information.

INHERIT Files

INHERIT files are files exported from any of the applications of the INHERIT sequence analysis system. After feature information has been added to a sequence, the Factura program can export the data as an INHERIT file.

These files contain only Sequence, Annotation and Feature Views. They do not contain electropherogram information.


2-6 Getting Started

Text Files

Text

files contain a string of characters that represent the sequence bases. An input sequence must be a text file or any of the standard text formats for sequences, such as Staden, GCG, or Fasta.

You cannot use formatted text files created by word processing programs as sequence files.

Sequences output by the Factura program as text files are created in this format for easy export to other applications, they do not contain electropherogram or feature table information.

About Processing a Sequence File

When you process a sequence using the program, you can save identified features such as vector, ambiguity, confidence range, heterozygote positions, in the Sample File feature table.

The original sequence data is maintained unmodified in the Sample File. A copy of the data is stored in the file as editable data. When you edit and save a sequence, only the editable data is changed. The Factura sequence windows display the editable data. The Settings window in the Factura program allows you to revert sequences to the original data for feature identification.

Note

If you choose to revert an edited file, the editable data is overwritten with original data, and any editing you have performed is lost.

Getting Started 2-7

How to Process Files

Overview

The two ways to process files are by:

� Dragging and dropping

� Choosing the Process Sequences command from the Factura menu

For information on the ways to create a new sequence file, see “How to Create a New Sequence File” on page 7-4.

Dragging andDropping

You can drag and drop one or more files onto the Factura program Icon or an alias of the icon to process sequence files automatically. The files are processed and the results reported in the Log window.

Note Do not drag and drop sequence files and settings files at the same time because the order of processing is not defined.

About Dragging and Dropping

If when you drop files onto the Factura icon the program is not active, then the program is started and processes the sequence files using the default settings or the parameters last saved to the Settings file (see “What Settings are Active?” on page 4-9). The processed files retain the same disk location they had prior to the drag and drop procedure.

2-8 Getting Started

You can also use drag and drop to load a settings file. When a settings file is dropped on the Factura icon the parameters from the dropped file are input to the Settings window and made active.

Note In order to process sample files, you need to setup the Factura program by creating vector pattern files (see “Creating Vector Patterns” on page 5-1) for identification of the vectors associated with your sequence files.

Processing byMenu Selection

To choose sequences for processing:

Step Action

1 Choose Process Sequences from the Factura menu. A File Selection dialog box appears (Figure 2-1).

Figure 2-1 File selection dialog box

2 Use the three File type checkbox boxes to choose any combination of the three types of Factura input sequence files to display in the File selection dialog box (see Figure 2-1).

You can then process only the files that are displayed.

3 Select one or all the files shown in the dialog box for processing.

Getting Started 2-9

Factura Windows

Introduction The Factura program, with its drag and drop feature, can simplify the sequence cleanup and identification process with large sets of files. After adding vector patterns and changing settings during the initial setup procedure, you need only select which sequence files to process and the program does the rest.

You can process, modify settings, view, edit, and print any sequence file by using the windows described below. More detailed information about these windows and their functions are presented later in this manual.

Log Window When the program is opened, the date and time, current settings, and the vector patterns found in the Vector Pattern folder are displayed in the Log window (Figure 2-2).

The accumulated data and results from each processed sequence file are also displayed in the Log window. For details on finding, saving, and printing the information in the Log window see “Using the Log Window” on page 3-1.


Figure 2-2 Log window at startup

2-10 Getting Started

Settings Window The Factura Settings window contains the parameters used to process the sequence files. The Factura program executes whatever parameters are currently input to the Settings window.

For descriptions and details on changing and saving the settings, see Chapter 4, “Using the Factura Settings Window.”

Create VectorPattern Window

The Factura program refers to a vector pattern in order to identify the vector as part of its process. Use the Create Vector Pattern window to create vector patterns and save them as vector pattern files.

Getting Started 2-11

For details on creating vector patterns, see Chapter 5, “Creating Vector Patterns.”

Sequence Window A sequence window displays the contents of a single sequence file. You can open more than one sequence window at a time, allowing you to view and compare multiple sequence files. Sample Files from ABI PRISM instruments can produce information in four views.

INHERIT and text files do not contain electropherogram information and do not produce an Electropherogram view.

The following table lists the four sequence views. For more information, see “Using the Sequence Window” on page 6-1.

This view Displays Use for

Sequence View list of individual bases. searching for specified patterns, add features, and edit the sequence.

Annotation View list of information stored in the sequence file and information about the run that produced the sequence data.

If the file is not a Sample File, basic sequence statistics are presented.

Feature View

Note Text files contain no feature information, but you can add features and export the file (saved) to INHERIT file format.

identified features in sequences produced by ABI PRISM Genetic Analyzers or DNA Sequencers.

adding, modifying, and removing features.

Features identified by the Factura program might include vector segments, ambiguous regions, and positions that represent multiple bases (IUB code calls).

Electropherogram View four-color picture of the signal data collected and analyzed to produce the DNA sequence.

editing individual bases in the sequence, and displaying the original data for comparison.


Sequence Window View Buttons

You can go from one sequence window view to another by clicking the appropriate button in the bottom left corner of the window. Figure 2-3 shows a blowup of the view buttons.

Figure 2-3 Sequence window view buttons

Feature

ElectropherogramSequence

AnnotationView View

View View

Getting Started 2-13

Example of Using Factura

Setting Up aSequence File

In order to enable the program’s ability to identify the vector (used in the collection process) and clean up the DNA insert sequence, you must create a vector pattern appropriate to your input data. For instructions on creating a vector pattern, see Chapter 5, “Creating Vector Patterns.”

Once you have created a vector pattern file, you should select the check box labeled Identify Vector Sequence in the Settings window.

Note The Identify Vector Sequence parameter is set to off in the default settings since vector identification does not take place until you create a vector pattern file and place it in the Vector Patterns folder.

For best results, modify the parameters in the Settings window to correspond to your input data (see Chapter 4, “Using the Factura Settings Window.”)

To setup the Factura program:


Step Action Result

1 Open the Factura program. The Factura Log and Settings windows appear on screen.

2 Create a vector pattern and save it to the Vector Patterns folder (inside the Factura applications folder).

For instructions see Chapter 5, “Creating Vector Patterns.”

The vector pattern is loaded and available for use by Factura.

3 If necessary, input or change parameters in the Settings window.

For instructions see Chapter 4, “Using the Factura Settings Window.”

Whatever parameters are displayed in the Settings window are the active settings.


Processing aSequence File

You can process sequence files by dragging and dropping them on the Factura program icon (or an alias). The Factura program automatically processes the files and displays the results in the Log window.

However, you may first want to setup the program for a particular run of files. The Factura program cannot identify the vector unless it can refer to a suitable vector pattern. In addition, you may want to change the default parameters in the Settings window.

To process a sequence file:

Step Action Result

1 Choose Process Sequences from the Factura menu.

A File Selection dialog box appears.

2 Choose a sequence file and click Process.

Factura processes the file and displays the results in the Log window.

Use a sequence window to view and edit the processed sequence file, or you can use the processed sequence as input to other applications.

Note If there is a problem, an explanatory error message appears in the Log window.

3 Choose Open from the File menu.

A sequence window is created for viewing and editing the file.

Any bases that Factura has identified as being in vector sequence or ambiguous regions or outside of the confidence range are greyed out.

This indicates they are not to be used for further analysis.

4 Click on the Feature view button.

The sequence window changes to show a feature table that contains features added by Factura.

Using the Log Window 3-1

Using the Log Window 3Overview

In This Chapter This chapter provides an explanation of the information displayed in the Log window, how to save the information, and how to print the information.


Topic See page

Overview of the Log Window 3-2


3

3-2 Using the Log Window

Overview of the Log Window

Description The Log window displays a running account of Factura settings and process. The contents of the Log window are saved in the Factura Log file. The Factura Log file is a text file created by the Factura program and is located in the application folder.

You can print the information directly from the Log window, open the Log file in a word processing application for editing and printing, or create an independent text file with the contents of the Log window.

The following is an example of the Log window.

Figure 3-1 Log window

1

2

3

4

5


The following table describes Figure 3-1.

Date and TimeOpened

When you open the Factura program, the Log windows displays the date and time, active settings, and a list of the loaded vector patterns.

The following is an example of the Log window at startup.


Item Description See page

1 Date and time that the program is opened.

“Date and Time Opened” on page 3-3

2 Settings at startup or if you choose Load Settings from the Factura menu.

3 Vector patterns loaded at startup or if you choose Reload Vector Patterns from the Factura menu.

4 Files processed and the results of the processing.

“Processing Results” on page 3-4

5 Date and time that the program is quit.

“Date and Time Quit” on page 3-5

Startup settings

Active settings

List of vectorpatterns loaded


Processing Results General

The Log window displays the results of Factura processing with an individual listing for each sequence processed.

When you select both Identify Vector Sequence and Log Search Statistics as parameters, the Log window displays the level of success that the program had in aligning the proximal and distal halves of each loaded vector pattern against the beginning and end of the sequence, respectively. For more information, see “About Vector Patterns” on page 5-2.

Interpreting the Results

By examining and scoring the number of base matches at the cut–site, the Factura program charts the biggest proximal and distal matches and highest proximal and distal scores (Figure 3-2).

The Log window also displays results data for each sequence file processed as shown in Figure 3-3.

Figure 3-2 Proximal and distal matches

Figure 3-3 Processed sequence results


These results are interpreted as follows:

Date and TimeQuit

When you quit the Factura program, the date and time are entered in the Log file.

The Log window displays this information the next time you open the program.

Note You can use the start and quit times displayed in the Log window to find the beginning and end of previous Factura sessions.

Field Description

Vector Range The portion or portions of the sequence data that correspond to the vector.

One or two vector ranges might exist for any one sequence.

Ambiguity Range

The portion or portions of the sequence data that contains more ambiguously called bases than the Identify Ambiguity settings will allow.

For more information about ambiguity, see “Identify Ambiguity” on page 4-5.

Confidence Range

The largest sequence fragment, as assigned in the Settings window, that you accept as containing good data.

This parameter addresses the fact that sequence data tends to deteriorate after a large number of bases are sequenced.

Clear Range A range of bases in the sequence data that represents clear data, that is, data not containing portions identified as vector or ambiguous and which is within the confidence range.

Clear Length The number of clear bases in the clear range.

%N The percentage of Ns (ambiguities) remaining in the clear data.

Original Length

The total length of the input sequence data.


Using the Log Window

Updating The Log window gets updated when you:

� Load a new settings file

� Create a new vector pattern file or reload all vector patterns

If you change or make entries in the Settings window, the new parameters become the active settings. The Log window, however, does not display the new parameters until the Factura program uses the data for processing, input, or when the program is quit and reopened.

Clearing The data in the Log file accumulates until you use the Clear Log command on the Factura menu.

Note When you choose Clear Log, the Log window is emptied and all the data in the Log file is deleted.

Editing The Log window displays the data contained in the Log file, and since it is a text file, you can use any word processing application to edit the file.

If the Log file is moved out of the application folder or thrown away, Factura creates a new (empty) Log file the next time it is opened.

Note The Log file is a text only file. If you open and edit it using a word processing application, you must save it as a text only file in order for the Factura program to use it.

You can use the Save a Copy As command to create a separate text file with the contents of the Log file, edit the file, and then store it using a different name, thereby leaving the original file intact.

Saving The information displayed in the Log window is automatically saved to the Factura Log file. Each session’s data is appended to the Log file, creating a history of Factura’s usage. Each time the program is opened, the Log window displays the Log file along with the new startup information.

To leave the original file intact, use the Save a Copy As command to create separate text files from the content of the Log window.



Printing To print the Factura Log window, when the Log window is the active window, choose Print from the File menu.

Note If you open the Log file in a word processing application, close the file in its original format so that the Factura program can continue to access and add to its contents.

Using the Factura Settings Window 4-1

Using the Factura Settings Window 4Overview

In This Chapter This chapter provides an explanation of the Factura™ Settings window and the parameter (settings) choices you can make.


Topic See page

About the Factura Settings Window 4-2

Working with Settings Files 4-9

4

4-2 Using the Factura Settings Window

About the Factura Settings Window

Factura SettingsWindow

The following is an example of the Factura Settings window with the default settings (Figure 4-1).

Figure 4-1 Factura Settings window with the default settings

The Factura Settings window contains the following fields.

Revert SampleFiles

Select this checkbox to maintain the original sequence data from ABI PRISM® collection and sequencing software in its unmodified state in a Sample File. A copy of the base calls is stored in the file as editable data.

If you choose to revert the sequences, then

� Base calls are overwritten with the original data

� Feature table is cleared

� Any editing is lost

When you save a sequence from the Sequence window, only the editable data is changed. This parameter in the Settings window allows you to revert sequences to the original data for feature identification.


This option is useful if your data was substantially edited and you want to return it to the original form before identifying features.

Identify VectorSequence

Select this checkbox to direct the Factura program to identify the vector sequence using the vector patterns that you create (see “Creating Vector Patterns” on page 5-1).

Note The default value of Identify Vector Sequence is off (unchecked), but after you have created vector pattern files that correspond to the sequences you are processing we recommend that you make this parameter active.

Table 4-1 lists the Identify Vector Sequence parameters.

Table 4-1 Identify Vector Sequence parameters.

Use this parameter... To specify...

Maximum permitted gaps

Note If you reduce this number, the identification is performed more quickly, but the sensitivity is lessened for noisy vectors.

the maximum number of gaps allowable in the overlapping sequences when the comparison is made between the vector sequence in the Sample File and the actual vector sequence data.

Minimum match between vector and sequence

how closely the vector data included in the Sample File must match actual vector sequence data to identify the vector.

For example, if a Sample File contains a vector that is 20 bases long before the insert, specifying a percentage of 90% would mean that a match of 18 bases would identify the vector, but a match of only 17 bases would fail.


IdentifyConfidence Range

Select this checkbox to use these parameters to adjust the length of the sequence fragment that you accept as containing good data.

Sequence data tends to deteriorate after a large number of bases are sequenced. The Identify Confidence Range parameters limit the range of the data to what is most likely accurate even if few ambiguities are identified.


Search for short inserts a more rigorous search for the vector, both at the 5' and 3' ends of sequences from Sample Files.

You should always select this checkbox if your sample data might contain short inserts (for example, <400 bases) in addition to long inserts (>800 bases).

Ignore leading Ns

Note We recommend that you always ignore the leading Ns in order to make a more reliable identification of the beginning of the sequence.

the program to ignore Ns at the beginning of a sequence when identifying the vector.

If this checkbox is selected, vector identification begins at the first set of three consecutive bases without ambiguity (intervening Ns) and ignores all bases prior to this.

Log search statistics that the program includes in the results a description of how well each vector pattern matches the processed sequences.

Table 4-1 Identify Vector Sequence parameters. (continued)



Identify Ambiguity Select this checkbox to enable identification of regions of unacceptably high ambiguity in the sequence. Deselecting the box turns off ambiguity identification.

Table 4-2 describes how each of the two fields work.

Table 4-2 Identify Ambiguity parameters:


Remove base from the beginning and end until no more than...

a sliding window of data.

� When you enter values in the first two fields, the program removes one base at a time from each end of the data (beginning and end) until less than the specified number of ambiguities remain in the specified beginning and ending ranges

� It does not check all possible windows, but stops after it finds a series of unambiguous bases that matches the parameter specified.

� Therefore it is possible to miss a region in the middle that has more ambiguity. If you know that substantial ambiguity exists in the middle of the sequence, you might specify a larger window size.

For more information on how the parameter functions, see Table 4-3.

Reject remaining region if it has more than...

the total percentage of ambiguity allowed in the data after the beginning and ending range specification (immediately above) is met.


How the Program Identifies Areas of Ambiguity

Table 4-3 describes the process of how the program identifies areas of ambiguity.

IdentifyIUB/Heterozygous

Bases

Select this checkbox to specify a threshold used to assign IUB codes (see “Table of IUB Codes” on page 4-7) to mixed base positions.

The program uses these ratios to compare the sizes of up to three peaks in the same location, as seen in the electropherogram.

Note In order to have the Factura program assign the IUB codes for locations that contain three peaks, you should set the threshold percentage of the ratio of the 3rd highest peak to the 2nd to 100%. This disables the assignment of *ABI_Stop features. Once you have disabled the assignment of *ABI_Stop features, the program uses the peak threshold to determine whether to assign IUB codes to the second and third bases.

Table 4-3 How the program identifies areas of ambiguity

Step Action

1 With the default setting, 1 out of 20 bases, the program examines the first and last 20 bases of the sequence.

2 If two Ns are located in either 20 base window (grouping), the program moves in one base and checks the new 20 base windows.

3 This continues until each window consists of 20 bases with no more than one ambiguity.

Note The default setting, 1 out of 20, is equivalent to specifying that the beginning and ending ranges of the data must contain no more than 5% ambiguity.

For example, in the figure below the window stops at each end when 20 unambiguous bases are located, and does not identify the ambiguous region (all Ns) in the middle of the sequence.

Specifying a larger window size such as 1 N out of 50 bases might identify the ambiguity.

3'5'NNNNNNN NN

20 bases20 bases


We suggest that you select the checkbox labeled Update edited bases to store the IUB codes in the Sample Files. If you deselect this option, you must manually edit the data in the Sample Files to clarify ambiguities.

Note IUB/heterozygote identification operates with sequences from ABI PRISM Sample Files, it requires four-color electropherogram data.

How this parameter works

The Factura program first determines if there are multiple peaks in the same location. If the ratio between the second highest peak and the highest peak is above the threshold percentage, an IUB code is assigned (see “Table of IUB Codes” on page 4-7). If not, the existing base assignment is retained.

When analyzing sequences, as in genomic DNA, you may expect to find at most two peaks (bases) in the same location. Therefore, if a third peak is found whose size is above the stop threshold percentage that was assigned in the Settings window, the program creates a ABI-Stop feature. This alerts the user to take a closer look at the location. If the third peak is below the stop threshold, an IUB code is assigned to the first two bases identified.

Table of IUB Codes

IUB (International Union of Biochemists) codes indicate the presence of multiple bases at a given position in a sequence.

Table 4-4 IUB codes

IUB Meaning Complement

A A T

C C G

G G C

T/U T A

M A or C K

R A or G Y

W A or T W

S C or G S

Y C or T R

K G or T M

V A or C or G B


Export ClearRange

Select this checkbox to export the clear range of bases, identified by the Factura processing, to a new text file. You can write the new data back to the Sample File used for input and create a new text file, or you can leave the original file as it was.

The text file is created in the same folder as the original Sample File and have the same name as the Sample File with a .seq suffix added.

Note You can use this checkbox for Sample and INHERIT files. The Factura program automatically creates a copy of the original sequence when processing a text file. The file copy is created in the same location with a .original suffix.

Write Back toSample Files

Select this checkbox to write back the processed information to the original Sample File.

The results of the processed sequence are documented in the Log window and saved to the Log file. If you want to use the original Sample File as input to other applications, you can choose to write the results back to the original file.

Note In order to preserve the original sequence listing of a text file, the Factura program automatically creates a copy of the original sequence when a text file is processed. The program cannot revert the original sequence listing. The file copy is created in the same location with the same name and a “.original” suffix.

H A or C or T D

D A or G or T H

B C or G or T V

Table 4-4 IUB codes (continued)

IUB Meaning Complement


Working with Settings Files

Introduction This section describes:

� What settings are active

� Using the default settings

� Creating a settings file

� Loading a settings file

� Printing a settings file

What Settings areActive?

The Factura Settings window is always open when the Factura program is in use. Whatever parameters are displayed in the Settings window are active and used by the Factura program to process the sequence files. Figure 4-1 on page 4-2 shows the Factura Settings window with the default settings.

At startup, the program accesses the data in the Settings file (located in the application folder, see “Inside the Factura Applications Folder” on page 2-3) and uses it as input for the Settings window.

As soon as the new settings are entered you can use them to process the sequence files. The settings in the Settings window at the time the Factura program is quit are automatically saved to the Settings file. These settings are loaded and active the next time the program is opened.

Using the DefaultSettings

Use the default settings to get the best results from a general sampling of sequence files.

Note Vector Identification is unchecked in the default settings. If you have created vector patterns for your sequence files, you must select this parameter to enable the vector identification process.

To revert to the default settings at any time, choose Default Settings from the Factura menu.



Creating a SettingsFile

You can create a settings file containing the parameters that you specify for the quality of the data you plan to analyze.

For information on loading the settings, see “Loading a Settings File.”

To create your own settings file:

Loading a SettingsFile

There are two ways to load settings:

� Choose the Load Settings command from the Factura menu

� Drag and drop a settings file to the Factura program icon

Note Since the Factura program processes files in alphabetical order, if you drag and drop a settings file at the same time as sequence files, this may result in some sequence files being processed before the new parameters are loaded.

Printing theSettings

To print the settings, print the Log window. The Log window displays the settings that are active when the program is opened and it shows the settings associated with each process run.

To print the Factura Log window, when the Log window is the active window, choose Print from the File menu.

For more information, see Chapter 3, “Using the Log Window.”

Step Action

1 Choose Save Settings from the Factura menu.

A directory dialog box appears.

2 Choose a name for the file and a location and click Save.

Creating Vector Patterns 5-1

Creating Vector Patterns 5Introduction

In This Chapter This chapter provides an explanation of how to create vector patterns, how and where to save the patterns as files, and how to view existing vector patterns.


Topic See page

About Vector Patterns 5-2

The Create Vector Pattern Window 5-3

How to Create and Edit Vector Patterns 5-4

Reloading Vector Pattern Files 5-7

5

5-2 Creating Vector Patterns

About Vector Patterns

What is a VectorPattern?

A vector pattern (Figure 5-1) contains the minimum amount of information the Factura program needs to identify the vector sequence. It consists of the bases between the primer and the cut-site, followed by a caret character (^) that denotes the cut-site, and up to 1000 bases after the cut-site. These vector patterns are stored as text files in the Vector Pattern folder.

Using the vector patterns, the program can identify the vector and eliminate its bases from the DNA insert sequence, leaving a clear (unambiguous) range of the insert bases.

How Is It Used? For each loaded vector pattern, the Factura program attempts a dovetail alignment of the proximal and distal vector sequences with the beginning and end of the sequence being processed (Figure 5-2).

Figure 5-2 Dovetail alignment of the proximal and distal vector sequences

Vector

Primer Sequence

Proximal vector sequence

Restriction enzyme recognition

Cut-siteDistal vector sequence

Figure 5-1 Vector pattern

Proximal vectorsequence

Distal vectorsequence

Sequence being processed


The Create Vector Pattern Window

About the Window Using the Create Vector Pattern window, you can create vector patterns the Factura program uses to identify vectors and process your sequence files. Vector, primer, and site sequences are combined and saved to a vector pattern file that enables the program’s vector identification function.

Folders containing vector, primer, and site sequences are located in the application folder. Using the Create Vector Pattern window you choose from these folders to create a vector pattern. This pattern is then saved to a text file and stored in the Vector Patterns folder, within the Factura application folder.

Note The Factura program cannot identify the vector and accurately identify the ambiguous regions within the insert without referring to vector patterns that you have created.


How to Create and Edit Vector Patterns

Creating a VectorPattern File

The process the Factura program uses depends in part on the identification of the vector, primer and site used in sample preparation. You create vector patterns using this information and save them to vector pattern files that the program uses in the vector identification process.

Note Vector pattern files are text files. Use a word processing application to open, edit, and print the files. Save the file as a text only file after editing.

To create a vector pattern file:

Step Action Result/Comment

1 Choose Create Vector Pattern from the Factura menu.

The Create Vector Pattern window appears on screen.

2 Click the Vector button. The standard Macintosh computer file selection dialog box appears.

3 Locate the Vectors folder inside the Factura application folder, select a vector, and click Open.

The name of the vector you selected appears in the text box next to the Vector button (Figure 5-3 on page 5-5).

4 Click the Primer button, locate the Primers folder inside the Factura application folder, and select a primer.

If you choose a primer that is not a part of the vector you chose, an error message appears. Click Primer again and make another choice.

Note If the Factura program cannot find the primer sequence in the vector, then the program searches for the primer and reverse complement of the cut-site in a reverse complement of the vector.

In some instances, where the primer but not the cut-site is found, you may have to manually complement the primer or cut-site sequence.

If you have any questions, please contact your Technical Support Specialist or Field Applications Specialist.

5 Click Open. The name of the primer you selected appears in the text box next to the Primer button (Figure 5-3 on page 5-5).



6 Click the Site button, locate the Sites folder inside the Factura application folder, and select a site.

If you choose a site that is not a part of the vector you chose, an error message appears. Click Site again and make another choice.

7 Click Open.

For information on editing the vector pattern, see “Editing a Vector Pattern” on page 5-6.

The following happens

� The name of the site you selected appears in the text box next to the Site button (Figure 5-3).

� The vector pattern appears in the editing portion of the window.

\

Figure 5-3 Create Vector Pattern window

8 Click Create. The standard Macintosh computer file selection dialog box appears.

9 Save the vector pattern to the Vector Patterns folder for loading for processing.

This folder is located in the Factura application folder.

To create a vector pattern file: (continued)



Editing a VectorPattern

Editing Using the Create Vector Pattern Window

Once you have designated a vector, a primer, and a site in the Create Vector Pattern window, the vector pattern is displayed in the editing portion of the window.

You can edit the standard vector pattern configurations in the Create Vector Pattern window prior to creating the vector pattern file.

Note The Factura program accesses only vector pattern files that are kept in the Vector Pattern folder.

To edit in the Create Vector Pattern window:

Editing Using Other Applications

Vector pattern files are text files. You can use any word processing application to open, view, and edit the vector pattern files. Save the files as text (text only) files so the Factura program can use them as vector patterns.

Step Action

1 Use any of the standard editing commands on the Macintosh Edit menu to edit the sequence.

2 After configuring the vector pattern to correspond with your data, click Create and save the file to the Vector Pattern folder inside the Factura application folder.

The vector pattern is automatically loaded and ready for use by the program.

A caret characterrepresents thecut-site

Edit portion ofthe window


Vector pattern files, used by the program for vector identification are located in the Vector Patterns folder that is in the Factura application folder.

Editing Using a Sequence Window

You can also use a sequence window to edit a vector pattern file.

Note Factura cannot use INHERIT files as vector pattern files.

Reloading Vector Pattern Files

Why Reloading isNecessary

If you drag vector pattern files to the Vector Pattern folder while the Factura program is open, you must reload the vector patterns to make them available for processing.

How to Reload Vector Patterns

Choose Reload Vector Patterns from the Factura menu.

To edit a vector pattern using a sequence window:

Step Action

1 Display the sequence in a sequence window and use

� Standard Edit menu commands to cut, copy, paste, and clear bases or ranges of the sequence in the active window.

� Select All command in the Edit menu to select the entire sequence (including marked features).

2 When you have completed editing the sequence, save the file to the Vector Patterns folder.

You can do this by choosing:

� Save a Copy As from the File menu and selecting the Vector Patterns folder from the directory dialog box that appears.

� Export from the File menu, Text from the submenu, and selecting the Vector Patterns folder from the directory dialog box that appears.

Using the Sequence Window 6-1

Using the Sequence Window 6Introduction

In This Chapter This chapter provides a description of the appearance of sequence windows, their four views, and how to change the appearance of the data within the sequence windows.


Topic See page

About the Sequence Window 6-2

How to Open the Sequence Window 6-3

The Sequence Window and Views 6-4

Changing the Appearance of the Sequence 6-9

Electropherogram View Options 6-15

6

6-2 Using the Sequence Window

About the Sequence Window

Uses of theWindow

You can use the Sequence windows for

� Creating new sequences

� Viewing and editing existing sequence files

� Comparing two or more sequences

Types ofInformation

Displayed

A sequence window has four views that display different kinds and formats of information.

See “The Sequence Window and Views” on page 6-4 for detailed descriptions of the four Sequence window views.

Opening MultipleWindows

You can open more than one Sequence window at a time. Each window has the name of the sequence (file) it contains. If you type or copy and paste a sequence to create a new sequence file, you can designate the name of the saved file.

The ability to view more than one sequence at the same time allows you to compare and edit sequences on screen as well as from hard copies.


The four Sequence window views are:

Use this view... To...

Sequence view present the listing of bases from the file data.

Annotation view display an annotation from an automated sequencer, if one is available.

Feature view convey the Factura-generated results to other analysis applications in the form of defined vector, ambiguity, confidence range, and IUB/heterozygote features.

Electropherogram view

display sequence data in electropherogram format.

Note Electropherogram view is available only for Sample Files produced by ABI PRISM genetic analysis instruments.


Sequence MenuCommands

The Sequence menu commands become active when a sequence window is active. The individual commands are enabled depending on the particular view being displayed. The Sequence menu commands allow you to change the visual display of the information and to mark sequence features of interest.

How to Open the Sequence Window

Opening theSequence Window

To open the Sequence window:

Step Action


A standard Macintosh file directory dialog box appears.

2 Choose the sequence file that you want to open and click Open.

The following happens:

� Sequence window appears in sequence view with the listing of the individual bases in the sequence

� Sequence menu commands become active when a sequence window is active

� Individual commands on the menu are enabled depending on the particular view being displayed


The Sequence Window and Views

Introduction A sequence window can display sequence information in four formats. Each format has its own sequence window view. Some buttons and displays are common to all views.

Sequence Window The following is an example of a Sequence window.

Table 6-1 lists the parts of the Sequence window.

Table 6-1 Parts of the Sequence window

Item Description

1 If you click the lock image, the sequence is protected from edits. Clicking the image toggles between lock and unlock.

You cannot Cut or Paste to the sequence as long as the lock is closed.

2 The horizontal line in this summary graphic represents the length of the sequence and reflects the cursor position as you move it to different places in the sequence.

1 2 3 4

6

5

7


Sequence View Sequence view is the default view of an opened sequence window in the Factura program. The center column contains the sequence list. The left and right columns show the base positions at the beginning and end of each row.

How to Display in Sequence View

To display in Sequence view from any of the other views, click the second view button.

3 Relative position of the cursor or a selected range of the sequence.

A horizontal box denotes a selected length of the sequence.

4 Locations of color-marked features and the orientation of the sequence

5 The arrowhead on the right end of the summary graphic indicates the orientation (direction) of the sequence.

6 The main (center) portion of the sequence window displays different types of information about the sequence file, depending on which view is displayed.

7 The four small view buttons on the left-lower corner of a sequence window are used to select the view with which to display the sequence data

If you have opened an INHERIT® or text file the Electropherogram button does not appear since those file formats do not contain electropherogram information.

For more information, see “Sequence Window View Buttons” on page 2-12.

Table 6-1 Parts of the Sequence window (continued)

Item Description

You can take the following action in sequence view:

To... See...

find a specified pattern “How to Search for Patterns in a Sequence” on page 8-15.

complement the sequence “Displaying the Complement” on page 6-9.

add or remove features “How to Add, Remove, or Change Features” on page 8-3.


Annotation View Annotation view shows information stored in the file about the run that produced the sequence data.

If the file is not from an ABI PRISM genetic analysis instrument Sample File, basic sequence file information is presented.

How to Display in Annotation View

Click the first view button to display Annotation view.

The following is an example of the sequence window in Annotation view.

Feature View Feature view displays Factura- and user-identified features. The Factura identified features are displayed after the sequence has been processed by the Factura program.

You can assign features active or hidden status using the Feature submenu on the Sequence menu.

For more information, see “How to Assign the Status to a Feature” on page 8-7.

edit the display of a sequence “Electropherogram View Options” on page 6-15.

You can take the following action in sequence view: (continued)

To... See...


How to Display in Feature View

To display Feature view, click the third button.

The following is an example of the sequence window in Feature view.

After you update the sequence files, the Factura program adds feature ranges to the view, identifying portions of the data that represent vector, ambiguity, and confidence range. When you use these sequences with other ABI PRISM data products, you can ignore or hide data in the vector and ambiguity ranges, thereby effectively eliminating poor quality data.

In Feature view you can take the following action.

Note Since the program does not save feature table information to text files, Feature view for text files does not display any features. If you added features to a sequence in a text file, export the file to inherit format to store and display the identified features.


Topic See page

How to Add, Remove, or Change Features 8-3

Changing the Applied Marking Style of a Feature 8-10

Triangle indicatinga hidden feature

Plus sign indicatingan active feature

Ratio ofpeak heights


ElectropherogramView

Electropherogram view is available only with ABI PRISM® Sample Files. It is helpful to compare several files of the same data in Electropherogram view if you want to edit ambiguously called bases.

How to Display in Electropherogam View

To display in Electropherogram view, click the right-most view button.

The following is an example of the sequence window in Electropherogram view.

The default view shows a range of bases in the region of the sequence where you have placed the insertion point. You can zoom in or out, change the horizontal and vertical ruler display, and selectively turn off the drawing of particular bases.

For information on how to change the display of information in Electropherogram view see “Using the Electropherogram View” on page 6-11.


Changing the Appearance of the Sequence

Displaying theComplement

You can display the complement of a sequence in all sequence window views using the complement selection on the Sequence menu.

Changing theAppearance

In Sequence view you can change the font size and style in which the sequence is displayed, as well as the grouping of the characters.

If you want to change the way features are marked in Sequence view, see “How to Change the Appearance of Features” on page 8-9.

To display the complement of a sequence:

Step Action

1 Make sure the relevant sequence window is active.

2 Choose Complement from the Sequence menu.

If you wish to return to the original data, choose Complement a second time.

When vector and ambiguity features have been identified, the characters in these features as well as feature locations in the Feature table are changed to reflect the complement.

The display of original data in the electropherogram also shows the complement sequence (see “Using the Electropherogram View” on page 6-11).

To change the font and grouping of a sequence:

Step Action

1 Make the sequence window active.

2 Choose Sequence View Options from the Sequence menu. The Display Options dialog box appears.


3 The following table lists the options in the Display Options dialog box. As you change a value, the changes appear in the Sample box.

4 Click OK to accept your choices.

To change the font and grouping of a sequence: (continued)

Step Action

Use this field... To...

Font pop-up menu choose Monaco (default) or Courier.

Both fonts have fixed-width letter.

Size pop-up menu select one of seven values in a range from 6 to 24 points.

Prefer outline fonts checkbox specify that dynamically scaled fonts are used.

You do not need to select this checkbox unless you plan to increase the font size substantially (to the point it would otherwise look jagged).

Group characters every text box

specify the number of characters displayed in each group in Sequence view.


Using the Electropherogram View

Introduction In the Electropherogram view, you can:

� Edit the list of the sequence bases by comparing it to the original data

� Change the scaling of the electropherogram

� Hide the display of certain bases

� Change the vertical scale displayed by the window

Editing the List ofthe Base Sequence

In Electropherogram view an editable list of the base sequence appears across the top of the window. You can display the original sequence data in order to compare it to the editable data.

To show the original sequence data in an electropherogram:

Step Action

1 Make sure you are in Electropherogram view in the sequence window.

2 Choose Electropherogram View from the Sequence menu and Show Original from the submenu.

Note If the complement sequence is displayed, the original form of the data also displays as a complement.

Figure 6-1 is an example of the Electropherogram view that shows a second line of data representing the original form of the data. The line below it represents the data you can edit.


Changing Scaling To change the scaling, choose Electropherogram View from the Sequence menu and the submenu lists the scaling options. As you choose the scaling commands, the horizontal scaling changes.

3 You can keep track of any edits that you make to the sequence by comparing the two lines of data.

Figure 6-1 Electropherogram view with original and editable data

To show the original sequence data in an electropherogram: (continued)

Step Action

Original

Editabledata

data


To Zoom In

You can either:

� Choose Zoom In from the submenu to show the data in greater detail, or

� Click one of the bases and choose Zoom In to expand just that area.

After you have used the command three times in succession, it becomes grayed out and you can use either Zoom Out or Actual Size to reduce the size.

To Zoom Out

You can either:

� Choose Zoom Out from the submenu to show the data in smaller detail, or

� Click one of the bases and choose Zoom Out to expand just that area.

After you have used the command three times in succession, it becomes grayed out and you can use either Zoom In or Actual Size to reduce the size.

range of bases


To View in Full View

Choose Full View from the submenu scaled so that the entire electropherogram fits within the Electropherogram view.

To Return to the Original Size

You can either:

� Choose Actual Size from the submenu, or

� You can also click one of the bases and choose Actual Size.


Electropherogram View Options

Introduction You can use the Electropherogram Display Options dialog box in order to emphasize a base or bases and to show the relative values between bases.

Displaying theDialog Box

To display the Electropherogram Display Options dialog box, choose Electropherogram View Option from the Sequence menu. The Electropherogram Display Options dialog box appears.

Displaying Bases You can choose to display any base or combination of bases in the electropherogram using the Show Data checkboxes.

Select and deselect the Show Data checkboxes to selectively turn off the display of one or more of the electropherograms for the four bases. The data shown on the line at the top of the Electropherogram view window is not affected.

Example

If you deselect A, the electropherogram might appear like that shown in Figure 6-2. On your color monitor the green portion of the electropherogram corresponding to A is no longer visible.


Figure 6-2 Electropherogram with “A” deselected in Show Data

Showing DataPoints

Use the checkbox labeled Show data points so that the window shows vertical and horizontal rulers with values and tick points. The checkbox is selected by default.

You have the following options:


“A” continues to appearin the data line

Electropherograms for onlythree bases (C, G, T) aredisplayed

Field Description

Show data points checkbox

If you are using relative values for the vertical scale (see Changing Scaling), the maximum vertical value is about 1200 full scale, so you may want to disable the display of data points.

Counts Per Tick Use to specify the horizontal and vertical indexing of the rulers on the electropherogram display.

The unit of measure (counts) is:

� Number of scans on the horizontal axis performed in the ABI Sequences to obtain the data

� Signal intensity on the vertical axis


Changing Scaling Use the Vertical Display box to change the scaling of the vertical display. The default is Show relative values.

If you click Show relative values, then the scale of the electropherogram display is compressed vertically so that the electropherogram fits within a standard size sequence window (see “Example of Recalibrated Vertical Scale” on page 6-18).

Clicking Show real values, displays the real scale of the data, as in Figure 6-3 (the window was stretched vertically to show a larger portion of the electropherogram).

Note You would have to stretch the electropherogram shown in Figure 6-3 quite a bit further vertically to show the tops of the highest peaks, which are at a scale value of approximately 1200.

Figure 6-3 Real Value electropherogram


Example of Recalibrated Vertical Scale

The Counts Per Tick value is not initially applied when the vertical display is set to Show relative values, as shown in Figure 6-2 on page 6-16. In this mode the default Counts Per Tick value (20) shows too many tick marks.

If you desire, you can change the Counts Per Tick value to recalibrate the vertical scale. For example, if you change the vertical Counts Per Tick value from 20 to 125, the vertical scale shown in Figure 6-4 results.

Figure 6-4 Electropherogram (Recalibrated Vertical Scale)

Working with Sequences 7-1

Working with Sequences 7Overview

In This Chapter This chapter provides descriptions of how to open, create, edit, save and print sequences from a sequence window.

Topics included in this chapter include the following:

Topic See page

Sequence Files Factura Recognizes 7-2

How to Open an Existing Sequence File 7-3

How to Create a New Sequence File 7-4

How to Change Sequences in Sequence View 7-7

How to Edit and Add Bases In Electropherogram View 7-9

How to Print 7-11

How to Save Files 7-14

How to Export Data 7-15

How to Close a Sequence Window 7-16

7

7-2 Working with Sequences

Sequence Files Factura Recognizes

Files Types The Factura program recognizes and opens three types of sequence files:

File type Description

Sample Files Files that contain sequence data in four different formats used to create the four different Sequence window views.

For more information, see “The Sequence Window and Views” on page 6-4.

INHERIT files Files that are exported from any of the applications of the INHERIT® sequence analysis system or created by Factura to retain feature information.

Since INHERIT files don’t contain electropherogram information they produce only Sequence, Annotation and Feature Views.

They do not contain electropherogram information.

Text files Files that contain the sequence string of bases.

You can use only text files or any of the standard text formats for sequences, such as Staden, GCG, and Fasta as sequence files.

The Factura program cannot use formatted text files as sequence files that are created by some word processors.


How to Open an Existing Sequence File

Opening a File To open an existing sequence file:

Step Action Result


A standard Macintosh computer file directory dialog box appears.

2 Choose the sequence file and click Open.

The opened sequence file appears in the Sequence view (default) window and lists the bases in the sequence.

The following is an example of a sequence window.


How to Create a New Sequence File

Overview Creating a new sequence file is a useful tool that enables you to view and edit both sequence information that you create and sequences copied from independent data. You can then save or export the data as an INHERIT or text formatted file.

! WARNING ! The Factura program saves files in the same format in which they were opened. If you created a sequence within an opened text file, you must export the file as an INHERIT file to retain any feature information that has been added. View the file in Annotation view to verify whether the file is a text file or an INHERIT file. If the view is empty, then it is a text file and the features will be lost. For more information, see “Factura Input and Output Sequence Files” on page 2-5.

Creating a NewSequence File

The following procedure describes how to use the New command to create a new sequence file. If you use the New command, then the file is opened and saved in INHERIT format.


To use the New command:


1 Choose New from the File menu.

An empty sequence window appears in Sequence view.

2 Place the insertion point in the central box and type a sequence.

3 Choose Save from the File menu to save the new sequence under a new file name.

The Factura program automatically saves new files as INHERIT files in order to retain any feature information that you created.

Note Exporting in text file format discards any feature information that you added.


Copying andPasting a Sequence

You can copy sequences from any text source and then use them as input to create a new file from within the Sequence window.


To use the Copy and Paste commands:

Step Action Result


A standard Macintosh computer file directory dialog box appears.

2 Choose the sequence file and click Open.

A sequence window opens containing the sequence data stored in the text file.

3 Choose New from the File menu.

A new, blank sequence window appears.

4 Click the sequence window containing the text file to make it active.

5 Choose Select All from the Edit menu.

The entire sequence is highlighted.

6 Choose Copy (Command-C) from the Edit menu.

7 Click the new (empty) sequence window to make it active.

8 Choose Paste (Command-V) from the Edit menu.

The copied text appears in the new sequence window.

9 Choose Save or Export from the File menu.

10 Enter a name for the new sequence and click Save (Command-S).


Exporting aSequence List

The following procedure describes how to use the Export command to generate a new sequence file using an existing sequence file. You can export a sequence list from any source and paste it into a sequence window.

To use the Export command:

Step Action

1 Display the sequence in the sequence view that you want to use to generate a new sequence.

2 Choose Export from the File menu and INHERIT from the submenu.

A directory dialog box appears.

3 Enter a file name, select a location to save the file, and click Save.


How to Change Sequences in Sequence View

Introduction You can perform the following functions in Sequence view:

� Edit a base in a sequence

� Add a base in a sequence

� Delete a base in a sequence

� Change a base in a sequence

You can edit individual bases in Sequence view or Electropherogram view. To remove or change features, use Feature view.

Editing a Base In Sequence View you can use the:

� Standard Edit menu commands to cut, copy, paste, and clear bases or ranges of the sequence in the active window

� Select All command in the Edit menu to select the entire sequence (including marked features)

Editing bases in Electropherogram View is more precise than editing in Sequence View. If you edit ABI PRISM® Sample Files in Sequence View, you should check the Electropherogram view when you are finished.

For information about how to edit bases in Electropherogram view see “How to Edit and Add Bases In Electropherogram View” on page 7-9.

Adding Bases The following procedure describes how to add a base or a range of bases to the sequence.

Note If you add bases in Sequence view and then change to Electropherogram view, the new bases are spaced as evenly as possible between the two previously existing bases.


To add a base or a ranges of bases:

Step Action

1 Place the insertion point at the position in the sequence where you wish to add a base.

2 Type or paste the characters you wish to insert.


Deleting Bases To delete a base or a range of bases from the sequence:

Changing Bases To correct or replace bases in the sequence:

Note You can also place the insertion point to the right of the character you wish to replace, press the delete key, then type the character you want in that position.

Step Action

1 Select the base or range of bases.

2 Press the Delete key or choose Clear from the Edit menu.

Step Action

1 Select the base you wish to change.

2 Type the new character you want in that position.


How to Edit and Add Bases In Electropherogram View

Introduction In Electropherogram view the Edit menu commands are not available and you can edit only one base at a time. You can add, delete, or change bases in much the same way as described for the Sequence view. However, the spacing of the characters is much more precise.i

Editing a Base The following procedures describe how to move the selection between bases and how to select a base and not a position.

Note Because the available base positions are so close together, it is possible to select a position very close to one of the bases when you are actually trying to select the base itself. If you do so, you might insert a character when you intend to change an existing character.

Moving the Selection Between Bases

The following procedure describes how to edit a single base. Multiple base positions (approximately ten) are available between the displayed bases in Electropherogram view, which means that the spacing of the characters is more precise.

To move the selection between bases:

Selecting a Base and Not a Position

The following procedure ensures that you have selected the base, not a position, only one pixel away. Once you have selected the base, you can delete it by pressing Delete or replace it by typing a new character.

Step Action

1 Place the insertion point between two characters and click.

A position is selected.

2 You can take the following action:

To move from... Press the...

base to base Left Arrow and Right Arrow keys.

position to position Option key while you use the Left Arrow and Right Arrow keys.


Note This procedure is useful because the available base positions are so close together, it is possible to select a position very close to one of the bases when you are actually trying to select the base itself. If you do so, you might insert a character when you intend to change an existing character.

Adding a Base You may wish to add bases to the sequence while looking at the electropherogram rather than add them directly to the list of bases in the Sequence view.

To select a base and not the position in Electropherogramview:

Step Action

1 Place the insertion point to the left or the right of the character you wish to select and click.

2 Press the Right Arrow key or Left Arrow key to move to the base you wish to select.

To add bases in the Electropherogram View:

Step Action

1 Place the insertion point between bases where you wish to insert the character and click.

2 To move the insertion point closer to one of the flanking bases, hold down the Option key and use the Left or Right Arrow key to move to the position where you wish to insert the base.

3 Type the new character.


How to Print

Introduction This section describes how to print:

� An electropherogram view in landscape orientation

� All views, including electropherograms, in portrait orientation

Printing anElectropherogram

The following procedure describes how to print an electropherogram.

To print an electropherogram in landscape orientation:

Step Action

1 With a sequence window active, choose Page Setup from the File menu.

The Page Setup dialog box appears.

2 Click landscape (horizontal) orientation.

3 Use the fields in the Electropherogram Settings box to fine tune how the electropherogram is printed.

They specify the number of times the electropherogram wraps down the page and the number of data points displayed within each wrap.

Landscape orientation



You can select the following options.

4 Choose Print from the File menu.

The Print Options dialog box appears.

5 Deselect the all the checkboxes except Electropherogram.

6 Select the checkbox labeled, Allow for 3-hole punch, if you want to punch holes in the page and file it in a three-ring binder.

7 Click Ok.

To print an electropherogram in landscape orientation: (continued)

Step Action

Select... To...

Single Page radio button print the entire electropherogram on one page.

Variable Size radio button print on several pages, depending on the settings in the two text entry fields.

Number of Panes per Page text entry box

enter the number of panes that you want printed on each page.

Number of Points per Pane enter the number of points (peaks) you want displayed in each pane.


Printing All Views To print all the views, including electropherograms:

Step Action

1 With a sequence window active, choose Print from the File menu.

The Print Options dialog box appears.

2 Select the checkboxes indicating the views you wish to print.

3 Select the checkbox labeled, Allow for 3-hole punch, if you want to punch holes in the page and file it in a three-ring binder.

4 Click Ok.

IF you select... THEN...

all four checkboxes � the first three views are printed in landscape on a single page

� the electropherogram prints on several pages

an individual view or only the first three views

They print in portrait orientation.


How to Save Files

Introduction This section describes how to save files in other formats and how to save changes made in the sequence window.

If you have edited any bases or marked features in the sequence window, save the changes before closing the sequence window. When you save changes, the original sequence remains unmodified. Feature changes are stored in the feature table, and edits are stored in the editable copy of the sequence.

Saving Files inOther Formats

The Factura program saves files in the same format in which they were opened.

The following table lists how to create and save files in the INHERIT format and how to change formats.

Note Since text files do not contain feature information, to save features that you have edited in a text file export the file in INHERIT format see “How to Export Data” on page 7-15.

Saving Changes inthe Sequence

Window

The following procedure describes how to save changes in the sequence window.

IF you want to... THEN use the...

create files in the INHERIT format New command on the File menu.

change formats Export submenu on the File menu to change formats.

To save changes made in the sequence window:

Step Action

1 If the window in which you made changes is not the active window, click it to make it active.

2 Choose Save (Command-S) from the File menu.

The changes you made in that sequence window are saved to the sequence file.


How to Export Data

ExportingSequence Data

You can export sequence data that you have opened, created, or edited in a sequence window. You can also save the data back to the original file (see “Saving Changes in the Sequence Window” on page 7-14).

3 If you click the close box in the sequence window without saving the changes, the following dialog box allows you to save them if you wish.

You can take the following action.

To save changes made in the sequence window: (continued)

Step Action

IF you want to... THEN click...

save the changes made in the sequence window

Save.

close the window without saving changes

Don’t Save.

cancel closing the window Cancel.

To export sequence data to a new file format:

Step Action

1 Choose Export from the File menu.The Export selection is only active when you are using the sequence window.

2 You can save files in INHERIT and Text formats.

Note If you have established features by creating and editing a sequence in an opened or created text file, export the file as an INHERIT file, not as a text file. Text files do not contain feature information.


How to Close a Sequence Window

Ways to Close aWindow

There are two ways to close a sequence window:

� Click the close box or

� Choose Close from the File menu while the window is active.

Note You should save any editing before you close the sequence window. See “How to Save Files” on page 7-14 for instructions on saving. To print any of the four views of the sequence window see “How to Print” on page 7-11.

If the SaveChanges Dialog

Box Appears

If you have modified the sequence and have not saved it, the following dialog box appears, allowing you to save the changes.

You can take the following action:

IF you want to... THEN click...

save the changes made in the sequence window

Save.

close the window without saving changes

Don’t Save.

cancel closing the window Cancel.

Using Sequence Features 8-1

Using Sequence Features 8Introduction

In This Chapter This chapter describes how to find, mark, format, and display specific bases or ranges of bases within a sequence. These features are designated by the Factura program process or created by you.


Topic See page

Feature Types 8-2

How to Add, Remove, or Change Features 8-3

How to Assign the Status to a Feature 8-7

How to Join Features in the Feature Table 8-8

How to Change the Appearance of Features 8-9

How to Change a Marking Style 8-11

How to Automatically Mark Features 8-14

How to Search for Patterns in a Sequence 8-15

8

8-2 Using Sequence Features

Feature Types

List of Features The process the Factura program uses automatically identifies and marks five types of features.

Note To comply with the standard GenBank feature table definition, feature names start with an asterisk (*).

Feature Type Description

*ABI_Vector Identifies bases as part of the vector.

*ABI_Limits When the checkbox labeled Identify Confidence Range is checked in the Factura Setting dialog box, this feature creates an active feature within the parameters defined in the Settings window.

*ABI_Ambiguity When checkbox labeled Identify Ambiguity is checked in the Factura Settings dialog box, ambiguous regions are marked as Ambiguity features.

*ABI_Multibase When more than one base has been identified in one location in the sequence this feature is marked.

*ABI_Stop Signifies a third peak appearing in the electropherogram at a location where only two bases were expected.


How to Add, Remove, or Change Features

Introduction After you have processed a sequence file you can add other specialized features, and change or delete the features identified by the Factura program.

Adding a Feature The following procedure describes how to a feature to the feature table.

To add a feature to the feature table:


1 Take the following action.

2 Choose Feature from the Sequence menu and Add from the submenu.

The Add/Edit Feature dialog box appears (Figure 8-1).

Figure 8-1 Add/Edit Feature dialog box

Note If you chose a range of characters in Sequence view, it is entered as a default in the Feature range table.

3 Type a name for the new feature (up to 15 characters long) in the Feature key entry field.

Note To comply with the GenBank feature table definition the name should start with an asterisk (*).

4 Enter text describing the new feature in the Description entry field.

IF you are in... THEN select a...

Feature view feature.

Sequence view range of characters.


5 Choose a marking style from the Style pop-up menu.

Using the Style pop-up menu, you can choose one of eight marking styles to help you identify your feature.

The color of the style you choose identifies your feature in Sequence view, Feature view, and the summary graphic in all views.

Note For information about defining the marking styles, see page 8-11.

6 Check the range of your new feature.

If you selected a range prior to opening the dialog box, it shows as the default range.

To change the range:

a. Select either the start or end value in the appropriate Feature range (s) entry field

b. Type a new value and click Replace

To add a feature to the feature table: (continued)




7 You can take the following action.

8 Type a name for the new feature (up to 15 characters long) in the Feature key entry field.


9 Enter text describing the new feature in the Description entry field.

10 When you are finished defining the feature, click OK.

To add a feature to the feature table: (continued)


To... Then...

add multiple ranges to the feature

for each new range

a. Enter the start and end value in the entry fields.

b. Click Add.

If you do not change the Feature key and Description fields, the new range is added to the same feature.

remove a range a. Select it in the feature window on the right side of the dialog box.

b. Click Delete.

replace a range a. Select the range you want to replace.

b. Type in a new range in the Feature Range (s) entry fields.

c. Click Replace.


Removing aFeature

To remove a feature from a sequence file:

Changing aFeature

To change a feature in the feature table:

Step Action

1 Make sure you are in Feature view.

2 Select the feature you wish to remove.

3 Choose Feature from the Sequence menu and Remove from the submenu.

Step Action


2 Select the feature you wish to change.

3 Choose Feature from the Sequence menu and Modify from the submenu.

The Add/Edit Feature dialog box (shown in Figure 8-1 on page 8-3) appears.

4 Make changes as described in “Adding a Feature” on page 8-3.

You can use the Edit/Feature dialog box to:

� Remove one or more of multiple ranges.

� Add additional ranges.

� Change the values of existing ranges or change the style that marks the feature.

� Change the name or description of the feature by typing new information in the Feature key or Description entry fields.

5 Click OK to implement the changes.


How to Assign the Status to a Feature

Assigning theStatus

To assign the status to a feature:

Step Action


2 Select the feature or features that you want to assign a status.

3 Choose Feature from the Sequence menu and the submenu appears.

For a list of the five types of features, See “Feature Types” on page 8-2.


Choose... To... Symbol displayed

Active create an active feature or range.

+

Note Greys out or hides all of the other bases in the sequence when displayed in sequence view.

Note Other ABI PRISM analysis software applications are able to ignore the hidden features.

Hidden grey-out bases or a range of bases.

Normal list the features in the Feature view that are neither active or hidden.

no symbol is displayed


How to Join Features in the Feature Table

Joining Features You can join two or more individual features in the feature table into a single feature with multiple ranges. The following procedure creates a new feature that contains all the ranges included in the individual features you select. The original features remain in the feature table.

If you wish to delete any of features, see “Removing a Feature” on page 8-6.

To join features in the feature table:

Step Action

1 In Feature view select the features you wish to join by clicking each while pressing the Command key (�).

To select contiguous features:

a. Click the first feature in the group.

b. Press the Shift key and click the last feature in the group.

2 Choose Feature from the Sequence menu and Join from the submenu. The Add/Edit Feature dialog box appears, with blank Feature key and Description entry fields.

The Feature Ranges box at the right shows all ranges from the features you selected.

3 Type a new name for the joined feature in the Feature key entry field.


4 Type text describing the joined feature in the Description entry field.

5 Choose a new marking style from the Style pull-down menu.

6 Click OK.


How to Change the Appearance of Features

Why Change theAppearance

To easily locate the features identified by the Factura program and the special features you add to sequence feature tables, you can mark them with defined styles.

The highlighting appears in

� Sequence view

� Feature view

� Summary graphic at the top of all views

The styles specify colors and borders that mark the features to identify them (the borders appear only in Sequence view).

You can change the way features are marked and the definitions of the marking styles when you are in the Feature view of a sequence window.

Table of DefaultMarking Styles

You can apply any of eight marking styles to a previously created feature (Table 8-1). Each default style has a defined color and border. You can assign new parameters to these styles using the Edit Mark Styles selection on the Sequence menu.


Table 8-1 Default marking styles:

Style Name Color Border

Blue Blue No Underline

*ABI_Multibase Red Light Underline

Green Bold Bright Green Heavy Underline

Gray Double Gray Double Underline

Brown Brown No Underline

D Green Single Dark Green Light Underline

D Blue Bold Dark Blue Heavy Underline

Purple Double Purple Double Underline


Changing theApplied MarkingStyle of a Feature

You can change and update the color and border assigned to a feature in the feature table. The change is displayed in the sequence file.

To change the marking style that is applied to a feature:

Step Action


2 Select the feature.

3 Choose Mark Style from the Sequence menu and the style that you want to apply from the submenu.


How to Change a Marking Style

Changing aMarking Style

You can change the color or border assigned to any of the eight marking styles that are assigned to identify a feature. You can do this any time a sequence window is active in any of the four views.

If you mark a feature with one of the styles and then edit the style, the change occurs immediately in the marked feature.

To edit the marking styles:


1 Make sure a sequence window is active.

2 Choose Edit Mark Styles from the Sequence menu.

The Edit Mark Styles dialog box appears (Figure 8-2).

Figure 8-2 Edit Mark Styles dialog box

3 In the Available Styles listed on the left, select the style you want to edit.

When the dialog box appears, the first style in the list is selected.

Before making any change, make sure that you have selected the style you want to edit.

If you make any entries or choices in the right side of the dialog box, they are applied to the style selected in the list on the left.




5 Click the radio button identifying the border of your choice.

The choices available to differentiate the display of a feature in Sequence view are:

6 When you have finished editing marking styles, click OK.

To edit the marking styles: (continued)


IF you want to... THEN...

change the name of the style selected in the list on the left of the dialog box

type a new name in the Name entry field.

assign a new color style click the color box and the color wheel dialog box appears.

See “Assigning New Colors” on page 8-13 for help assigning and changing colors to a marking style.

Click this radio button... To insert...

no underline under the marked feature (Default).

a line under the feature.

a bold line under the feature.

double line under the feature


Assigning NewColors

You can assign new colors to the mark styles by using the color wheel dialog box.

How to Display the Color Wheel

To display the color wheel dialog box, click the color box in the Edit Mark Styles dialog box (See Figure 8-2 on page 8-11).

The following is an example of the color wheel dialog box.

How to Assign a New Color

Table 8-2 lists the ways that you can assign a new color.

Table 8-2 Ways to assign a new color


To use the scrolling fields:

1 Place the cursor on one of the up or down arrows and hold the mouse button.

This scrolls through the available colors and color parameters.

The upper half of the box shows the changes and the lower half of the box retains the original color until you assign a new color by clicking OK.

2 When you are satisfied with your new color, click OK.

Original color New color

Scrolling fields


How to Automatically Mark Features

AutomaticallyMarking a Feature

You can have the Factura program automatically mark features as they are identified by renaming the marking styles with feature names.

To use the color wheel:

1 Place the insert point on the desired color in the wheel and click.

The new color appears in the upper half of the color box.

2 You can continue moving the insertion point around and clicking until you obtain the desired color.

3 When you are satisfied with your new color, click OK.

Table 8-2 Ways to assign a new color (continued)


To automatically mark a feature:

Step Action

1 Choose Edit Mark Styles from the Sequence menu. The Edit Mark Styles dialog box appears.

2 Select the marking style that you wish to use for the feature.

3 Type the name of the feature in the Name entry field.

Note Enter the name exactly the same as the feature name.

4 Click OK.


How to Search for Patterns in a Sequence

Table of SearchingOptions

The following table lists the searching options that you can use to search for patterns in a sequence.

Note For information on how to use the Find and Find Again commands in the Electropherogram view, see “Searching in Electropherogam View.”

Searching for aSpecified Sequence

When in Sequence view, you can search for a specified sequence of bases or amino acids in a sequence by using the Find command.

To search for... See this topic...

a particular pattern of bases in the Sequence view of an active window

“Searching for a Specified Sequence” on page 8-15.

the same pattern again as many times as necessary

“Finding the Next Occurrence” on page 8-18.

combinations of the bases using Generalized Expression Patterns (Grep)

“Searching for Multiple Base Combinations” on page 8-18.

a specific numeric (offset) position in the sequence list of bases

“Using the Offset Find Option” on page 8-19.

To search for a specified sequence:

Step Action Result

1 Make sure that you are in Sequence view in the sequence window.

Note If you want to find a pattern in the valid (clear) range, place the insertion point just before this range in the sequence.

Note The search begins at the cursor position.

If the pattern is before the cursor, it is only found if Wrap around is turned on (see “Wrap around checkbox” on page 8-17).


2 Choose Find from the Edit menu.

The Find dialog box appears.

For information on the fields, see “Find Dialog Box Fields” on page 8-17.

3 In the Find what? entry field, enter the sequence of characters you wish to locate.

4 Click Find to perform the search.

The first instance of the specified pattern is highlighted and its position is marked in the summary graphic at the top of the sequence window (Figure 8-3).

Figure 8-3 First instance of the specified pattern

To search for a specified sequence: (continued)

Step Action Result

Summary graphic


Find Dialog Box Fields

The following table describes the radio button and checkboxes in Find dialog box.


Table 8-3 Options in the Find dialog box

Field Description

Literal radio button Search for patterns that match exactly what you have typed.

IUPAC/IUB radio button If you have entered a IUB character as part of the pattern.

The Find command locates all possible matches. For example, if you entered TAR, the Find command locates TAG and TAA.

Grep radio button Search for a regular expression pattern.

Table 8-4 on page 8-18 describes some of the expressions and how they function.

Offset radio button Move the cursor to the position or range of the positions you specify.

For more information, see “Using the Offset Find Option” on page 8-19.

Case sensitive checkbox Select if you want either upper- or lower-case variants of a letter recognized as different symbols.

Wrap around checkbox Select if you want the search to start again at the beginning of the sequence after it has reached the end.

If the Wrap around checkbox is not selected, then the search stops at the end of the sequence.


Finding the NextOccurrence

To find the next occurrence of the specified pattern, choose Find Again (Command-G). Choosing this command, you bypass the dialog box and use the pattern defined in the previous Find command.

Each time you use the Find Again command, the next occurrence of the specified pattern is located.

Searching inElectropherogam

View

To use the Find and Find Again commands in Electropherogram View:

Searching forMultiple BaseCombinations

You can search for multiple base combinations, by clicking Grep (Generalized Regular Expression Patterns) in the Find dialog box. Table 8-4 lists the Generalized Regular Expression Patterns.


Step Action

1 Use the Find command in Sequence view.

2 When the pattern is highlighted, switch to Electropherogram View.

The electropherogram displays the range of bases in the highlighted region of the sequence.

Table 8-4 Generalized Regular Expression Patterns

Expression Match Performed Example

[a] (brackets) Any character inside the brackets

AA[AC][GT] matches AAAG, AAAT, AACG, or AACT.

[AGC] matches A, G or C.

[l^l] (brackets with ^ asfirst character inside)

Any character EXCEPT the character(s) inside the brackets

A[^AG]C matches ACC or ATC.

* after character Zero or more such characters

AT[CG]*T matches ATT or ATCT or ATGGT, and so on.

. (period) Any character AA.A matches AAAA, AACA, AAGA, AATA, AANA, and so on.

– (dash) enclosed by brackets

A range of characters AA[A–z] matches AAA, AAC, AAG, AAz, and so on.


Using the OffsetFind Option

The following are two examples of using the Offset Find option.

Entering a string of numbers

If you enter 123 in the Find what? entry field, with Offset selected, the cursor is positioned after the 123rd base in the sequence. Figure 8-4 shows where the insertion point is positioned.

Figure 8-4 Example of a single position found by Find using the Offset option

Entering a range

If you enter the range 123…250 (press Option-semicolon to create the ellipsis), Figure 8-5 shows how the range of bases is highlighted after the Find is completed.

Note Make sure to use the special ellipsis character, created by typing Option-semicolon (;). The range finding function does not work by typing three periods.

Insertion point


Figure 8-5 Example of range found by Find using the Offset option

Summary graphic shows the relativeposition of the highlighted range

Using Factura for UNIX A-1

Using Factura for UNIX AIntroduction

In This Appendix This appendix contains a description of the commands, environment variables, directories, and files related to the Factura 2.1 for the UNIX operating system. There is also information on how to process sequences from the BioLIMS® database.

Topics in this appendix include the following:

Topic See page

About Factura for UNIX A-2

Installation A-3

About the Factura Settings File A-7

How to Change the Factura Settings File A-9

How to Create Vector Patterns A-11

Types of Input Files Supported A-13

How to Process a Sequence File A-14

What is the Output? A-16

Using the BioLIMS Database A-18

A

A-2 Using Factura for UNIX

About Factura for UNIX

Similarities toMacintosh Version

The UNIX and Macintosh computer versions of the Factura 2.1 software use identical algorithms to identify features. The definitions and explanations of algorithm settings and results found in the main text of this manual are applicable to both versions of Factura. For more information on the output sequence files, see “Factura Input and Output Sequence Files” on page 2-5.

For more information on the settings, see Chapter 4, “Using the Factura Settings Window,” and for more information on the results, see “Overview of the Log Window” on page 3-2.

CommandFormats

To display the command line usage, type factura without specifying a sample file, for example

% factura

The following is displayed:


Installation

Copying Files fromCD-ROM

The following steps describe how to copy the Factura for UNIX application files from the CD-ROM.

Note You must have root user permission to execute the following commands.

continued on next page9

Step Action

1 Log in as the root user.

2 Insert the Factura for UNIX CD-ROM into the CD-ROM drive and type:

# cd /chrom/cdrom0

where 0 is the number of the CD-ROM drive.

3 To copy the application to a directory of your choice, type:

# cp -r factura installdir

where installdir is the directory where you want to install Factura for UNIX.

4 To see the files in the Factura install directory, type:

# ls

The following files are listed:

� factura.settings

� Read_Me_First

� sites/

� vectors/

� factura

� primers/

� seqedranks

� vectorpatterns


Running From aWorking Directory

If you want to run Factura for UNIX from a working directory that is different from the installation directory, then you need to set the following three environment variables:

� FACTURA_DOVETAIL_SCORES_FILE

� FACTURA_PATTERN_DIR

� FACTURA_SETTINGS_FILE

To set these environment variables:

Step Action

1 Specify the path to the file that contains dovetail scores by setting the FACTURA_DOVETAIL_SCORES_FILE environment variable.

This example shows the FACTURA_DOVETAIL_SCORES_FILE environment variable pointing to the file called, Dovetail_Scores, in the Factura install directory:

setenv FACTURA_DOVETAIL_SCORES_FILE $HOME/Factura/Dovetail_Scores

If the environment variable, FACTURA_DOVETAIL_SCORES_FILE, is not defined, then Factura for UNIX looks for the file, seqedranks, that resides in the working directory.

2 Specify the path to the pattern directory by setting the FACTURA_PATTERN_DIR environment variable.

This example shows the FACTURA_PATTERN_DIR environment variable pointing to the pattern directory in the Factura install directory:

setenv FACTURA_PATTERN_DIR $HOME/Factura/vectorpatterns/

If you have not defined the environment variable, FACTURA_PATTERN_DIR, then the program goes to the default directory, which is ./vectorpatterns/.


Other OptionalEnvironment

Variables

There are three more optional environment variables that you can set in order to run Factura for UNIX. Edit your .cshrc or .profile file to set the following environment variables:

� FACTURA_VECTOR_DIR

� FACTURA_PRIMER_DIR

� FACTURA_SITE_DIR

To set these optional environment variables:

3 Specify the path to the settings file by setting the FACTURA_SETTINGS_FILE environment variable.

This example shows the FACTURA_SETTINGS_FILE environment variable pointing to the setting file ./factura.settings in the Factura install directory:

setenv FACTURA_SETTINGS_FILE $HOME/Factura/factua.settings

For more information about the Factura settings file, see “About the Factura Settings File” on page A-7.

Step Action

Step Action

1 Specify the path to the vector directory by setting the FACTURA_VECTOR_DIR environment variable.

This example shows the FACTURA_VECTOR_DIR environment variable pointing to the vector directory in the Factura directory:

setenv FACTURA_VECTOR_DIR $HOME/Factura/vectors/

If you have not defined the environment variable, FACTURA_VECTOR_DIR, then the program goes to the default directory, which is ./vectors/.

2 Specify the path to the primer directory by setting the FACTURA_PRIMER_DIR environment variable.

This example shows the FACTURA_PRIMER_DIR environment variable pointing to the primer directory in the Factura directory:

setenv FACTURA_PRIMER_DIR $HOME/Factura/primers/

If you have not defined the environment directory, FACTURA_PRIMER_DIR, then the program goes to the default directory, which is ./primers/.


3 Specify the path to the site directory by setting the FACTURA_SITE_DIR environment variable.

This example shows the FACTURA_SITE_DIR environment variable pointing to the site directory in the Factura directory:

setenv FACTURA_SITE_DIR $HOME/Factura/sites/

If you have not defined the environment directory, FACTURA_SITES_DIR, then the program goes to the default directory, which is ./sites/.

Step Action


About the Factura Settings File

What the File Does The format of the settings file for Factura for UNIX is the same as the Macintosh computer version of the Factura program (for details, see “About the Factura Settings Window” on page 4-2).Factura for UNIX uses the settings file to specify the parameters of the algorithms.

The following table lists how Factura for UNIX uses the settings file:

The file only has to list those settings that explicitly need to be changed. The settings used when processing a sequence depends on all the settings files that you have loaded since starting the application (see “Specifying Multiple Settings Files” on page A-15).

Table of Settings Table A-1 describes how the boolean and integer settings work. Table A-2 lists each of the algorithms, the setting name, setting type, default value, and value range (if appropriate).

IF the... THEN Factura for UNIX...

settings file is not specified in the command line

checks the environment variable FACTURA_SETTINGS_FILE.

environment variable is not defined uses the default file, which is ./factura.settings.

default file does not exist creates a new settings file and saves it in the directory where you are working.

Table A-1 How the settings work

IF there is a line in the settings file for an algorithm... THEN the value is...

any boolean value for that algorithm that is not listed

turned off.

any boolean value for that algorithm that is listed

turned on.

any integer parameter value for that algorithm that is not listed

unchanged.


Table A-2 The settings file

Algorithm name Setting nameSettingtype

Default value

Value range

RevertBases dontExecute boolean off

VectorID dontExecute boolean on

maxGaps integer 20 0...32767

min%Match integer 80 1...32767

shortInserts boolean off

ignoreleadingNs boolean on

logSeachStats boolean off

ConfidenceRangeID

dontExecute boolean off

minValidBase integer 1 1...32767

maxValidBase integer 650 1...32767

AmbiguityID dontExecute boolean off

windowSize integer 20 1...32767

maxNsPerWindow integer 1 0...32767

max%NsInClearRange

integer 5 0...32767

IubID dontExecute boolean off

threshold% integer 50 0...100

stopLevel integer 50 0...100

updateEditedBases boolean on

ExportSamplesAs Text

dontExecute boolean on

WriteBack dontExecute boolean off


How to Change the Factura Settings File

What Can YouChange

The parameters that you can change are shown in Table A-2 on page A-8.

Creating YourOwn Settings File

To create your own Factura settings file, use a text editor to change the values of the algorithms in the file according to the quality of the data that you plan to analyze.

Note It is recommended that you retain a copy of the original Factura settings file.

Each of the settings consists of an algorithm name followed by a colon, and then a list of settings separated by commas. Each setting consists of a setting name followed optionally by an equal sign and a setting value. For example,

VectorID:maxGaps=20,min%Match=80,ignoreleadingNs, logSearchStats,shortInserts;

Ways to Changethe File

You can edit the Factura settings file as follows:

Examples ofSettings Files

The following are two examples of settings files.

All Settings On

The following is an example of a settings file with all processes turned on and all parameters assigned to the default values.

RevertBases:;

VectorID:maxGaps=20,min%Match=80,ignoreleadingNs, logSearchStats,shortInserts;

To... Then...

turn all the settings off insert the line, don’t execute, after the semicolon and before the first parameter

turn off specific parameters delete the parameter from the list

change a parameter value change the value


ConfidenceRangeID:minValidBase=21,maxValidBase=550;

AmbiguityID:windowSize=20,maxNsPerWindow=1,max%NsIn ClearRange=5;

IubID;threshold%=50,stopLevel=50,updateEditedBases;

ExportSampleAsText:;

WriteBack:;

All Settings Off

The following is an example of a setting file with all processes turned off.

RevertBases:dontExecute;

VectorID:dontExecute,maxGaps=20,min%Match=80, ignoreleadingNs,logSearchStats,shortInserts;

ConfidenceRangeID:dontExecute,minValidBase=21, maxValidBase=550;

AmbiguityID:dontExecute,windowSize=20, maxNsPerWindow=1,max%NsInClearRange=5;

IubID:dontExecute,threshold%=50,stopLevel=50, updateEditedBases;

ExportSampleAsText:dontExecute;

WriteBack:dontExecute;


How to Create Vector Patterns

About VectorPatterns

Using vector patterns, Factura for UNIX can identify the insert vector and eliminate its bases from the DNA sequence, leaving a clear range of bases.

Vector pattern files are created by specifying a vector, a primer, and a site (cut-site), and saving the calculated pattern to a text file.

Note Vector identification cannot take place until you have created one or more applicable vector pattern file(s) and stored it in a directory. The program accesses the directory either by means of the environment variable FACTURA_PATTERN_DIR or by using the default directory ./vectorpatterns/.

Note The default setting for the FACTURA_PATTERN_DIR is disabled.

Factura for UNIX compares all the vector patterns in the vector pattern directory to the sequence being processed. This eliminates the need for you to assign a specific vector pattern for each sample run.

You should create a new vector pattern file when the sequence being processed is thought to contain a vector/primer/cut-site combination that does not already exist in the vector pattern directory.

Creating a VectorPattern

To create a single pattern file, you need to know the vector, primer, and cut-site names.

The command line to create a vector pattern is:

% factura -vector VectorFile -primer PrimerFile -site SiteFile [-pattern PatternFile]

File names are required for the VectorFile, PrimerFile, and SiteFile. Naming the PatternFile is optional.



Relative Paths General

If the file name for the VectorFile, PrimerFile, or SiteFile is a relative file name, that is, relative to a directory, then the path of the directory that is pointed to by the corresponding environment variable is appended to the beginning of the file name.

If the environment variable is not defined, then the program uses the default directories, respectively (Table A-3).

Examples

For example, if the VectorFile is a relative file name, Factura for UNIX looks for this file in the directory specified by the FACTURA_VECTOR_DIR environment variable and appends the VectorFile name to the file name. If the environment variable is not defined, then Factura for UNIX looks for the vector file in the ./vectors directory.

IncorrectlyCreated Vector

Patterns Message

If you try to create a vector pattern by incorrectly selecting a primer or site that is not a part of the vector sequence, an error message appears.

The following is an example of an error message.

Could not create vector pattern from the following components:

File Name of vector: Vectors/BLUE

File Name of Primer: Primers/KS

File Name of Site: Sites/Aat

Table A-3 Default directories

./vectors/ ./sites/

./primers/ ./vectorpatterns/


Types of Input Files Supported

Input Files The following types of input files are supported:

� Sample Files (a specific data file created by ABI PRISM sequencing and analysis instruments. The format of the file is called ABIF.)

� INHERIT files

� text files

� Pearson files

The Sample and INHERIT® files are standard formats for data created by Applied Biosystems instruments and collection software.

For more information, see “Factura Input and Output Sequence Files” on page 2-5.


How to Process a Sequence File\\

Processing aSequence File

To process a sequence file type:

Factura {[-s settings_file] sequence_file}

The following table describes the command line options:

Examples You can process a sequence file as follows:

Specifying a Settings File

To specify a settings file:

Factura -s my_settings C057d03f


Where... Designates...

settings_file

Note If you do not specify a settings file, Factura for UNIX checks the contents of the FACTURA_SETTINGS_FILE environment variable.

the file name of the text file containing the algorithms settings (see “About the Factura Settings File” on page A-7).

sequence_file the file name of the sequence input file to be processed (see “Types of Input Files Supported” on page A-13).

You can type multiple sequence files or the name of multiple directories to process all the files in the directory recursively.


my_settings a file that you specify.

C057d03f the name of a sequence file.


Specifying Multiple Settings Files

To specify multiple settings files:

Factura -s my_settings1 C057d03r -s my_settings2 C057d06r

In this case, the second sequence file is processed using the second settings file.

Specifying Multiple Sequence Files

To specify multiple sequence files:

Factura C057d03a C057d03b C057d03c

where the sequence files are separated by a space

Specifying a Directory Name

To specify a directory name:

Factura mydirectory

where mydirectory is the name of a directory

Note The directory can contain subdirectories and Factura for UNIX processes all the contents recursively.


What is the Output?

Files Factura for UNIX may change the name of the original file, create a new ABIF file, or create a text file with the clear range, depending on the settings of the WriteBack option and the ExportSamplesAsText option.

Processing the File This section describes how to process ABIF files and how to process text files.

Processing ABIF Files

The following table describes the options for processing ABIF files:

Processing Text files

For text files, if the WriteBack option is on, then Factura for UNIX saves the original file as filename.original and the processed file as filename.

Display Parameters and results of the process the Factura for UNIX program uses are sent to the console:

� Date and time that the program is opened

� Settings at startup or reloaded

� Vector patterns loaded at startup or reloaded

� Files processed and the results of the processing

� Date and time that the program is quit

For more information see “Overview of the Log Window” on page 3-2.


IF... THEN Factura for UNIX...

WriteBack option is on saves the original file as filename.original and the processed file as filename.

ExportSamplesAsText option is on saves the clear range to a text file called filename.seq.


ProcessingExample

The following is an example of a processed sequence file.


Using the BioLIMS Database

Introduction This section describes how to use Factura for UNIX to process sequences contained in the BioLIMS database.

For more information on the BioLIMS database, see Appendix B, “Using the BioLIMS Database.”

The DatabaseConnection File

In Factura for UNIX, the BioLIMS database connection information is stored in a four-line text file which is pointed to by the environment variable BIOLIMS_CONF.

The form of the file is

database account user namedatabase account passworddatabase nameserver name

An Example For example, the following database connection file would permit a user named george with password george1 to connect to the BioLIMS database sfdb on the server called SYBASE:

georgegeorge1sfdbSYBASE

Editing theDatabase

Connection File

When the BioLIMS applications are installed, a default database connection file called biolims.conf is placed in the Factura directory along with the Factura for UNIX executable. Alternatively, you can find the biolims.conf file in the Factura directory on the BioLIMS CD-ROM. You, or your system administrator, should copy this file into your account where you can edit it, replacing the default information with that appropriate to your BioLIMS database, server, and user account.



EnvironmentVariables

There are three environment variables that you need to set in order to use Factura for UNIX to process sequences from the BioLIMS database. You should edit your .cshrc file or your .profile file to set the BIOLIMS_CONF, the LD_LIBRARY_PATH, and the SYBASE environment variables as follows.

Specifying the BIOLIMS_CONF Variable

Specify the path name to the database connection file using the BIOLIMS_CONF environment variable.

This example points to a database connection file, biolims.conf, in the user’s own Factura directory:

setenv BIOLIMS_CONF ~/factura/biolims.conf

If you move or rename the database connection file, remember to edit your .cshrc or .profile file to point to the correct file name and location.

Note If the BIOLIMS_CONF environment variable is not set, the existence of a file named biolims.conf in the current directory is assumed.

Specifying the LD_LIBRARY_PATH Variable

You need to include the directory of the Sybase SQL Server shared library file (libsybdb.so) in the library path list. When the BioLIMS applications are installed, this file is placed in the lib directory in the sybase account.

Specifying the SYBASE Variable

You need to set the SYBASE environment variable to point to the directory that contains the interfaces file. When the BioLIMS applications are installed, the interfaces file is placed in the top level of the sybase account.



ProcessingSequences

The command line to process BioLIMS database sequences using Factura for UNIX is:

% factura {[-s Settings][-database][-col Collection][-seq DBSeq]}




-s settings a file name of a text file that contains the algorithms settings.

For more information about the settings files, see “About the Factura Settings File” on page A-7.

- database the BioLIMS database.

The program processes all the sequences in the database that have the status = analysis.

- col Collection the name of a database collection.

The program processes all the sequences in the named collection.

-seq DBSeq the name of a database sequence.

The program processes the named sequence.


ProcessingExample

The following is a partial example of the output results of processing a collection.

Using the BioLIMS Database B-1

Using the BioLIMS Database BIntroduction

In This Appendix This appendix describes how to access the BioLIMS database, how to set the preferences, and how to open or process a sequence that is located in the BioLIMS database.

Topics in this appendix include the following:

Topic See page

About the BioLIMS Database B-2

Installing the BioLIMS Client Package, Including Factura B-3

How to Configure for Server Connection B-10

How to Access the BioLIMS Database B-12

How to Set the BioLIMS Preferences B-14

Working with Sequences B-16

How to Use the Sequence Chooser Window B-17

B

B-2 Using the BioLIMS Database

About the BioLIMS Database

In the Factura 2.1 program, we have introduced support for selecting, viewing, editing, and processing sequences from the BioLIMS database.

The BioLIMS system provides a relational database for sequences created by ABI PRISM® analysis software. This database accommodates multiple users and versions while preserving the original data.

The Factura program’s user interface has changed to accommodate accessing the BioLIMS database. Once you have accessed the BioLIMS database, you can display the Sequence Chooser window by choosing the Open command from the File menu or by choosing Process Sequences command from the Factura menu.


Installing the BioLIMS Client Package, Including Factura

Introduction The Factura software is shipped on a CD-ROM disc. A separate disc is shipped as part of the BioLIMS Client Package and as part of the Instrument Package. One BioLIMS Client Package is required for each Macintosh computer on which the Factura software is installed.

This section describes the following:

Before Installing � Check that you have at least 56MB of free disk space to accommodate the BioLIMS applications.

� Quit all open applications.

� Turn off any virus protection software that you may have running.

Installing theBioLIMS Client

Follow these steps to install the entire BioLIMS Client Package onto your Macintosh computer.

Topic See

Complete installation of the BioLIMS Client Package

“Installing the BioLIMS Client” on page B-3.

Custom installation, e.g., installing the Factura software

“Doing a Custom Installation” on page B-4.

Removal of the installation “Removing the Installed Package” on page B-5.

To install the BioLIMS Client Package:

Step Action

1 Insert the BioLIMS Client Package CD-ROM disc.

The BioLIMS Client Package window opens automatically.

2 Double-click the BioLIMS Client Installer icon in the BioLIMS Client Package window.

3 Click Continue.

4 This dialog box contains important information that you should read.

After you have read it, click Continue to open the BioLIMS Client Installer window. You may print or save the contents if you want.


Doing a CustomInstallation

You may not want to install all of the BioLIMS Client Package. For example, you might want to install only the Factura software on your Macintosh computer.

5 To install the whole BioLIMS Client Package, use the default Easy Install described here.

For information about

� Custom installation, see “Doing a Custom Installation” on page B-4.

� Removing an installed package, see “Removing the Installed Package” on page B-5.

6 Use the Switch Disk button or the Install Location pop-up menu to choose the disk on which to install the BioLIMS Client Package.

If the software cannot be installed on the chosen disk, a warning appears in the Installer window.

7 Choose the Select Folder item on the Install Location pop-up menu.

A Macintosh browser box appears.

8 Use the browser box to select a folder in which to install the BioLIMS Client Package applications.

9 Click Install to begin the installation.

10 At the conclusion of the installation, and you should Restart your computer.

To install the BioLIMS Client Package: (continued)

Step Action

To do a custom installation:

Step Action

1 Follow steps 1 to 4 in the procedure “Installing the BioLIMS Client” on page B-3.

2 Select Custom Install from the pop-up menu at the top left of the window.

3 Check the names of all the applications that you want to install.

For information about the individual applications, click the information button to the right of the application name to display an information dialog box.


Removing theInstalled Package

This section describes how to remove the BioLIMS Client Package from your Macintosh computer. The Remove process deletes all the applications installed in the BioLIMS folder and also the files and folders placed in the System folder by the installer.

Note If you have moved BioLIMS files or folders from their original installed locations, they may not be found and deleted by the remove operation. Also, any files that have been added to the application folders, such as those created when the applications are run, are not deleted by the remove operation.

IMPORTANT If you have installed both the BioLIMS Instrument Package and the BioLIMS Client Package on the same Macintosh computer, you should not use Remove unless you intend to delete both the Client and the Instrument Packages. This is because the Remove process deletes files common to both packages, including files that are in the System Folder.

4 Use the Switch Disk button or the Install Location pop-up menu to choose the disk on which to install the selected applications.

Be sure that there is enough space on the disk to accommodate your chosen applications. The Installer window reports both the space available on the disk and the approximate disk space required for the selected applications.



6 Use the browser box to select a folder in which to install the selected applications.

7 Click Install to begin the installation of the selected applications.

8 At the conclusion of the installation, you should Restart your computer.

To do a custom installation: (continued)

Step Action

To removing the BioLIMS Client Package files:

Step Action

1 Follow steps 1 to 4 in the procedure “Installing the BioLIMS Client” on page B-3.

2 Select Remove from the pop-up menu at the top left of the window.




Files Installed This section lists the files that are installed in the:

� BioLIMS folder when you install the BioLIMS Client Package or the Instrument Package

� System folder

BioLIMS Folder

IMPORTANT Before running the software for the first time, read the About text file for the application. Important information not contained in the manual may be found in the About text file.

The following is the open BioLIMS folder following a BioLIMS Client Package installation. Table B-1 lists the files that are installed when you install the BioLIMS Client Package.

4 Use the browser box to locate the folder that contains the BioLIMS folder.

5 Click Remove to begin the removal of the BioLIMS Client Package applications on your disk.

6 At the conclusion of the remove operation, an alert box appears with the message whether or not the remove was successful.

Note If files have been moved or added to the BioLIMS folder, the remove operation will be reported as unsuccessful; you should then examine and delete the remaining files in the BioLIMS folder yourself.

To removing the BioLIMS Client Package files: (continued)

Step Action


The following is the open BioLIMS folder following a BioLIMS Instrument Package installation. Table B-2 lists the files that are installed when you install the Instrument Package.

Table B-1 BioLIMS Client Package files:

This folder… Contains the…

Sequencing Analysis

� Sequencing Analysis and Basecaller applications

� About Sequencing Analysis text file

� Other folders associated with the Sequencing Analysis application

Factura � Factura software

� About Factura text file

� Other files and folders associated with the Factura software

AutoAssembler � AutoAssembler software

� About AutoAssembler text file

� Engines folder

� Assembly Data folder

BioLIMS Extras � Sample2DB, Collections Manager, and SimpleText software

� About Sample2DB and About Collections Manager text files

� Scripts folder

� Sybase folder containing the interfaces and other database-related files


System Folder

Table B-3 lists the files placed in the Macintosh System Folder

Table B-2 The BioLIMS Instrument applications are placed in four folders in the main BioLIMS folder

This folder… Contains the…

Sequencing Analysis

� Sequencing Analysis and Basecaller software

� About Sequencing Analysis text file

� Other folders associated with the Sequencing Analysis software

Factura � Factura software

� About Factura text file

� Other files and folders associated with the Factura software

Data Collection data collection software for three instrument configurations:

� ABI PRISM 377 DNA Sequencer

� ABI PRISM 377 DNA Sequencer with XL Upgrade

� ABI 373 DNA Sequencer with XL Upgrade

BioLIMS Extras � SimpleText application

� Scripts folder

� Sybase folder that contains the interfaces and other database-related files

Table B-3 Files placed in the Macintosh System Folder

Item Folder Location Description

Sybase Config Control Panels Sybase control panel (see step 5 on page B-11)

libblk Extensions Sybase library extension file

libcomn Extensions Sybase library extension file

libcs Extensions Sybase library extension file

libct Extensions Sybase library extension file

libctb Extensions Sybase library extension file

libintl Extensions Sybase library extension file

libsybdb Extensions Sybase library extension file


libtcl Extensions Sybase library extension file

libtcp Extensions Sybase library extension file

SequenceChooserLib Extensions BioLIMS library extension file

ABI Folder System Folder Mobility, comb, & matrix files

Table B-3 Files placed in the Macintosh System Folder

Item Folder Location Description


How to Configure for Server Connection

Configuring forServer Connection

Follow the steps below to configure your Macintosh computer for connection to the Sybase server.

! WARNING ! Any time you change the BioLIMS database server name, its IP address or host and domain name, or the port number, you need to repeat this procedure.

To configure for Sybase SQL Server connection:

Step Action

1 Find the interfaces file in the Sybase folder in the BioLIMS Extras folder.

2 Open the file with SimpleText or a similar text editing application.

3 Find the lines:

SYBASE

query MacTCP mac_ether neuron.apldbio.com 2500

and edit them:

� Replace SYBASE by the name of the database server

� Replace neuron.apldbio.com with the IP address or host and domain name of the server machine

� Replace 2500 with the port number

You can find this information in the interfaces file on the Sybase server, or your BioLIMS database administrator can provide you with the information.

If you have access to more than one server, duplicate the two lines and edit them for the other server(s). For example, for two servers, one called SYBASE and one called SERVER2, the interfaces file might look like this:

SYBASE

query MacTCP mac_ether neuron.apldbio.com 2500

SERVER2

query MacTCP mac_ether 192.135.191.128 2025

4 Save and close the interfaces file.


5 Open the SybaseConfig control panel. This control panel is found in the Control Panels folder in the System folder.

6 The first time you open the SybaseConfig control panel, a file browser opens automatically. If a file browser does not open immediately, click the Interfaces Files button to open one.

7 Use the file browser to locate and open the interfaces file that you edited in the steps above.

8 Set the Default Language pop-up menu to us_english.

9 Close the SybaseConfig control panel.

To configure for Sybase SQL Server connection: (continued)

Step Action


How to Access the BioLIMS Database

AccessingBioLIMS

The following procedure describes how to access the BioLIMS database by completing the BioLIMS Access dialog box.

Before you can work with the Factura software, you must establish a connection to the BioLIMS database. This connection is done using the BioLIMS Access dialog box.

To access the BioLIMS database:

Step Action

1 Choose BioLIMS Access from the File menu. The BioLIMS Access dialog box appears.

2 In the text boxes, enter

� Your user name on the server

� The password for your server account

� The name of the database on the server (You may have access to more than one database on the server.)

� The server name

IMPORTANT All these text boxes are case sensitive.

3 Click the checkbox labeled Save Password if you want your password saved so that you need not enter it every time you open the connection.

4 If you want the database to open automatically when you start the Factura program, click the checkbox labeled Open on Launch.


5 If you intend to use more than one database or user account, enter an alias for this BioLIMS session information.

Use the pop-up menu to change, add, or remove aliases.

If you have more than one alias, click the checkbox labeled Make Default to choose which one appears when you first open the Edit Session dialog box.

6 Click Open to open the connection to the database.

7 Take one of the following actions:

To access the BioLIMS database: (continued)

Step Action

IF the logon was... THEN...

successful the fields in the dialog box gray out and the message changes to the following: This connection is currently open

Go to “How to Set the BioLIMS Preferences” on page B-14

unsuccessful an alert dialog box appears.

Check that:

� All the logon information was entered correctly and in the correct case

� Your interfaces file is correctly configured

� If the connection is still not open, consult your BioLIMS database administrator or the BioLIMS System Administration manual.

For more information, see “How to Configure for Server Connection” on page B-10


How to Set the BioLIMS Preferences

Setting theBioLIMS

Preferences

This section describes how to set the BioLIMS preferences using the Preferences dialog box. You use this dialog box to choose the kind and frequency of BioLIMS processing.

To set the BioLIMS preferences:

Step Action

1 Choose Preferences from the File menu. The Preferences dialog box appears.


IF you choose... THEN...

Process Entire Database any sequence in the database with the status set to analysis will be automatically processed when this command is chosen from the Factura menu.

Process a Sequence or Collection

Note If this menu item is chosen, then the Sequence Chooser window is only presented once, regardless of the setting of the second pop-up menu.

the Sequence Chooser is displayed when this command is chosen from the Factura menu.

For information on using the Sequence Chooser window, see page B-17

The selections are processed at the interval selected from the BioLIMS Preferences.


3 Use the second pop-up menu to choose how often to process sequences from the BioLIMS database.

The menu choices are:

� Once

� At one minute intervals

� At 10 minutes intervals

� At 30 minutes intervals

� At 1 hour intervals



4 Click OK.


To set the BioLIMS preferences: (continued)

Step Action

IF you want to... THEN see...

process BioLIMS sequences “Working with Sequences” on page B-16.

open the Sequence Chooser window

“How to Use the Sequence Chooser Window” on page B-17.


Working with Sequences

Viewing andEditing

To view and edit a sequence, choose Open from the File menu. The Sequence Chooser window appears (see “How to Use the Sequence Chooser Window” on page B-17). You can select a sequence, or sequences, or a collection of sequences, which are displayed in the Sequence View window.

For more information on using the Sequence View window, see Chapter 6, “Using the Sequence Window.”

Processing To process sequences:

Step Action

1 Select Process Sequences from the Factura menu and the following happens.

For more information on setting the preferences, see “How to Set the BioLIMS Preferences” on page B-14.

2 The processing results are displayed in the Log window. There is an individual listing for each sequence processed.

For more information on the using the Log window, see “Overview of the Log Window” on page 3-2.

IF have preferences set to... THEN...

Process Entire Database the Factura progress dialog appears

As the sequences are processed, the progress bar fills up from the left to the right.

Process a Sequence or Collection

the Sequence Chooser window appears (see “How to Use the Sequence Chooser Window” on page B-17).

Select either sequences or collections to process and click Select.


How to Use the Sequence Chooser Window

About theSequence Chooser

Window

The Sequence Chooser window is common to the following BioLIMS applications.

� AutoAssembler™

� Factura™

� Sample 2DB

� Sequencing Analysis

Using the Sequence Chooser, you can search the BioLIMS database for specific collections and sequences. Table B-5 on page B-20 lists the five collection criteria and Table B-6 on page B-21 lists the nine sequence criteria by which you can search.

In This Section Topics in this section includes the following:


For this topic See page

Displaying the Window B-18

Parts of the Window B-19

Collection Search Criteria B-20

Sequence Search Criteria B-21

Searching the BioLIMS Database B-23


Displaying theWindow

The two ways to display the Sequence Chooser window are by:

� Choosing Open from the File menu.

� Choosing Process Sequences from the Factura menu.

The Sequence Chooser window appears (Figure B-1).

Figure B-1 Sequence Chooser window


Status line

Search results

Split bar

Sequence searchcriteria pop-up menusand text boxes

Collection searchcriteria pop-up menusand text boxes

Criteria pop-up menus Search button


Parts of theWindow

Table B-4 describes the parts of the Sequence Chooser window that were labeled in Figure B-1.

Table B-4 Sequence Chooser window parts

Item Description

Criteria pop-up menu

Use this pop-up menu to specify the search criteria visible on the Sequence Chooser.

Note If you only intend to use a subset of criteria, setting only those visible helps reduce clutter in the window.

However, the search results are the same whether a criterion is invisible or blank and visible.

Search button Click this button to query the BioLIMS database.

Collection search criteria pop-up menus and text boxes

Use these pop-up menus and text boxes to define the collection criteria of the search.

IMPORTANT Only those collections that match each and every criterion you specify are returned. That is, search criteria are combined using the logical AND operation.

For more information, see “Collection Search Criteria” on page B-20.

Sequence search criteria pop-up menu and text boxes

Use these pop-up menus and text boxes to define the sequence criteria of the search.

IMPORTANT A collection is returned if one or more of the sequences contained in it fulfill all of the specified sequence criteria.

For more information, see “Sequence Search Criteria” on page B-21.

Split bar Drag this bar to alter the relative amount of space allocated to the top and bottom portions of the Sequence Chooser window.

Search results After a successful query, found collections are listed in this area as Name, Modification date, and Creator.


Collection SearchCriteria

Table B-5 shows the collection search criteria. The collections returned by the Sequence Chooser must match all of the collection criteria and contain at least one sequence that matches all of the sequence criteria.

Status line Error messages and other important information is reported here.

For example, the Status Line lists how many collections were returned in a search.

Table B-4 Sequence Chooser window parts (continued)

Item Description

Table B-5 Allowed collection search criteria

CriterionPop-up Menu Choices Allowed Text Description

Collection Creator

� is

� starts with

� ends with

� contains

up to 255 characters

Name of the creator/owner of the collection

Collection Name � is

� starts with

� ends with

� contains

up to 255 characters

Name of the collection

Collection Type � any

� run

� project

� other

NA Collection type.

Default is any menu item.

Creation Date � is any

� is

� is before

� is after

� is between

date only—set with arrow buttons

Date the collection was created


Sequence SearchCriteria

Table B-6 shows the sequence search criteria. The collections returned by the Sequence Chooser must contain at least one sequence that matches all of the specified sequence criteria.

Modification Date

� is any

� is

� is before

� is after

� is between


Date the collection was last modified

Table B-5 Allowed collection search criteria (continued)


Table B-6 Sequence search criteria


Sequence Creator

� is

� starts with

� ends with

� contains

up to 255 characters including letters, numbers, and punctuation

Name of the person responsible for the run

Sequence Name

� is

� starts with

� ends with

� contains


Name of the sequence

Sample Name � is

� starts with

� ends with

� contains


Sample name from the Sample Sheet

Gel Path � is

� starts with

� ends with

� contains


The full path name to the original gel file, e.g., Hard Disk:Data:GelRuns:L28t



Length � is any

� is

� is before

� is after

� is between

number The length of the most recent version of the sequence in the database

Status � any

� nascent

� prepare

� collect

� analysis

� cleanup

� assembly

NA Status of the sequence. The pop-up menu lists the six stages of collection and analysis

Instrumentation � any

� gel

� capillary

NA Whether the sample was run on a gel or capillary instrument

Start Collect Time

� is any

� is

� is before

� is after

� is between


Date data collection began

End Collect Time

� is any

� is

� is before

� is after

� is between


Date data collection ended

Table B-6 Sequence search criteria (continued)



Searching theBioLIMS Database

Follow these steps to use the Sequence Chooser to search the BioLIMS database for specific collections and sequences.

To search the BioLIMS database:

Step Action

1 Use the items from the Find Collection with Criteria pop-up menu to define your search.

Note To list all of the items in the BioLIMS database, perform the search with no criteria specified. For large databases, this process may be slow.

2 To use the pop-up menu:

Note As you choose items from the pop-up menu, a black dot appears next to the item on the menu and the item is added to either the collection search criteria or the sequence search criteria section of the window.

Choose menu items... To define the search by...

above the horizontal line collection criteria

below the horizontal line sequence criteria


The following is an example of the Sequence Chooser window showing four collection search criteria and five sequence search criteria.

3 Use the pop-up menus and text fields to define your search query. Refer to Table B-5 on page B-20 and Table B-6 on page B-21 for details about the search criteria. When you are satisfied with the search, click Search.

The results of the search appear in the lower portion of the window.

Note Collections returned by the Sequence Chooser must match all of the collection criteria and contain at least one sequence that matches all of the sequence criteria.

4 To view the sequences contained in the collections, click the small triangle to the left of the collection name.

5 Select sequences and or collections.

To search the BioLIMS database: (continued)

Step Action

smalltriangle



To search the BioLIMS database: (continued)

Step Action

IF you click... THEN...

Open the sequence or collection is displayed in a Sequence View window.

For information, see Chapter 6, “Using the Sequence Window.”

Process the sequence or collection is processed.

For more information, see “Working with Sequences” on page B-16.

Cancel the Sequence Chooser window is closed.

Technical Support C-1

Technical Support C

ContactingTechnical Support

You can contact Applied Biosystems for technical support by telephone or fax, by e-mail, or through the Internet. You can order Applied Biosystems user documents, MSDSs, certificates of analysis, and other related documents 24 hours a day. In addition, you can download documents in PDF format from the Applied Biosystems Web site (please see the section “To Obtain Documents on Demand” following the telephone information below).

To ContactTechnical Support

by E-Mail

Contact technical support by e-mail for help in the following product areas:

Product Area E-mail address

Genetic Analysis (DNA Sequencing)

[email protected]

Sequence Detection Systems and PCR

[email protected]

Protein Sequencing, Peptide and DNA Synthesis

[email protected]

Biochromatography, PerSeptive DNA, PNA and Peptide Synthesis systems, CytoFluor®, FMAT™, Voyager™, and Mariner™ Mass Spectrometers

[email protected]

Applied Biosystems/MDS Sciex [email protected]

Chemiluminescence (Tropix) [email protected]

C

C-2 Technical Support

Hours forTelephone

Technical Support

In the United States and Canada, technical support is available at the following times:

To ContactTechnical Support

by Telephone orFax

In North America

To contact Applied Biosystems Technical Support, use the telephone or fax numbers given below. (To open a service call for other support needs, or in case of an emergency, dial 1-800-831-6844 and press 1.)

Product Hours

Chemiluminescence 8:30 a.m. to 5:30 p.m. Eastern Time

Framingham support 8:00 a.m. to 6:00 p.m. Eastern Time

All Other Products 5:30 a.m. to 5:00 p.m. Pacific Time

Product orProduct Area

TelephoneDial...

FaxDial...

ABI PRISM® 3700 DNA Analyzer

1-800-831-6844,then press 8

1-650-638-5981

DNA Synthesis 1-800-831-6844,then press 21

1-650-638-5981

Fluorescent DNA Sequencing

1-800-831-6844,then press 22

1-650-638-5981

Fluorescent Fragment Analysis (includes GeneScan® applications)

1-800-831-6844,then press 23

1-650-638-5981

Integrated Thermal Cyclers (ABI PRISM® 877 and Catalyst 800 instruments)

1-800-831-6844,then press 24

1-650-638-5981

ABI PRISM® 3100 Genetic Analyzer

1-800-831-6844,then press 26

1-650-638-5981

BioInformatics (includes BioLIMS™, BioMerge™, and SQL GT™ applications)

1-800-831-6844,then press 25

1-505-982-7690

Peptide Synthesis (433 and 43X Systems)

1-800-831-6844,then press 31

1-650-638-5981

Protein Sequencing (Procise‚ Protein Sequencing Systems)

1-800-831-6844,then press 32

1-650-638-5981


PCR and Sequence Detection

1-800-762-4001,then press 1 for PCR,2 for the 7700 or 5700,6 for the 6700or dial 1-800-831-6844, then press 5

1-240-453-4613

Voyager‰ MALDI-TOF Biospectrometry and Mariner‰ ESI-TOF Mass Spectrometry Workstations

1-800-899-5858,then press 13

1-508-383-7855

Biochromatography (BioCAD‚ Workstations and Poros‚ Perfusion Chromatography Products)

1-800-899-5858,then press 14

1-508-383-7855

Expedite‰ Nucleic acid Synthesis Systems

1-800-899-5858,then press 15

1-508-383-7855

Peptide Synthesis (Pioneer‰ and 9050 Plus Peptide Synthesizers)

1-800-899-5858,then press 15

1-508-383-7855

PNA Custom and Synthesis

1-800-899-5858,then press 15

1-508-383-7855

FMAT‰ 8100 HTS System and Cytofluor‚ 4000 Fluorescence Plate Reader

1-800-899-5858,then press 16

1-508-383-7855

Chemiluminescence (Tropix)

1-800-542-2369 (U.S. only),or 1-781-271-0045

1-781-275-8581

Applied Biosystems/MDS Sciex

1-800-952-4716 1-650-638-6223

Product orProduct Area

TelephoneDial...

FaxDial...

Outside North America

RegionTelephoneDial...

FaxDial...

Africa and the Middle East

Africa (English Speaking) and West Asia (Fairlands, South Africa)

27 11 478 0411 27 11 478 0349

South Africa (Johannesburg)

27 11 478 0411 27 11 478 0349

Middle Eastern Countries and North Africa (Monza, Italia)

39 (0)39 8389 481 39 (0)39 8389 493

Eastern Asia, China, Oceania

Australia (Scoresby, Victoria)

61 3 9730 8600 61 3 9730 8799

China (Beijing) 86 10 64106608 86 10 64106617

Hong Kong 852 2756 6928 852 2756 6968

Korea (Seoul) 82 2 593 6470/6471 82 2 593 6472

Malaysia (Petaling Jaya) 60 3 758 8268 60 3 754 9043

Singapore 65 896 2168 65 896 2147

Taiwan (Taipei Hsien) 886 2 2358 2838 886 2 2358 2839

Thailand (Bangkok) 66 2 719 6405 66 2 319 9788

Europe

Austria (Wien) 43 (0)1 867 35 75 0 43 (0)1 867 35 75 11

Belgium 32 (0)2 712 5555 32 (0)2 712 5516

Czech Republic and Slovakia (Praha)

420 2 61 222 164 420 2 61 222 168

Denmark (Naerum) 45 45 58 60 00 45 45 58 60 01

Finland (Espoo) 358 (0)9 251 24 250 358 (0)9 251 24 243

France (Paris) 33 (0)1 69 59 85 85 33 (0)1 69 59 85 00

Germany (Weiterstadt) 49 (0) 6150 101 0 49 (0) 6150 101 101

Hungary (Budapest) 36 (0)1 270 8398 36 (0)1 270 8288

Italy (Milano) 39 (0)39 83891 39 (0)39 838 9492

Norway (Oslo) 47 23 12 06 05 47 23 12 05 75

Poland, Lithuania, Latvia, and Estonia (Warszawa)

48 (22) 866 40 10 48 (22) 866 40 20


Portugal (Lisboa) 351 (0)22 605 33 14 351 (0)22 605 33 15

Russia (Moskva) 7 095 935 8888 7 095 564 8787

South East Europe (Zagreb, Croatia)

385 1 34 91 927 385 1 34 91 840

Spain (Tres Cantos) 34 (0)91 806 1210 34 (0)91 806 1206

Sweden (Stockholm) 46 (0)8 619 4400 46 (0)8 619 4401

Switzerland (Rotkreuz) 41 (0)41 799 7777 41 (0)41 790 0676

The Netherlands (Nieuwerkerk a/d IJssel)

31 (0)180 331400 31 (0)180 331409

United Kingdom (Warrington, Cheshire)

44 (0)1925 825650 44 (0)1925 282502

All other countries not listed (Warrington, UK)

44 (0)1925 282481 44 (0)1925 282509

Japan

Japan (Hacchobori, Chuo-Ku, Tokyo)

81 3 5566 6230 81 3 5566 6507

Latin America

Del.A. Obregon, Mexico 305-670-4350 305-670-4349

RegionTelephoneDial...

FaxDial...

C-6 Technical Support

To ReachTechnical Support

Through theInternet

We strongly encourage you to visit our Web site for answers to frequently asked questions and for more information about our products. You can also order technical documents or an index of available documents and have them faxed or e-mailed to you through our site. The Applied Biosystems Web site address is

http://www.appliedbiosystems.com/techsupp

To submit technical questions from North America or Europe:

Step Action

1 Access the Applied Biosystems Technical Support Web site.

2 Under the Troubleshooting heading, click Support Request Forms, then select the relevant support region for the product area of interest.

3 Enter the requested information and your question in the displayed form, then click Ask Us RIGHT NOW (blue button with yellow text).

4 Enter the required information in the next form (if you have not already done so), then click Ask Us RIGHT NOW.

You will receive an e-mail reply to your question from one of our technical experts within 24 to 48 hours.


To ObtainDocuments on

Demand

Free, 24-hour access to Applied Biosystems technical documents, including MSDSs, is available by fax or e-mail or by download from our Web site.

To order documents... Then...

by index number

a. Access the Applied Biosystems Technical Support Web site at http://www.appliedbiosystems.com/techsupp

b. Click the Index link for the document type you want, then find the document you want and record the index number.

c. Use the index number when requesting documents following the procedures below.

by phone for fax delivery

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b. Follow the voice instructions to order the documents you want.

Note There is a limit of five documents per request.

through the Internet for fax or e-mail delivery

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License and Warranty D-1

License and Warranty DSoftware License and Limited Product Warranty

SOFTWARE LICENSE and LIMITED PRODUCT WARRANTY

PURCHASER, CAREFULLY READ THE FOLLOWING TERMS AND CONDITIONS (THE “AGREE-MENT”), WHICH APPLY TO THE SOFTWARE ENCLOSED (THE “SOFTWARE”). YOUR OPENINGOF THIS PACKAGE INDICATES YOUR ACCEPTANCE OF THEM. IF YOU DO NOT ACCEPT THEM,PROMPTLY RETURN THE COMPLETE PACKAGE, AND YOUR MONEY WILL BE RETURNED. THELAW PROVIDES FOR CIVIL AND CRIMINAL PENALTIES FOR ANYONE IN VIOLATION OF THELAWS OF COPYRIGHT.

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LIMITED WARRANTYFor a period of ninety (90) days after purchase of the SOFTWARE, Applied Biosystems warrants thatthe SOFTWARE will function substantially as described in the documentation supplied by Applied Bio-systems with the SOFTWARE. If you discover an error which causes substantial deviation from thatdocumentation, send a written notification to Applied Biosystems. Upon receiving such notification, ifApplied Biosystems is able to reproduce that error reliably at its facility, then Applied Biosystems will doone of the following at its sole option: (i) correct the error in a subsequent release of the SOFTWARE,which shall be supplied to you free of charge or (ii) accept a return of the SOFTWARE from the pur-chaser, and refund your purchase price of the SOFTWARE. Applied Biosystems does not warrant thatthe SOFTWARE will meet your requirements, or will be error-free, or will conform exactly to the docu-mentation. Any sample or model used in connection with this Agreement is for illustrative purposesonly, is not part of the basis of the bargain, and is not to be construed as a warranty that the JSOFT-WARE will conform to the sample or model.

LIMITATION OF LIABILITYEXCEPT AS SPECIFICALLY STATED IN THIS AGREEMENT, THE SOFTWARE IS PROVIDED ANDLICENSES “AS IS.” THIS WARRANTY IS GIVEN IN LIEU OF ALL OTHER WARRANTIES,EXPRESSED OR IMPLIED, INCLUDING THOSE OF MERCHANTABILITY OR FITNESS FOR A PAR-

D

D-2 License and Warranty

TICULAR PURPOSE. NOTWITHSTANDING ANY FAILURE OF THE CENTRAL PURPOSE OF ANYLIMITED REMEDY, APPLIED BIOSYSTEMS LIABILITY FOR BREACH OF WARRANTY SHALL BELIMITED TO A REFUND OF THE PURCHASE PRICE FOR SUCH PRODUCT. IN NO EVENT WILLAPPLIED BIOSYSTEMS BE LIABLE FOR ANY DAMAGES, INCLUDING INCIDENTAL OR CONSE-QUENTIAL DAMAGES, EVEN IF APPLIED BIOSYSTEMS HAS BEEN ADVISED OF THE POSSIBIL-ITY OF SUCH DAMAGES.

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Index-1

Index

Symbols%N field 3-5

Aactive settings 4-9Active status, assigning to feature 8-7adding

a range of bases to a sequence using Sequence view 7-7

base in Electropherogram view 7-10features 8-3 to 8-5multiple ranges to features 8-5

Ambiguity Range field 3-5ambiguous bases, editing using

Electropherogram View 6-8Annotation view, description 6-6AppleScripts, about 2-4arrow keys, moving in electropherograms 7-9

Bbases

editing in Electropherogram view 7-9 to 7-10

moving the selection between bases 7-9

selecting a base and not a position 7-9 to 7-10

editing in Sequence view 7-7 to 7-8changing bases in the sequence 7-8deleting a range of bases 7-8

BioLIMS Client package, installing B-3 to B-4BioLIMS database B-2 to B-16

about the database B-2accessing BioLIMS B-12 to B-13configuring the server connection B-10 to

B-11

installing B-3 to B-9before installing B-3BioLIMS Client package B-3 to B-4files installed B-6 to B-9performing custom installation

B-4 to B-5removing installed package B-5 to B-6

Sequence Chooser window B-17 to B-21displaying the window B-18parts of the window B-19 to B-20using B-23 to B-25

setting the search interval B-14 to B-15borders, using to mark features 8-9

Ccase sensitive searches 8-17Clear Length field 3-5Clear Log command 3-6Clear Range field 3-5closing, sequence window 7-16color

assigning new colors to marking style 8-13 to 8-14

using to mark features 8-9Command key, using to join features 8-8complement, displaying 6-9Confidence Range field 3-5Copy command, using to create new sequence

file 7-5Create Vector Pattern window

description of use 2-10, 5-3using to create vector pattern 5-4 to 5-5using to edit vector patterns 5-6 to 5-7

creatingnew sequence files 7-4 to 7-6settings file 4-10

custom installation B-4 to B-5

Index-2

customer support C-1 to C-7documents on demand C-7

Ddefault settings, using 4-9deleting

features 8-6installed package B-5 to B-6range of bases from the sequence 7-8

DNA technical support 1-5dragging and dropping files, processing files 2-7

Eeasy install (installing BioLIMS Client

package) B-3 to B-4Edit Mark Styles command, using to assign new

styles 8-9editing

in Electropherogram view 7-9 to 7-10in Sequence view 7-7 to 7-8Log window 3-6protecting sequence from edits 6-4vector patterns 5-6 to 5-7

Electropherogram view 6-11 to 6-14changing scaling 6-12 to 6-14description of view 6-8editing 7-9 to 7-10

by moving the selection between bases 7-9

by selecting a base and not a position 7-9 to 7-10

editing the list of base sequence 6-11 to 6-12

printing 7-11 to 7-12selecting a base 7-9 to 7-10turning off display 6-15using the Electropherogram Display Options

dialog box 6-15 to 6-18using the Find and Find Again

commands 8-18ellipsis (…), using for entering a range 8-19environment variables, setting A-5Export Clear Range checkbox 4-8Export command

exporting sequence data 7-15using to create new sequence file 7-6

FFactura

applications folder, contents of 2-3 to 2-4installing

BioLIMS Client package B-4 to B-5Macintosh version 2-2UNIX version A-3 to A-6

Log, contents of 2-4setup, described 1-4

factura settings file, for the UNIX versionchanging for UNIX platform A-9 to A-10creating user specified settings A-9example A-9 to A-10See Also Factura Settings window

Factura Settings window 4-2 to 4-10active settings, what they are 4-9contents of 2-4creating settings file 4-10description of fields 4-2 to 4-8description of use 2-10loading settings 4-10printing settings 4-10using default settings 4-9

Feature view, description of 6-6 to 6-7features

adding multiple ranges 8-5adding to a sequence 8-3 to 8-5assigning status to a feature 8-7automatically marking features 8-14changing a feature 8-6changing appearance 8-9 to 8-10

of applied marking style 8-10table of marking styles 8-9

changing marking style 8-11 to 8-14assigning new colors 8-13 to 8-14

GenBank naming convention 8-8joining 8-8removing 8-6types, table of 8-2

file types recognized by Factura 7-2files

from installation B-6 to B-9Find Again command, using 8-18Find command, using 8-15 to 8-20

entering a range using ellipsis 8-19searching for a specified sequence 8-15 to

8-16

Index-3

searching for multiple base combinations 8-18

using the Find Again command 8-18using the Offset Find Option 8-19 to 8-20

font, changing 6-9 to 6-10

GGenBank, naming convention 8-8Generalized Regular Expression Patterns (Grep)

searching for General regular expression patterns search 8-17

searching for multiple base combinations 8-18

Hhelp C-1 to C-7

documents on demand C-7Hidden status, assigning to feature 8-7

IIdentify Ambiguity checkbox 4-5 to 4-6

how parameter works 4-6Identify Confidence Range checkbox 4-4Identify IUB/Heterozygous Bases checkbox 4-6

to 4-8how the parameter works 4-7IUB codes, table of 4-7

Identify Vector Sequence checkbox 4-3 to 4-4INHERIT files, recognized by Factura 2-5, 7-2input files 2-5 to 2-6installing

BioLIMS B-3 to B-9before installing B-3BioLIMS Client package B-3 to B-4files installed B-6 to B-9performing custom install B-4 to B-5removing installed package B-5 to B-6

Macintosh version 2-2UNIX version A-3 to A-6

Instrument package, installing B-3 to B-9International Union of Biochemists. See IUB

codesinternet address C-6IUB codes

assigning 4-6searching for IUB character 8-17

table of 4-7IUPAC/IUB character search 8-17

Jjoining features 8-8

Lleft-arrow key

moving in electropherograms 7-9license D-1 to D-2literals, searching for 8-17loading a settings file 4-10lock image 6-4Log file, creating duplicate of file 3-6Log window 2-9

contents of 3-2 to 3-5using 3-6 to 3-7See Also Log file

Mmarking features 8-9 to 8-10

automatically marking features 8-14changing applied marking style of a

feature 8-10changing marking style 8-11 to 8-14

assigning new colors 8-13 to 8-14table of marking styles 8-9

NNew Sequence command 7-4Normal status, assigning to feature 8-7

Ooffsets

example using 8-19 to 8-20searching for 8-17

Option keyusing to create ellipsis 8-19using to move cursor in

electropherograms 7-9original data

displaying in electropherogram view 6-11 to 6-12

Original Length field 3-5output files 2-5 to 2-6

Index-4

PPrimers folder, contents of 2-3printing 7-11 to 7-13

all views 7-13electropherograms 7-11 to 7-12Log window 3-7settings file 4-10

Process Sequences menu command 2-8processing files 2-7 to 2-8

about processing a sequence file 2-6dragging and dropping files 2-7identifying the vector sequence 4-3 to 4-4results shown in Log window 3-4 to 3-5types of input files supported A-13using menu selection 2-8using the UNIX platform

displaying the results A-16

Rrange, adding multiple ranges to features 8-5Reload Vector Pattern command 5-7removing

installed package B-5 to B-6Revert Sample Files checkbox 4-2right-arrow key, moving in

electropherograms 7-9

Ssample files

description of 2-5recognized by Factura 7-2viewing information from run 6-6

sample procedure 2-13 to 2-14Save a Copy As command, using for Log

window 3-6saving

changes in the sequence window 7-14 to 7-15

information in the Log window 3-6searching 8-15 to 8-20

for multiple base combinations 8-18for specified sequence 8-15 to 8-16using the Find Again command 8-18using the Offset Find Options 8-19 to 8-20See Also BioLIMS database

Select All command 7-5

Sequence Chooser window (BioLIMS) B-17 to B-21

displaying the window B-18parts of the window B-19 to B-20using B-23 to B-25

sequence files 2-5 to 2-6about processing a sequence file 2-6maintaining original files 4-2what is a sequence file 2-5See Also sequences

Sequence menu, about 6-3Sequence view

description 6-5 to 6-6using to edit bases 7-7 to 7-8

Sequence View Options command 6-9sequence window

changing font and grouping of sequence 6-9 to 6-10

closing 7-16creating new sequence file 7-4 to 7-6description 2-11displaying the complement 6-9exporting sequences 7-15opening an existing sequence 7-3opening the window 6-3printing all views 7-13types of information displayed 6-2using to edit vector pattern 5-7window and views 6-4 to 6-8

Annotation view 6-6Electropherogram view 6-8Feature view 6-6 to 6-7Sequence view 6-5 to 6-6

sequences 8-15 to 8-20changing font and grouping of

sequence 6-9 to 6-10creating a new sequence file 7-4 to 7-6displaying the complement 6-9editing in Electropherogram view 7-9 to

7-10editing in Sequence view 7-7 to 7-8exporting 7-15files Factura recognizes 7-2locking 6-4opening an existing sequence 7-3searching for multiple base

combinations 8-18

Index-5

searching for specified sequence 8-15 to 8-16

See Also searchingSites folder, contents of 2-3software license D-1 to D-2status, assigning. See features, assigning status

to a featureStyle pop-up menu, using to mark features 8-4summary graphic 6-4

displaying marking style 8-4showing search results 8-16

support. See technical support

Ttables

default marking styles 8-9feature types 8-2

technical support C-1 to C-7documents on demand C-7hours, what information to provide 1-5, C-2

text files, recognized by Factura 2-6, 7-2

UUNIX Factura, using A-2 to A-17

changing settings file A-9 to A-10creating user specified settings A-9example of A-9 to A-10

displaying command formats A-2installing A-3 to A-6

copying application files from CD-ROM A-3

running from a working directory A-4setting other environment variables A-5

parameter formats A-9results A-16similarities to Macintosh version A-2types of input files supported A-13vector patterns A-11 to A-12

about A-11could not create vector pattern error

message A-12

Vvector patterns 5-2 to 5-7

about the Create Vector Pattern window 5-3creating a vector pattern file 5-4 to 5-5

editing 5-6 to 5-7for Factura to access vector patterns 2-3on the UNIX platform A-11 to A-12reloading vector patterns 5-7Vector Pattern window, about 2-10what is a vector pattern 5-2

Vector Range field 3-5Vectors folder.See vector patternsviewing

Full View (electropherogram) 6-14returning to original size

(electropherogram) 6-14zooming electropherogram 6-13 to 6-14

views, associated with file types 7-2

Wwarranty D-1 to D-2windows 2-9 to 2-12

Create Vector Pattern windowdescription of use 2-10

Factura Settings window 4-2 to 4-10description of fields 4-2 to 4-8description of use 2-10working with 4-9 to 4-10

Log windowclearing contents 3-6contents of 3-2 to 3-5description of use 2-9

sequence windowsdescription 2-11

sequence windows, description 2-11See Also sequence window

word processing applicationusing to edit vector file 5-6

world wide web address C-6wrap around searches 8-17Write Back to Sample Files checkbox 4-8

Zzooming electropherogram 6-13 to 6-14

Headquarters850 Lincoln Centre DriveFoster City, CA 94404 USAPhone: +1 650.638.5800Toll Free: +1 800.345.5224Fax: +1 650.638.5884

Worldwide Sales OfficesApplied Biosystems vast distribution and service network, composed of highly trained support and applications personnel, reaches into 150 countries on six continents. For international office locations, please call our local office or refer to our web site at www.appliedbiosystems.com.

www.appliedbiosystems.com

Applera Corporation is committed to providing the world’s leading technology and information for life scientists. Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses.

Printed in the USA, 01/2001Part Number 904946B

an Applera business

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