MicroSEQ ID Analysis Software Version 2.2. Getting Started Guide

Transcription

1 MicroSEQ ID Analysis Software Version 2.2 Getting Started Guide

2 For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. Information in this document is subject to change without notice. APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. TO THE FULLEST EXTENT ALLOWED BY LAW, IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF, WHETHER OR NOT FORESEEABLE AND WHETHER OR NOT APPLIED BIOSYSTEMS IS ADVISED OF THE POSSIBILITY OF SUCH DAMAGES. NOTICE TO PURCHASER:DISCLAIMER OF LICENSE 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 Life Technologies Corporation, either expressly or by estoppel. TRADEMARKS Trademarks of Life Technologies Corporation and its affiliates include: Applied Biosystems, AB (Design), AB Logo, ABI PRISM, BigDye, GeneMapper, MicroSEQ, KB, POP-6, SeqScape, Variant Reporter. BLAST is a trademark of the National Library of Medicine. Microsoft, Vista, and Windows are trademarks of the Microsoft Corporation. All other trademarks are the sole property of their respective owners Life Technologies Corporation. All rights reserved. Part Number Rev. A 01/2010

3 Contents Preface vii How to Use This Guide vii How To Obtain Support vii Chapter 1 Software Basics Start Here: Getting the Most out of This Guide What is the MicroSEQ ID Analysis Software? Version 2.2 Features User Groups and Privileges User Workflow Tutorial Data Software Structure Software Toolbars Glossary File-Naming Conventions For More Information Chapter 2 Administrator: Set Up the Software Before You Begin Installing the Software Setting Up General Defaults Setting Up User Accounts Setting Up Authentication Setting Up the Audit Trail Setting Up Electronic Signatures Chapter 3 Scientist: Set Up Analysis Information Before You Begin Creating a Custom Library Creating a Master Analysis Protocol Creating a Custom Report Template Chapter 4 Analyst: Set Up a Project Before You Begin Customizing the Display Settings iii

4 Contents Creating a Project Analyzing the Project Data Evaluating Results: Analysis QC Report Evaluating Results: Library Search Report Creating a Phylogenetic Tree Editing Data in the Analyzed Project Exporting or Printing Reports For More Information Appendix A Using Autoanalysis with Applied Biosystems 3130 Series Genetic Analyzers Integrating MicroSEQ and Data Collection Software Automatically Exporting Projects After Autoanalysis Creating Required Files in the Data Collection Software Scheduling and Starting a Run Launching Autoanalysis Manager Appendix B Using Autoanalysis with Applied Biosystems 3500 Series Genetic Analyzers Integrating MicroSEQ and Data Collection Software Preparing the MicroSEQ ID Analysis Software Automatically Exporting Projects After Autoanalysis Preparing the 3500 Series Data Collection Software New concepts for 3130 Series Genetic Analyzer Users Creating a MicroSEQ ID secondary analysis protocol for autoanalysis configuration 110 Create a plate template for autoanalysis from the MSID plate template provided Creating a Plate for Autoanalysis Creating a Plate From a Template Assigning plate contents Starting an Autoanalysis Run Starting the Autoanalysis Manager Linking the Plate and Starting the Run Appendix C Electronic Signatures About Electronic Signatures for Users Electronically Signing Your Work Viewing and Verifying Electronic Signatures Appendix D Software Warranty Information Computer Configuration Limited Product Warranty iv

5 Contents Documentation Related documentation Obtaining information from the Help system Send us your comments Index v

6 Contents vi

7 Preface How to Use This Guide Text conventions This guide uses the following conventions: Bold text indicates user action. For example: Type 0, then press Enter for each of the remaining fields. Italic text indicates new or important words and is also used for emphasis. For example: Before analyzing, always prepare fresh matrix. A right arrow symbol ( ) separates successive commands you select from a dropdown or shortcut menu. For example: Select File Open Spot Set. Right-click the sample row, then select View Filter View All Runs. User attention words Two user attention words appear in Applied Biosystems user documentation. Each word implies a particular level of observation or action as described below: Note: Provides information that may be of interest or help but is not critical to the use of the product. IMPORTANT! Provides information that is necessary for proper instrument operation, accurate chemistry kit use, or safe use of a chemical. How To Obtain Support For the latest services and support information for all locations, go to: At the Applied Biosystems web site, you can: Access worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities. Search through frequently asked questions (FAQs). Submit a question directly to Technical Support. Order Applied Biosystems user documents, MSDSs, certificates of analysis, and other related documents. Download PDF documents. Obtain information about customer training. Download software updates and patches. vii

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9 Chapter 1 1 Software Basics Software Basics Software Basics This chapter covers: Start Here: Getting the Most out of This Guide What is the MicroSEQ ID Analysis Software? Version 2.2 Features User Groups and Privileges User Workflow Tutorial Data Software Structure Software Toolbars Glossary File-Naming Conventions For More Information Scientist: Set Up Analysis Information Analyst: Set Up a Project 1

10 1 Step 1 Software Basics Start Here: Getting the Most out of This Guide The Applied Biosystems MicroSEQ ID Analysis Software Version 2.2 Getting Started Guide is an installation guide and tutorial. It provides: Installation instructions to help the Administrator set up the MicroSEQ ID Analysis software. Brief, step-by-step tasks to help users quickly learn the MicroSEQ ID Analysis software. The tasks use tutorial data that is supplied on the software CD-ROM. To get the most out of this Getting Started Guide, Applied Biosystems recommends that you: Step 1 Read this Software Basics chapter. Step 2 Determine which user group you belong to. See User Groups and Privileges on page 4. Step 3 Work through the tutorial tasks appropriate for your user group. See User Workflow on page 5. What is the MicroSEQ ID Analysis Software? MicroSEQ ID Analysis software v2.2 is a tool for microbial identification of bacteria and fungi. The software analyzes data generated using a MicroSEQ sequencing kit and an Applied Biosystems capillary-based genetic analyzer. The MicroSEQ ID Analysis software: 1. Compares the sequence of unknown isolate data to the sequences of known bacteria or fungi stored in a library. 2. Generates a final identification list of organisms that are the closest matches to the unknown. 3. Reports the percent similarity that reflects how closely the unknown isolate matches the library sequence. Note: The MicroSEQ ID system workflow is optimized for 3130 and 3500 Series Genetic Analyzers using POP-6 polymer and a 50-cm array. For instructions on setting up analysis on a 3730 Series Genetic Analyzer, and/or using POP-7 polymer, refer to the instrument user guide provided with your analyzer. 2

11 Software Basics 1 Version 2.2 Features The basic functions of the MicroSEQ ID Analysis software have not changed in version 2.2. Version 2.2 supports.ab1 files generated by an Applied Biosystems 3500/3500xL (3500 Series) Genetic Analyzer, and it displays the following new information in the Annotation tab of the Sample View: Re-Injection (RInj) Assay Name (AsyN) Assay Version (AsyV) Version 2.2 is compatible with the following versions of data collection and secondary analysis software: MicroSEQ ID Analysis Software Installation Type Computer/Data Collection (DC) Software Version Secondary Analysis Software Version (or later) Supported SeqA Seqscape Variant Reporter Gene- Mapper New v2.2 Standalone v5.4 v2.7 v1.1 v Series DC v1.0 New v2.2 Upgrade v Series DC v3.0 v5.3.1 v2.5 and 2.6 N/A v4.0 New v2.2 Upgrade v Series DC v3.0 v5.3.1 v2.5 and N/A v Autoanalysis not supported 3

12 1 Step 3 Software Basics User Groups and Privileges Three user groups are defined in the software: Administrator, Scientist, and Analyst. Each of these user groups has the privileges listed in the table below. User Group Administrator Scientist Analyst Privileges Install and start the software Set up defaults Set up users Set up authentication settings Set up auditing Set up electronic signatures All Scientist privileges All Analyst privileges Create libraries Create analysis protocols Set up custom reports All Analyst privileges Create a project Analyze the project Evaluate the results Edit data in the analyzed project Export or print reports 4

13 Software Basics 1 User Workflow The workflow below provides an overview of the tasks presented in this Getting Started Guide. The tasks use tutorial data (see page 6) that is supplied on the software CD- ROM. IMPORTANT! Each task should be performed in the order given by the specified user group. The Administrator and Scientist must perform their tasks before the Analyst can set up a project. Install and start the software. Set up general defaults. Administrator: Set up the software Set up users. Set up authentication. see Chapter 2 Set up auditing. Set up electronic signatures. Scientist: Set up reference data Create libraries. Create analysis protocols. Create custom reports. see Chapter 3 Create a project. Analyze the project. Analyst: Set up a project Evaluate the results. Edit data in the analyzed project. see Chapter 4 Electronically sign your work. Export or print reports. 5

14 1 Step 3 Software Basics Tutorial Data When you perform the tasks in this Getting Started Guide, you will use tutorial data that is supplied on the MicroSEQ ID Analysis software CD-ROM. The tutorial data is in the following installation drive location: drive letter:\appliedbiosystems\microseqid\tutorial Data\Bacterial500Samples Table 1 defines the two types of files provided and their folder names, as well as the specific file names and extensions. Table 1 Tutorial data referenced in this guide File Type Folder Name File Name and Extension Library Bacterial500CustomLibrary Tutorial_Bacterial500_Lib_v2.0.fsta Sample file 500Ecoli _A01_MSID_50cm_POP6_std.ab _A07_MSID_50cm_POP6_std.ab1 Note: The tutorial data was electrophoresed on an Applied Biosystems 3130 Genetic Analyzer running POP-6 polymer on a 50-cm array using the standard run (SR) protocol. The unknown isolates were sequenced with BigDye Terminator v1.1 chemistry. 6

15 Software Basics 1 Software Structure To perform the tasks in this Getting Started Guide, you will access three windows from the MicroSEQ ID Analysis software main window: MicroSeqID Manager window Options window Project window MicroSeq ID Analysis Software: Main Window Options Window MicroSeqID Manager Project Window General Defaults Projects Project Navigator Report Manager User Accounts Authentication Audit Trail Electronic Signature Libraries Analysis Protocols Custom Reports Specimen View Sample View Analysis QC Report Library Search Report Audit Trail Report Electronic Signature History Report Custom Report Create and edit settings Manage analysis information View project data View, export, or print results Options Window Access the Options window by clicking Tools Options. Use the Options window to create and/or edit: General defaults, which include: Display reports after analysis Export reports after analysis Export Projects after Autoanalysis Specify a directory to which reports are automatically exported User accounts Authentication Audit trails Electronic signatures 7

16 1 MicroSeqID Manager Window Software Basics MicroSeqID Manager Window Access the MicroSeqID Manager window by clicking Tools MicroSeqID Manager Use the MicroSeqID Manager window to enter and manage the information necessary to perform analyses on your projects: Edit, delete, duplicate, import, and export projects Create, edit, delete, duplicate, import, and export libraries Create, edit, delete, duplicate, import, and export master analysis protocols Create, edit, delete, duplicate, import, and export customized reports Project Window Access the Project window by clicking File Open Project and selecting an existing project. Use the Project window to: Open a project Search for a project Import specimens/sample files Edit data using the Properties and Settings of the Project or Specimen View, export, or print project data (Project Navigator, Specimen View, or Sample View) Access the Analysis Protocol Viewer to view analysis information Access the Report Manager to view, export, or print project analysis results 8

17 Software Basics 1 Software Toolbars The MicroSEQ ID Analysis software toolbars display buttons for software functions that you are likely to use often. Refer to the following diagrams (pages 9 to 11) for the names, descriptions, and keyboard shortcuts for each button. General Toolbar The General toolbar contains general processing tools for creating projects. New Project Ctrl+N Creates a new project Import Samples To Project Ctrl+M Save Project Ctrl+S Saves a modified project Find Again F3 Finds a selected sequence again Print Ctrl+P Delete Deletes a selected object MicroSeqID Manager Opens the MicroSeqID Manager window Undo Base Change Ctrl+Z Open Project Ctrl+O Opens a window to select and open a project Find Ctrl+F Finds a selected string/iub code in a sequence New Specimen Opens a new specimen 9

18 1 Analysis Toolbar Software Basics Analysis Toolbar The Analysis toolbar contains analysis tools for the projects you create. Analyze Samples Ctrl+R BLAST button Opens to NCBI s BLAST search tool web page Opens Applied Biosystems Products & Services web page Analysis Protocol Viewer Displays the analysis protocol Report Manager Ctrl+1 Opens the Report Manager window The BLAST button links you to the National Center for Biotechnology Information (NCBI) website that maintains the Basic Local Alignment Search Tool (BLAST ). Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res Sep 1;25(17): Review. 10

19 Software Basics 1 Display Toolbar The Display toolbar contains viewing options for the projects you create. Display Settings Ctrl+Y Opens the Display Settings window for the project Show/Hide sample QV Ctrl+K Show/Hide Electropherogram In Specimen Assembly view Inverse View of the electropherogram Full View of Electropherogram Ctrl+] View Aligned EP Zoom Out Ctrl+- Show/Hide consensus QV View Original Sequence In Sample Electropherogram view Zoom In Ctrl+= View Column Selector Show/Hide All Electropherograms 11

20 1 Display Toolbar Software Basics Glossary Term Definition Aligned consensus sequence Analysis protocol Clear range Consensus quality value (consensus QV) Consensus sequence Kit Project Sample file Sample quality value (sample QV) Specimen Specimen score Start and End delimiters Unknown isolate The consensus sequence after pairwise alignment with the library sequence. May contain gaps. A file that specifies the parameters for basecalling and data analysis. The region of the sequence that is basecalled and used to generate the consensus sequence. The clear range is determined by settings in the analysis protocol. A per-base estimate of the accuracy of the consensus-calling algorithm. The sequence assembled by the MicroSEQ ID Analysis software from all sample sequences in the specimen that meet the clear range and filtering thresholds. The consensus sequence is compared to a library of sequences to generate the closest matches. One of the MicroSEQ chemistry kits supported by the software: MicroSEQ S rdna Bacterial Identification Kit MicroSEQ Full Gene 16S rdna Bacterial Identification Kit MicroSEQ D2 rdna Fungal Identification Kit A container for related specimens. All analysis in the MicroSEQ ID Analysis software occurs in a project. A data file generated by a sequencer. It is part of a set of data files that are assembled by the MicroSEQ ID Analysis software to generate the consensus sequence. A per-base estimate of basecaller accuracy. A container that holds the sample data (sample files) from a single bacterial or fungal unknown isolate. Average consensus QV. Project setting parameters that determine the portion of the sample or sample file name used to create the specimen name. Characters that follow the Start delimiter and precede the End delimiter are used for the specimen name. For Example, if the sample file name is MSID_A03_C_781F.ab1 and you specify _ (underscore) as the Start delimiter and _ (underscore) as the End delimiter, the specimen name created is A03. Bacterial or fungal DNA isolated from a single colony or pure culture. % Match Indicates how closely the unknown isolate matches the top library sequence (Top Match). %Match = ( 1 - (The sum of mismatch penalties) / (The sum of the quality values for all the bases in the aligned consensus sequence) ) X 100 See % Match on page 72 for more information. 12

21 Software Basics 1 File-Naming Conventions The following are not allowed in user names or file names: Spaces \ / : *? < > Diacritical characters. Diacritical characters are found in some non-english alphabets. An example of a French diacritical character is Ç (C cedilla). If you use a space or any of these characters in a user name or file name, an error message is displayed. You must remove the invalid character to continue. For More Information Online Help For more detailed descriptions of the information and tasks contained in this Getting Started Guide, refer to the MicroSEQ ID Analysis software Online Help. The MicroSEQ ID Analysis software Online Help is supplied on the software CD- ROM. When the MicroSEQ ID Analysis software is installed and running, press F1, or select Help Contents & Index. Context-Sensitive Help The MicroSeq ID Analysis Software Online Help provides context-sensitive help for most windows in the software, as well as more general information about the software and procedures for common tasks. To access the context-sensitive help in the MicroSEQ ID Analysis software: Click to display information about the window or dialog box you are viewing. Note: The is not present in all windows and dialog boxes. 13

22 1 Context-Sensitive Help Software Basics 14

23 Chapter 2 2 Administrator: Set Up the Software Software Basics This chapter covers: Before You Begin Installing the Software Setting Up General Defaults Setting Up User Accounts Setting Up Authentication Setting Up the Audit Trail Setting Up Electronic Signatures Administrator: Set Up the Software Scientist: Set Up Analysis Information Analyst: Set Up a Project 15

24 2 Review Requirements and Workflow Administrator: Set Up the Software Before You Begin Review Requirements and Workflow User Privileges IMPORTANT! You must have Administrator privileges to perform the tasks in this chapter. An Administrator must install the MicroSEQ ID Analysis software and use it for the first time. The Administrator can set up the software for the Analyst, Scientist, or other Administrator users. Administrator Workflow Assign Administrator privileges to yourself when you install the software. The workflow below provides an overview of the Administrator tasks presented in this Getting Started Guide. IMPORTANT! Perform each task in the order given. Install and start the software. (pages 16 to 21) Set up general defaults. (page 23) Administrator: Set up the software Set up users. (page 25) Set up authentication. (page 27) Set up auditing. (page 28) Enable electronic signatures. (page 31) Registering the Software License and Warranty Before you begin, read Appendix D, Software Warranty Information. Appendix D explains your rights and responsibilities regarding the MicroSEQ ID Analysis software. During the installation process, you must accept the terms and conditions of the Software License Agreement before the software can be installed. Register the Software To register your copy of the MicroSEQ ID Analysis software, complete the registration card (included in this software package) and return it to Applied Biosystems. Registering the software enables Applied Biosystems to send you notification of software updates and any other future information that may be specific to MicroSEQ ID Analysis software owners. 16

25 Administrator: Set Up the Software 2 IMPORTANT! Your product registration number is located on the registration card. Be sure to record the number here before you return the registration card. Registration Number: Checking Hardware and Software Requirements Data Collection Software Autoanalysis of data from Applied Biosystems 3130/3130xl Series Genetic Analyzers (3130 Series) requires Data Collection Software version 3.0. Autoanalysis of data from Applied Biosystems 3500/3500xL Genetic Analyzers (3500 Series) requires 3500 Data Collection Software version 1.0 or later. Minimum System Requirements The table below summarizes the minimum system requirements for running the MicroSEQ ID Analysis software on your instrument or analysis computer. Note: In general, the more memory, the larger the screen size, and the more processing power in your system, the better the software performance. Minimum system requirements System Component CPU CD-ROM drive Operating system Regional and language options RAM Disk space Monitor Ethernet card Minimum Requirements 733 MHz or faster with an Intel Pentium III or IV processor Note: The software does not run on computers with a dual processor or with an Intel Xeon chip set. Any Microsoft Windows 2000 OS SP 3 or 4 or Microsoft Windows XP OS SP 1, 2, or 3 or Microsoft Windows Vista SP1 English (United States) XP Control Panel Regional and Language Options Region Options Vista Control Panel Regional and Language Options Administrative Change system locale 256 MB, 512 MB recommended 2 GB Storage requirements depend primarily on the quantity of data to be generated and stored. It is common to store many MicroSEQ ID Analysis software project files on the analysis computer. Because MicroSEQ ID Analysis software stores data files in the area where the program is installed, you should install the software on a partition with enough space for the projects and their files. A 17-inch monitor or larger is recommended A monitor of resolution is recommended. 10/100 Network Interface Card (NIC) with RWU 17

26 2 Checking Hardware and Software Requirements Administrator: Set Up the Software Hard Drive Partitions The installer defaults to the following location for the MicroSEQ ID Analysis software files: drive letter:\appliedbiosystems\microseqid IMPORTANT! Applied Biosystems strongly recommends that you install the MicroSEQ ID Analysis software on a drive other than the C drive, where the operating system is typically installed. Various memory-related issues have been observed when <10% of the C drive capacity is available. The MicroSEQ ID Analysis software and other applications may exhibit erratic behavior if <200 MB (~10%) of the drive is not free. This erratic behavior may also occur if either drive C or D is severely fragmented. The drive letter is determined by the following conditions: Table 2 Drive letter conditions If the computer The installer selects drive Is not connected to a genetic analyzer D (default) or C (if D drive is not available) Has Data Collection software that is connected to a 3500, 3130, or 3730 Series Genetic Analyzer E 18

27 Administrator: Set Up the Software 2 Installing the Software Log On Requirements To install the MicroSEQ ID Analysis Software, you must: Log on to the local computer (not a network domain) Use an Administrator account (unrestricted access) Note: After the software is installed, you can run the MicroSEQ ID Analysis Software without using an Administrator account. Verifying User Accounts Windows Vista 1. On the desktop, click Control Panel. 2. Double-click Classic View. 3. Double-click User Accounts. 4. In the Users tab, verify that the user account belongs to the Administrators group and the domain name is the same as the computer name. Belongs to the Administrators Domain is the same as the computer name 19

28 2 Preparing for Installation Administrator: Set Up the Software Windows XP 1. On the desktop, select Start Control Panel User Accounts. 2. In the Control Panel window, double-click. 3. In the Users tab, verify that the user account belongs to the Administrators group and the domain name is the same as the computer name. Belongs to the Administrators Domain is the same as the computer name Preparing for Installation IMPORTANT! If you will perform autoanalysis, install the software on the 3130 Series or 3500 Series Genetic Analyzer computer. To prepare for the installation: 1. Ensure that your system meets the minimum requirements (see Checking Hardware and Software Requirements on page 17). 2. Log in to Windows with local administrator privileges. 3. Check that you have at least 2 GB of free disk space to accommodate the MicroSEQ ID Analysis software, and sufficient space for all projects and their sample files. 4. If you will use the autoanalysis feature, start Data Collection Software before installing the MicroSEQ ID Analysis software: 3130 Series Genetic Analyzers Start the Data Collection Software Version 3.0. In the Service Console window, deselect Instrument Service and Viewer by right-clicking over the icons and selecting Stop. The two icons turn red. Note: Before stopping Instrument Service in the console, be sure that there are no runs in progress. If necessary, select Pause in the Data Collection Viewer and wait until the current run stops. 20

29 Administrator: Set Up the Software Series Genetic Analyzers Start the 3500 Data Collection Software Version 1.0. Make sure the Server Monitor is running. Installing the Software To install the MicroSEQ ID Analysis software: 1. Insert the MicroSEQ ID Analysis Software v2.2 CD into the computer CD-ROM drive. Note: If the installer does not start automatically, navigate to the CD-ROM drive and double-click setup.exe. 2. Follow the instructions to install the software. 3. When installation reaches the InstallShield Wizard Complete window, click Finish. After the software is installed, the Administrator must log into the software for the first time. After the initial login, the software can be set up for additional users. Upgrading to v2.2 Installation You can upgrade from any earlier version of MicroSEQ ID Analysis Software. However, v1.0 libraries are not supported. Upgrade Effect when Accessing v1.0 Libraries MicroSEQ ID Analysis Software does not support the v1.0 AB Bacterial 500, Bacterial Full Gene, and Fungal libraries. Analysis Protocols and Software v1.0 Data Files Level Project Specimen Sample Viewing Software v1.0 Analysis Protocols in Software v2.2 You can view your Analysis Protocols at this level. View the specimen analysis protocol by viewing it at the sample level. View v1.0 analysis protocols assigned at the sample level. Option: View the sample analysis protocol and copy the settings manually into the new analysis protocol. Applying Analysis Protocols to v1.0 files Your assigned analysis protocol at the project level applies to all the specimens in the project, unless the specimen has been assigned a specimen-level analysis protocol. Your assigned analysis protocol at the specimen level applies to all the samples in the specimen. You cannot apply an analysis protocol at the sample level. 21

30 2 Starting the Software for the First Time Administrator: Set Up the Software Starting the Software for the First Time The MicroSEQ ID Analysis software requires a user login process. Logging in with a user name allows MicroSEQ ID Analysis software to track each user s interactions with each project. To start the software for the first time: 1. Double-click the MicroSEQ ID Analysis desktop shortcut. 2. In the MicroSEQ ID Analysis Registration dialog box, enter all the information in the text fields. The User Name (login name) and password must be 6 to 15 characters long. Note: Enter a User Name that contains only alphanumeric characters. This field must contain characters that conform with the Microsoft Windows file system. Refer to File-Naming Conventions on page 13. The first user created is automatically assigned Administrator privileges. 3. Enter the registration code from the registration card you received with your software. 4. Click OK. While the program is loading, the splash screen appears. When the program is finished loading, the Login dialog box opens. 5. Enter your user name and password again. 6. Click OK to open the main MicroSEQ ID Analysis window. 22

31 Administrator: Set Up the Software 2 Setting Up General Defaults As a convenience, set up the general defaults to: Display reports after analysis Export reports after analysis Specify a directory to which reports are automatically exported Note: If you do not specify a directory, the default directory opens to C:\. To set up the general defaults: 1. In the MicroSEQ ID Analysis main window, select Tools Options to open the Options dialog box. 2. Select the General tab. 23

32 2 Starting the Software for the First Time Administrator: Set Up the Software Default character is a hyphen 3. Select the Display Reports after Analysis check box. 4. Select Export Reports after Analysis check box, then select the format in which to export them from the Format drop-down list. Note: Export Project after Auto-Analysis is an option for Applied Biosystems 3130 Series and 3500 Series Genetic Analyzer users. See Using Autoanalysis with Applied Biosystems 3130 Series Genetic Analyzers on page 89 or Using Autoanalysis with Applied Biosystems 3500 Series Genetic Analyzers on page 105 for more information. 5. Set the directory path to use as the Default Path for Import/Export: a. Click Browse to open the Import/Export Default Directory Location browser. b. Navigate to the desired directory. c. Click Set Directory. 6. Click OK to save your defaults and close the Options dialog box. 24

33 Administrator: Set Up the Software 2 Setting Up User Accounts Because the MicroSEQ ID Analysis software tracks the projects and settings for each user, Applied Biosystems recommends that you create a user account for each individual that uses MicroSEQ ID Analysis software on the computer. The Users tab allows exporting of user names and access privileges for these users. Creating New Users To create new users: 1. In the MicroSEQ ID Analysis main window, select Tools Options to open the Options dialog box. 2. Select the Users tab. 3. Click New to open the User Management dialog box. 4. Enter the appropriate User Name, First Name, Last Name, and Password. Note: Enter a User Name that contains only alphanumeric characters. This field must contain characters that conform with the Microsoft Windows file system. Refer to File-Naming Conventions on page

34 2 Changing User Information Administrator: Set Up the Software 5. Determine the user s privileges and then select from the User Group drop-down list: Administrator, Scientist, or Analyst. 6. Click OK. The new user appears in the list in the Users tab. 7. Click OK to close the Options dialog box. New users can log in after you exit the MicroSEQ ID Analysis software and restart the application. Changing User Information To change any of the information for a user: 1. In the MicroSEQ ID Analysis main window, select Tools Options to open the Options dialog box. 2. Select the Users tab. 3. Double-click the appropriate name, or highlight it and click Open, to open the User Management dialog box. 4. Change or correct the user information as desired. Note: You cannot delete a user from the database. To prevent further use of a user account, select the Inactive check box. Note: If a user tries to log into an account and types the wrong password too many times, the account is locked. To unlock the account, click the Unlock button. 5. Click OK. The user information is updated in the Users tab. Click OK to close the Options dialog box. 26

35 Administrator: Set Up the Software 2 Importing/ Exporting User Accounts To set up a default directory for importing/ exporting, see page 24. You can import or export user accounts from one computer to another. For example, an Administrator can set up user accounts for many users, then select all the user account files and export them to other systems that use the MicroSEQ ID Analysis software. For more information on importing/exporting user accounts, refer to the MicroSEQ ID Analysis software Online Help. Press F1, or select Help Contents & Index. Go to the Index tab and type user account, then select export or import. Setting Up Authentication The MicroSEQ ID Analysis software includes authentication features to assist with 21 CFR part 11 for electronic records requirements and ensures user integrity. To set up authentication: 1. In the MicroSEQ ID Analysis main window, select Tools Options to open the Options dialog box. 2. Select the Authentication tab. Note: The settings shown below are the defaults for authentication. 3. Complete the fields in the Authentication Settings section: Field Lockout user after invalid login attempts Within minutes Entry The number of times a user can enter an incorrect password or user name before being locked out of the software. The default lockout time is 3 minutes. The number of minutes a user has to enter the maximum number of login attempts before being locked out of the software. The default is 1 minute. 27

36 2 Importing/ Exporting Authentication Settings Administrator: Set Up the Software Field Maintain lockout for minutes Timeout Feature On Automatic timeout after minutes Allow Password to Expire Change password every days The number of minutes that must elapse before the user can log in again after being locked out of the software. Enter a number or accept the default of 3 minutes. Click to enable the timeout feature. Entry The number of minutes that must elapse before the software times out. The default is 30 minutes. Click to require a change in password at regular intervals, set in the Change Password every days field. The number of days before the user must enter a new password. The default is 90 days. Importing/ Exporting Authentication Settings To set up a default directory for importing/ exporting, see page 24. You can import or export the authentication settings from one computer to another. For example, an Administrator may want to set up authentication information for many users, then select all the files and export them to other systems using MicroSEQ ID Analysis software. For more information on importing/exporting Authentication settings, refer to the MicroSEQ ID Analysis software Online Help. Press F1, or select Help Contents & Index. Go to the Index tab and type Authentication settings, then select export or import. Continue with Setting Up the Audit Trail. Setting Up the Audit Trail The MicroSEQ ID Analysis software includes audit trail features to assist with 21 CFR part 11 for electronic records requirements and tracks user edits to consensus sequences. To set up the audit trail: 1. In the MicroSEQ ID Analysis main window, select Tools Options to open the Options dialog box. 2. Select the Audit Trail tab. 28

37 Administrator: Set Up the Software 2 3. In the Audit Trail tab, select one of the following: Audit Trail On When a user makes a change to a project, the software displays a dialog box that requires the user to select the reason for the change. The software logs the change, the user making the change, and the time and date the change was made in the Audit Trail. Silent Audit Trail On When a user makes a change to a project, no dialog box is displayed. However, the software still logs the change, the user making the change, and the time and date the change was made in the Audit Trail. Click New to add a new reason to the Audit Reason list. See Adding a New Reason to the Reason List. Click Open to edit a reason. See Inactivating or Editing a Reason. Adding a New Reason to the Reason List To add a new reason to the audit reason list: 1. Select Tools Options and select the Audit Trail tab. 2. Click New to open the Audit Reason Editor. 3. In the Reason field, type a reason for a change. For this Getting Started Guide, type Base Change. 4. In the Description field, type a description of where and when to use the reason. 5. Click OK. The new reason appears in the Audit Reason section of the Options dialog box. 29

38 2 Inactivating or Editing a Reason Administrator: Set Up the Software 6. Click OK to save the audit settings. Inactivating or Editing a Reason To inactivate or edit a reason: 1. Select Tools Options and select the Audit Trail tab. 2. Select the reason that you wish to inactivate or edit. 3. Click Open to open the Audit Reason Editor and make your changes and/or select Inactive. 4. Click OK. Accessing Application Audit Report To view the audit trail history of all the users at the application level, rather than in a specific project, select Tools Application Audit Report to open the Application Level Audit Report window. For information on the Report Settings button, see Changing the Report Settings. Changing the Report Settings Change a report s display settings using Report Settings located on the Report Manager, Application Level Audit Report, and Custom Report Manager windows. To change the report settings: 1. Click Report Settings to open the Reports Settings dialog box. 2. Set the options in the Report Display Settings tab as desired: Horizontal Scrolling When enabled, displays a scroll bar at the bottom of the Report Manager window. Font Size Wrap Text When enabled, multiple lines of text are displayed in a column. When disabled, only one line of text is displayed in a column. 30

39 Administrator: Set Up the Software 2 3. Optional: In the Concise Alignment Settings tab, select the number of top matches. 4. Click OK to enable the options. Setting Up Electronic Signatures MicroSEQ ID Analysis Software allows you to electronically sign when saving, printing, and/or exporting a report or project by using the Electronic Signature (esig) Software utility. You can also sign at any other time. Users belonging to the Administrator group set up, monitor, and maintain the electronic signature system. This feature is turned off by default. Enabling Electronic Signatures To use the electronic signature feature, you must enable it for the application and for each user who you want to have signature privileges. To set up your system for electronic signatures: 1. Log in as an Administrator. 2. Select Tools Options. 3. Select the Electronic Signature tab. 4. Select Enable Electronic Signature to enable the action list and the items in the Prompt verification dialog pane. Note: An e-sig prompt is not displayed when projects are exported during autoanalysis. 31

40 2 Enabling Electronic Signatures Administrator: Set Up the Software 5. Select the events in the Prompt verification dialog section to display an electronic signature dialog box when an action is required: Save/Save As Project Print Export Report Export Project 6. Select the Users tab. 7. Select a user, then double-click to open the User Management dialog box. 8. Select Enable Electronic Signature, then click OK. Note: In the User tab s Electronic Signature column, the box is now selected for that user. 32

41 Administrator: Set Up the Software 2 9. Repeat steps 7 to 8 for each user that requires electronic signature privileges. 10. Click OK to save. Inactivating a User 1. Log in as an Administrator. 2. Select Tools Options, then the Users tab. 3. Double-click on the user name to open the User Management dialog box. 4. Select Inactive, then click OK. A checkmark displays in the User Inactive column for the selected user. Creating Actions for an Electronic Signature To create a new action for electronic signatures: 1. Log in as Administrator. 2. Select Tools Options. 3. Select the Electronic Signature tab. 4. Click New to open the Electronic Signature Action dialog box. 33

42 2 Modifying Actions for an Electronic Signature Administrator: Set Up the Software 5. Enter the information for the following fields, then click OK: Action The name of the action or task to be performed. Meaning The meaning of the action. Challenge The legal implications of the action. Note: If you select Inactive, the action becomes inactive and is gray in the action list in the Electronic Signature tab. In addition, the action no longer appears in the drop-down list of actions for an electronic signature. Modifying Actions for an Electronic Signature To add or modify the actions for electronic signatures: 1. Log in as Administrator. 2. Select Tools Options. 3. Select the Electronic Signature tab. 4. To modify an action in the Action List, double-click the action row to open the Electronic Signature Action dialog box. 34

43 Administrator: Set Up the Software 2 5. Enter the information for the following fields, then click OK: Action The name of the action or task to be performed. Meaning The meaning of the action. Challenge The legal implications of the action. Note: If you select Inactive, the action becomes inactive and is gray in the action list in the Electronic Signature tab. In addition, the action no longer appears in the drop-down list of actions for an electronic signature. 35

44 2 Modifying Actions for an Electronic Signature Administrator: Set Up the Software 36

45 Chapter 3 3 Scientist: Set Up Analysis Information Software Basics This chapter covers: Before You Begin Creating a Custom Library Creating a Master Analysis Protocol Creating a Custom Report Template Scientist: Set Up Analysis Information Scientist: Set Up Analysis Information Analyst: Set Up a Project 37

46 3 Review Requirements and Workflow Scientist: Set Up Analysis Information Before You Begin Review Requirements and Workflow User Privileges IMPORTANT! You must have Administrator or Scientist privileges to perform the tasks in this chapter. While both Administrators and Scientists can set up the analysis information (that is, set up libraries and analysis protocols) for the MicroSEQ ID Analysis software, to get the most out of this Getting Started Guide, a Scientist should perform the tasks in this chapter. User Accounts Before you can log in to the software for the first time, an Administrator must create a user account for you (Chapter 2). Scientist Workflow The workflow below provides an overview of the Scientist tasks presented in this Getting Started Guide. IMPORTANT! Perform each task in the order given. Create libraries. (page 40) Scientist: Set up reference data Create analysis protocols. (page 45) Create custom reports. (page 51) Logging In Once your user account has been created, log into the software. To log in to the software: 1. Start the MicroSEQ ID Analysis software by double-clicking the desktop shortcut. The Log In dialog box opens, showing the last user s name. 38

47 Scientist: Set Up Analysis Information 3 2. Enter your user name and password, then click OK. Prompting the Audit Reason Dialog Box If your administrator has enabled the audit trail function, then an event such as adding or deleting libraries or analysis protocols, prompts the Audit Reason dialog box to open. 1. Select a reason for the event from the drop-down list. (In this example, consensus substitution is the event.) 2. Optional: Enter a description. 3. Click OK. Your action is recorded in the Audit Trail Report. 39

48 3 Prompting the Audit Reason Dialog Box Scientist: Set Up Analysis Information Creating a Custom Library During project analysis, the MicroSEQ ID Analysis software compares the consensus sequence to library sequences in order to generate a list of the closest matches. You may use Applied Biosystems custom or proprietary libraries or you can create your own custom library. Create a custom library using tutorial data and a tutorial library specifically created for the tutorial procedures. To create a custom library: 1. In the MicroSEQ ID Analysis main window, open the MicroSeqID Manager window: Select Tools MicroSeqID Manager, or click (MicroSeqID Manager). 2. Select the Libraries tab. 3. Click New to open the Library Editor, then select the General tab. 4. Enter information in the General tab as follows: In the Name field, type GSGTutorialLibrary (spaces aren t allowed; use the underscore character). From the Kit drop-down list, select Bacterial500Kit. In the Comments field, type Custom tutorial library for the Getting Started Guide (Bacterial 500 Kit). Your General tab should look like this: 40

49 Scientist: Set Up Analysis Information 3 5. Select the Entries tab, then click Import to open the Import Library Sequence Entries browser. 6. Import the library file: a. In the Files of type field, make sure FASTA format (*.fsta or *.fasta, depending on your file extension) is selected. b. Navigate to the installation drive location: drive letter:\appliedbiosystems\microseqid\tutorial Data\ Bacterial500Samples\Bacterial500CustomLibrary Note: When creating your own custom libraries, you can import information from any location. 41

50 3 Prompting the Audit Reason Dialog Box Scientist: Set Up Analysis Information c. Select Tutorial_Bacterial500_Lib v2.0.fsta. d. Click Import. 7. Click OK to acknowledge the import. The sequences in the GSGTutorialLibrary appear in the Entries tab of the Library Editor. Note: After the library file is imported, you can double-click any sequence entry to open the Library Sequence Entry Viewer and view the entire sequence. The sequence is read-only, but you can edit all other fields. 8. Click OK to save the new library and close the Library Editor. The new library appears in the Libraries tab of the MicroSeqID Manager window. 42

51 Scientist: Set Up Analysis Information 3 Accessing GenBank Sequence Information Import a GenBank sequence to a custom library or as a text specimen by changing the GenBank format to a FASTA file format. To access a GenBank file: 1. Enter NCBI in your Internet browser search field to display the National Center for Biotechnology Information home page. 2. Click All Databases to open the Entrez cross-database search page. 3. In Search across databases, enter the genus species of interest and click Go. 4. Select Nucleotide: sequence database (GenBank). 5. Select the report that includes the 16s rrna sequence and the Nucleotide information page opens. 6. In the Display drop-down menu, select FASTA. 7. In the Send to drop-down menu, select File to save the FASTA (*.fasta) file to your computer. Importing a FASTA File into a New Library To import a FASTA file into a New Library: 1. Select Tools MicroSeqID Manager, or click (MicroSeqID Manager) then select the Libraries tab. 2. Click New to open the Library Editor, then select the General tab. 3. Enter information in the General tab. 4. Select the Entries tab, then click Import to open the Import Library Sequence Entries browser. 43

52 3 Importing FASTA File into a Custom Library Scientist: Set Up Analysis Information 5. Import the file: Navigate to the file and select it. In the Files of type drop-down list, select the appropriate FASTA format (*.fsta /*.fasta). 6. Click Import. 7. Click OK, then click Close. Importing FASTA File into a Custom Library To import a FASTA file into a Custom Library: 1. Select Tools MicroSeqID Manager, or click (MicroSeqID Manager) then select the Libraries tab. 2. Select the library and double-click to open the Library Editor. Go to step 4 in Importing a FASTA File into a New Library, to complete the sequence import. Adding a Consensus Sequence to A Custom Library To add a consensus sequence to a custom library: 1. In the Project Navigator pane, right-click the Specimen. 2. Select Export Consensus Sequence To Library to open the Add Consensus Sequence to Library dialog box. 3. Select either: New Library, then enter an appropriate name for the custom library, then click OK to open the Library Editor dialog box. Existing Library, then select from the drop-down list, then click OK to open the Library Editor dialog box. 4. In the General tab, make changes to the name, kit, or add comments. 5. Select the Entries tab and double-click the entry name to open the Library Sequence Entry Viewer dialog box. 6. Fill in the appropriate information to the following fields: Entry Name: (automatically brought in) Comment Genus Species: Subspecies Source Entry Number 44

53 Scientist: Set Up Analysis Information 3 Type IJSB Ref GenBank Ref # Synonyms 7. Click OK, then click OK to close the Libraries tab. Creating a Master Analysis Protocol A master analysis protocol is a collection of settings that determine how the MicroSEQ ID Analysis software: Calls pure and mixed bases Identifies poor-quality data to be excluded from the clear range Filters data used to generate the consensus sequence Creating a master analysis protocol requires that you specify: Basecalling settings Mixed Bases settings Clear Range setting Filter settings For this tutorial, use the specifications given below to create the GSG_AnalysisProtocol. The Analyst will select the GSG_AnalysisProtocol when setting up a project in Chapter 4. When you create your own master analysis protocols, you can choose different specifications. Using the MicroSeqID Manager IMPORTANT! To create a master analysis protocol, you must access the Analysis Protocol Editor from the MicroSeqID Manager. This selection ensures that the protocol will be available for selection in other projects. The master analysis protocol is not applied to any projects that are reanalyzed. Opening a New Master Analysis Protocol To open a new master analysis protocol: 1. In the MicroSEQ ID Analysis main window, click (MicroSeqID Manager) to open the MicroSeqID Manager window. 45

54 3 Opening a New Master Analysis Protocol Scientist: Set Up Analysis Information 2. Select the Analysis Protocols tab. 3. Click New to open the Analysis Protocol Editor, then select the General tab. 4. In the Name field, type GSG_AnalysisProtocol (spaces are not allowed; use the underscore character). 5. In the Comments field, type New master analysis protocol for the Getting Started Guide. 6. Continue with Specifying Basecalling Settings below. Do not click OK until you complete all the tabs in the Analysis Protocol Editor. 46

55 Scientist: Set Up Analysis Information 3 Specifying Basecalling Settings The Basecalling tab has settings that determine how the software identifies sequence bases according to the run conditions applied at sample collection. To specify the basecalling settings: 1. In the Analysis Protocol Editor, select the Basecalling tab. 2. In the Basecalling section, enter information as follows: In the Basecaller drop-down list, select KB.bcp. In the DyeSet/Primer drop-down list, select KB_3130_POP6BDTv1.mob. 3. In the Ending Base section, select the At PCR Stop check box. 4. Check that the completed Basecalling tab looks like the one shown below. In the Basecalling tab, the Processed Data and Quality Threshold sections are only available when the KB basecaller is selected. 5. Continue with Specifying Mixed Bases Settings below. Do not click OK until you complete all the tabs in the Analysis Protocol Editor. Specifying Mixed Bases Settings Individual sequence positions are considered for mixed base identification only if the second peak height is more than a certain percentage of the main peak height. Set the percentage according to sample type, reaction kit and purification reaction, and expected or acceptable percentage. To specify the mixed base settings: 1. In the Analysis Protocol Editor, select the Mixed Bases tab. 2. Select the Use Mixed Base Identification check box to generate calls according to the international standard IUB code for heterozygous positions. 47

56 3 Specifying Clear Range Settings Scientist: Set Up Analysis Information 3. Use the default secondary peak threshold value of 25%. 4. Check that the completed Mixed Bases tab looks like the one shown below. 5. Continue with Specifying Clear Range Settings. Do not click OK until you complete all the tabs in the Analysis Protocol Editor. Specifying Clear Range Settings You can specify as the clear range the part of the sequence that has the highest quality (that is, has the fewest errors and ambiguities and has confident basecalls and good spacing). When you specify the clear range, you also can include only data corresponding to your consensus sequence and exclude data from the outlying 5 or 3 regions. To specify the clear range: 1. In the Analysis Protocol Editor, select the Clear Range tab. 2. Select Use quality values. 3. Select the default criterion for the clear range: fewer than 4 bases out of 20 with QVs less than 20. Note: Because the MicroSEQ ID Analysis software generates a quality value (QV) for each base, you can select a region of the sequence where a specified number of bases have QVs above a certain level. A QV of 20 = 1% probability of basecall error. 48

57 Scientist: Set Up Analysis Information 3 4. Check that the completed Clear Range tab looks like the one shown below. When you create your own analysis protocols, you can apply more than one Clear Range Method. The methods are applied in order and the smallest clear range is generated. Continue with Specifying Filter Settings. IMPORTANT! Do not click OK until you complete all the tabs in the Analysis Protocol Editor. Specifying Filter Settings Filter settings specify the criteria for rejecting sample sequences from a specimen assembly. Although sequences that do not meet the minimum and maximum standards you set here are not assembled, you can view them, modify their analysis settings, and reanalyze them from within the project. To specify the filter settings: 1. In the Analysis Protocol Editor, select the Filter tab. 49

58 3 Specifying Filter Settings Scientist: Set Up Analysis Information 2. Use the default filter settings, as described in the table below. Default Setting Maximum Mixed Bases (%) = 20.0 Maximum Ns (%) = 10.0 Minimum Clear Length (bp) = 50 Minimum Sample Score = 20 Description Total maximum percentage of mixed bases that can occur in the clear range of a sample sequence. If a sample clear range includes more than the specified percentage of mixed bases, the filtering step in analysis fails and is reported in the Analysis QC Report. Total maximum percentage of ambiguous bases (Ns) that can occur in the clear range of a sample sequence. If a sample clear range includes more than the specified percentage of Ns, the filtering step in analysis fails and is reported in the Analysis QC Report. Minimum number of bases required in the clear range of a sample sequence. If a sample clear range contains fewer than the specified number of bases, the filtering step in analysis fails and is reported in the Analysis QC Report. Minimum Sample Score (average of all sample QVs in the clear range) required. The range is 1 to 50. If a sample clear range is lower than the specified score, the filtering step in analysis fails and is reported in the Analysis QC Report. 3. Check that the completed Filter tab looks like the one shown below. 4. Click OK to save the new analysis protocol and close the Analysis Protocol Editor. The GSG_AnalysisProtocol appears in the Analysis Protocols tab of the MicroSeqID Manager window. 50

59 Scientist: Set Up Analysis Information 3 Creating a Custom Report Template Overview The MSID Set Up Analysis.fm generates the AB Standard Report that has four default reports: Analysis QC Report Library Search Report Audit Trail Report Electronic Signature Report You can select any of the categories and their items as shown below to create your own customized report. Categories to add to a customized report Categories Selectable items Summary Analysis QC Report Library Search Report Audit Trail Report Electronic Signature Report Annotation Assembly View Page Break Project Information Specimen Information Sample Information Specimen Analysis legend Specimen Analysis table Sample Analysis Library Table Concise Alignment Table Hit List table Phylogenetic Tree Library Search Table Application Level Audit Trail Project Level Audit Trail Electronic Signature Analysis Protocol Info Instrument Info Plate Info Plate Owner Info Run Info Results Group Info Results Group Owner Info Sample Info Specimen Info Assembly Table Insert a page break 51

60 3 Naming the Custom Report Scientist: Set Up Analysis Information Naming the Custom Report To name a custom report: 1. Select Tools MicroSeqID Manager and select the Custom Reports tab. 2. Click New to open the General tab of the Custom Report Editor dialog box. 3. In the Name field, type GSG_CustomReport (spaces are not allowed; use the underscore character). 4. In the Comments field, type New custom report for the Getting Started Guide. Do not click OK at this time. Custom Report Editor Use the Custom Report Editor dialog box to create your custom report. 52

61 Scientist: Set Up Analysis Information 3 Add a section to the report Section preview pane Item title Delete a selected Enter a section name that displays below your custom report folder Enter an item title for the section report Choose a category Select an item from the category Add an item Delete an item Custom Report Editor dialog box Adding Sections to the Custom Report For this tutorial, your final custom report will display the library items of hit list and concise alignment and the summary items of project and specimen information in the GSG_CustomReport. Consider naming a section from the Choose Category list. You can then add the various items associated with the category. See page Select the Editor tab. 2. In the Section Name field, type Library Search Report that simultaneously displays below your Custom Report folder. 3. Add Summary as a section: a. Click to the right of the Custom Report folder pane. b. In the Section Name field, type Summary and your new section is added below your last selected section. 53

62 3 Scientist: Set Up Analysis Information 4. Add Electronic Signature Report as a section: You may want to have a unique section name when items from different categories are added to the same section. a. Click to the right of the Custom Report folder pane. b. In the Section Name field, type Electronic Signature Report. 5. Select Electronic_Signature_Report and click to move this section up. (Clicking would move the section down.) 6. Delete a section: a. Select Electronic_Signature_Report under the Custom Report folder. b. Click to the right of the Custom Report folder pane. Adding Items to the Library Section Consider naming an item title from the item list. You can right-click on a table header and select the columns to display in the Report Manager. 1. In the Choose Category drop-down list, select Library Search Report to access the library items in the items drop-down list below Choose Category. 2. Add Hit List Table as an item: a. Select Hit List Table from the drop-down list that simultaneously displays the table in the right-side preview pane. b. In the Item Title field, type Hit List Table. Hit List Table replaces New Item Title in the Items in Section pane and displays in the right-side preview pane. 3. Add Concise Alignment Table as an item: a. Click to the right of the Items in Section pane. b. In the Choose Category drop-down list, select Library Search Report. c. Select Concise Alignment Table from the drop-down list. d. In the Item Title field, type Concise Alignment Table. Adding a Page Break 1. Add a page break without entering a title: a. Add a new item title by clicking to the right of the Items in Report pane. 54

63 Scientist: Set Up Analysis Information 3 b. In the Items Title field, select New Item Title, and press Backspace or Delete. New Item Title is deleted but position 3 is retained in the Items in Section pane. c. Select Page Break from the Choose Category drop-down list. Adding Items to the Summary Section 1. Select Summary Report under the CustomReport folder. 2. In the Choose Category drop-down list, select Summary. 3. Add Project Information as an item: a. Select Project Information from the item drop-down list. b. In the Item Title field, type Project Information. 4. Add Specimen Information as an item: a. Click to the right of the Items in Section pane. b. In the Choose Category drop-down list, select Summary. c. Select Specimen Information from the item drop-down list. d. In the Item Title field, type Specimen Information. 5. Optional: Change the report settings. See Changing the Report Settings on page Click OK to save the custom report. The GSG_CustomReport displays in the Custom Reports table. 55

64 3 Changing the Report Settings Scientist: Set Up Analysis Information Changing the Report Settings You may want to change a report s display settings. This option, located on the Report Manager window and the Application Level Audit Report is accessible to all users. To change the report settings: 1. Click Report Settings to open the Reports Settings dialog box. 2. Set the options in the Report Display Settings tab as desired: Horizontal Scrolling When enabled, displays a scroll bar at the bottom of the Report Manager window. Font Size Wrap Text When enabled, multiple lines of text are displayed in a column. When disabled, only one line of text is displayed in a column. 3. Optional: In the Concise Alignment Settings tab, select the number of top matches. 4. Click OK to enable the options. 5. Click OK to save the custom report template. 56

65 Chapter 4 4 Analyst: Set Up a Project Software Basics This chapter covers: Before You Begin Customizing the Display Settings Creating a Project Analyzing the Project Data Evaluating Results: Analysis QC Report Evaluating Results: Library Search Report Creating a Phylogenetic Tree Editing Data in the Analyzed Project Exporting or Printing Reports For More Information Analyst: Set Up a Project Scientist: Set Up Analysis Information Analyst: Set Up a Project 57

66 4 Review Requirements and Workflow Analyst: Set Up a Project Before You Begin Review Requirements and Workflow User Privileges You may have Administrator, Scientist, or Analyst privileges to perform the tasks in this chapter. While any user group can set up a project in the MicroSEQ ID Analysis software, to get the most out of this Getting Started Guide, an Analyst should perform the tasks in this chapter. User Accounts Before you can log in to the software for the first time, an Administrator must create a user account for you (Chapter 2). Analysis Information Before you can set up a project for this Getting Started Guide, a Scientist must perform the task in Chapter 3 to create a master analysis protocol (page 37). Analyst Workflow The workflow below provides an overview of the analyst tasks presented in this Getting Started Guide. IMPORTANT! Perform each task in the order given. Create a project. (page 61) Analyze the project. (page 66). Analyst: Set up a project Evaluate the results. (pages 70 and 74) Edit data in the analyzed project. (page 82) Electronically sign your work. (page 128) Export or print reports. (page 87) Logging In Once your user account has been created, log into the software. To log in to the software: 1. Start the MicroSeq ID Analysis software by double-clicking the desktop shortcut. The Log In dialog box opens, showing the last user s name. 58

67 Analyst: Set Up a Project 4 2. Enter your user name and password, then click OK. Customizing the Display Settings Display settings control how projects are visually displayed when they are first opened after analysis is complete. Each user can customize his or her own display settings. To customize the display settings: 1. Open a project or create a project to enable display settings. 2. In the MicroSeq ID Analysis main window, select Analysis > Display Settings to open the Display Settings window. 3. Select the General tab. 4. In the Comments field, type My display settings. 5. Select the Bases tab and choose the font, scale, and colors as desired. 59

68 4 Logging In Analyst: Set Up a Project 6. Select the Electropherogram tab and choose the scaling and axes as desired. 7. Select the Views tab and choose the General, Sample, and Specimen Views as desired. 60

69 Analyst: Set Up a Project 4 8. Click Save to save your display settings and close the Display Settings window. You will see the new display settings the next time you open an analyzed project. You may change your display settings at any time. Creating a Project Creating a project requires that you: Select project settings Create new specimens and import sample files View the project setup summary Selecting Project Settings To select project settings: 1. In the MicroSeq ID Analysis main window, select File New Project. The New Project Wizard opens with the Select Project Settings dialog box displayed. 2. In the Project Name field, type Tutorial500KitProject. 61

70 4 Selecting Project Settings Analyst: Set Up a Project 3. In the Matches to Display field, select 5. Note: Matches to Display refers to the number of library matches that will be displayed in the Library Search Results Project View. When you create your own projects, you can choose to display 3 to 20 matches. 4. In the Kit field, select Bacterial500Kit. Note: Kit refers to the MicroSEQ chemistry kit used to generate the sample data in the project. The Bacterial500Kit is the MicroSEQ S rdna Bacterial Identification Kit. 5. In the Analysis Protocol field, select GSG_AnalysisProtocol. Note: The GSG_AnalysisProtocol is the analysis protocol the Scientist created in Chapter In the Project Analysis Report field, select GSG_Custom_Report. 7. In the Libraries Available field, select Tutorial_Bacterial500_Lib_v2.0 a library specifically created for tutorial data. Note: Do not use the tutorial libraries for your projects other than analyzing tutorial project data. Note: Only those libraries that specify the selected kit (step 4 above) are listed. Note: When you create your own projects, you may search the Applied Biosystems libraries and any of your own custom libraries at the same time. For optimal performance, however, Applied Biosystems recommends searching the Applied Biosystems libraries first. 8. Check that the completed Select Project Settings dialog box looks like the one shown below. 62

71 Analyst: Set Up a Project 4 9. Click Next>> to continue with Creating a New Specimen and Import Sample Files, below. Do not click Finish at this time. Creating a New Specimen and Import Sample Files In the MicroSEQ ID Analysis software, a specimen refers to a container that holds the sample data (sample files) from a single bacterial or fungal unknown isolate. Create a new specimen, then import sample files into the specimen. You can import sample files individually or import an entire sample folder. Sample files imported into a specimen are in the.ab1 file format and are unassembled. There are two sample files for use with this Getting Started Guide: _A01_MSID_50cm_POP6_std.ab _A07_MSID_50cm_POP6_std.ab1 To create specimens and import sample files: 1. In the Create Specimens dialog box, click New Specimen. 63

72 4 Creating a New Specimen and Import Sample Files Analyst: Set Up a Project A new specimen (Specimen1) is added to the Samples to Add section. You will rename the specimen after project analysis; see step 2 on page 67. Note: The default specimen name (Specimen1) can only be changed in the Project Navigator pane after the project has been created. Note: To assign the name automatically, refer to the MicroSEQ ID Analysis software Online Help. Press F1, or select Help Contents & Index. Go to the Index tab and type new project, then select New Project - Create Specimens Dialog Box. 2. In the Look in field drop-down list, navigate to and select the 500Ecoli sample folder: drive letter:\appliedbiosystems\microseqid\tutorial Data\Bacterial500Samples\ 500Ecoli 3. Add the samples by selecting: _A01_MSID_50cm_POP6_std.ab1, then click Add Sample _A07_MSID_50cm_POP6_std.ab1, then click Add Sample. The sample files display under Specimen1 in the Samples To Add section. 64

73 Analyst: Set Up a Project 4 4. Continue with Viewing the Project Setup Summary on page 65. Viewing the Project Setup Summary 1. In the Create Specimens dialog box of the New Project Wizard, click Next. The Finish dialog box appears, showing the project setup summary. 2. Review the summary to make sure all information has been entered correctly. 3. If you need to make any changes: a. Click Back to return to the appropriate dialog box (Create Specimens or Select Project Settings). b. Make your corrections. c. Click Next to open the Finish dialog box again. 4. Continue with Analyzing the Project Data on page

74 4 Analyzing 3500.ab1 files Analyst: Set Up a Project Analyzing the Project Data After specifying the project settings and creating a new specimen, analyze the project data. Analyzing 3500.ab1 files The 3500 Series Genetic Analyzer applies a basecalling protocol to the.ab1 files it generates. When you analyze these sample files, the MicroSeq ID Analysis Software applies an analysis protocol (which uses the same KB Basecaller v1.4.1) and basecalls the data again. Options for Analyzing You can analyze the project data: Now, by clicking Finish and Analyze, or At a later time, by clicking Finish Analyze at a later time Analyze now New Project Wizard dialog box For this Getting Started Guide, follow the procedure below to analyze the project data now. For information on analyzing project data at a later time, refer to the MicroSEQ ID Analysis software Online Help. Press F1, or select Help Contents & Index. Go to the Index tab and type analyzing data, then select analyzing. Finish and Analyze the Project Data To finish and analyze the project data: 1. In the Finish dialog box of the New Project Wizard, click Finish and Analyze. An Analysis Status message appears as the software analyzes the data. 66

75 Analyst: Set Up a Project 4 To display the Analysis QC Report automatically after analysis: Select Tools Options General, then select the Display Reports after Analysis check box. When analysis is complete, the Project window opens. 2. In the Project Navigator pane, change the default specimen name: a. Double-click the default specimen name (Specimen1). b. Type Unknown_1. c. Press Enter. IMPORTANT! If you do not press Enter after changing the specimen name, the change is not applied. 3. In the Project Navigator pane, select: a. The project name (Tutorial500KitProject) to view the Library Search Results (that is, the % Match results for all the specimens). b. The specimen name (Unknown_1) to view the Layout and Assembly Views for that specimen. 67

76 4 Adding FASTA File as a Text Specimen Analyst: Set Up a Project c. Click (sample QV) to view the QV bars. Adding FASTA File as a Text Specimen In addition to using the.ab1 (sample) file format, you can import text sequences in FASTA file format, to a project. The analysis of text sequences displays the comparison of the sequence(s) with the library entries selected in the project. Note: The Analysis QC report (page 70) for a text specimen, displays NA for Basecalling, Filter, and Assembly, and a value 0 for # Samples and Specimen Score. To add a FASTA file as a text specimen: 1. Open a project. 2. Select File Import Text Specimen to open the Import Text Specimen dialog box. 3. Click Browse to browse for the.fasta/.fsta file and select the file. 4. Click OK. Viewing the Tutorial Custom Report To view the tutorial custom report for this project: 1. In the Project Navigator pane of the Project window, select the project name (Tutorial500KitProject). Project name 68

77 Analyst: Set Up a Project 4 Alternatively, click (Report Manager) to open Report Manager. 2. Select Analysis Report Manager to open the Report Manager for the Tutorial500KitProject. 3. Click each section under the GSG_Custom_Report folder to view the report items in the right-side preview pane. 4. Optional: Change the Report Settings. See Changing the Report Settings on page Continue with Evaluating Results: Analysis QC Report on page

78 4 Viewing the Analysis QC Report Analyst: Set Up a Project Evaluating Results: Analysis QC Report To verify that the sample files passed each step of the analysis process, evaluate the information provided in the Analysis QC Report. Viewing the Analysis QC Report To change from a custom report setting to the AB Standard Report setting, use the Properties and Settings dialog box in the following procedure. To view the Analysis QC Report: 1. In the Project Navigator pane of the Project window, select Tutorial500KitProject, then right-click to select Properties and Settings. Project name 2. In the Report Settings pane, select AB Standard Report. 3. Click OK. 4. Select Analysis Report Manager. If desired, you can display the Project View beneath the Analysis QC Report. From the toolbar, select Window Tile. 5. In the Reports navigation pane, select Analysis QC Report. 6. Review the data in the Specimen Analysis section. See Interpreting the Specimen Analysis Section on page Review the data (if any) in the Sample Analysis section. See Interpreting the Sample Analysis Section on page Continue with Evaluating Results: Library Search Report on page 74. Interpreting the Specimen Analysis Section The Specimen Analysis section displays a table that lists each specimen: Column Description Specimen The name of the specimen. The specimen name is hyperlinked to the Specimen View. # Samples The number of sample files in the specimen. Basecalling, Filter, and Assembly Specimen Score The status of these analysis steps. See Analysis Quality on page 71. Average consensus quality value (QV). See Specimen Score on page

79 Analyst: Set Up a Project 4 Column Description Top Match The name of the identified microorganism (library sequence) with the highest % Match. % Match Indicates how closely the unknown isolate matches the top library sequence (Top Match). See % Match on page 72. Consensus Length Library Entry Length Comments The number of bases of the consensus sequence. The number of bases of the library sequence entry. Enter any comments for this specimen. Analysis Quality The quality of the analysis for each analysis step (basecalling, filtering, and assembling) is indicated by the icons displayed in the Basecalling, Filter, and Assembly columns. Analysis quality icons Icon Green square Yellow triangle Red octagon Description Successful Analysis: All of the sample files in the specimen successfully completed the analysis step. Quality Alert: The quality data is poor. Look at sample preparation or electrophoresis problems. Failed Analysis: Check error messages. None of the sample files in the specimen successfully completed the analysis step. Symbol Linked to The Sample Analysis section of the Analysis QC Report. If any specimen displays the or icon, clicking the icon links you to the affected sample files in the Sample Analysis section. See Interpreting the Sample Analysis Section on page 73 for more information. To see the Sample View of a Navigator. Specimen Score sample file, select the sample name in the Project The Specimen Score is the average consensus QV (that is, the average QV of all base QVs in the specimen). To obtain the Specimen Score, per-base sample QVs are calibrated on a scale corresponding to: QV = 10log 10 (P e ) Where: P e is the probability of error of the basecall 71

80 4 Interpreting the Specimen Analysis Section Analyst: Set Up a Project The range of a QV is 1 (low confidence) to 50 (high confidence). Values above 30 indicate high-quality results. Note: Mixed bases are common in many bacterial species with multiple copies of the rrna gene. Some bacterial species have mixed bases and the QV may be lower even though the sequences are of high quality. The following table shows the probability of basecall errors for several QVs. This table provides an abbreviated list only. To view the probability of basecall errors for all QVs (ranging from 1 to 50), refer to the Online Help. Press F1, or select Help Contents & Index. Go to the Index tab and type quality value. Probability of basecall errors QV Pe QV Pe 10 10% % 20 1% % % % % % Denotes commonly used cutoff values for Low Confidence, Medium Confidence, and High Confidence ranges. % Match The % Match indicates how closely the unknown isolate matches the top library sequence (Top Match). The % Match is determined as follows: %Match = ( 1 - (The sum of mismatch penalties) / (The sum of the quality values for all the bases in the aligned consensus sequence) ) X 100 Mismatch penalty = (The mismatch factor) X ( The quality value of each mismatched base in the aligned consensus sequence) Mismatch factors are assigned: 0.5 for half-match of a mixed base aligned with a pure base and the mixed base contains the pure base 1.0 for mismatch The % match calculation uses quality values to weight the likelihood of a true mismatch. For example, a low quality mismatched base has less weight than a high quality mismatched base when the two are at the same position. 72

81 Analyst: Set Up a Project 4 Interpreting the Sample Analysis Section If the Specimen Analysis section displays the or icon for any specimen, clicking the icon links you to the affected sample files in the Sample Analysis section. In the Sample Analysis section, the report specifies for each failed sample file: Column Sample Step Description Description The name of the failed sample file The analysis step at which the failure occurred: basecalling, filtering, or assembling A detailed description of the particular analysis failure In the figure below for example, clicking (System Error icon) under the Filter column for Specimen1 links you to the Sample Analysis section. The Sample Analysis section displays the failed sample file (MicroSeq041703_D03_H_535R_07) and a description of the failure. Description of failure for Specimen1 Hyperlinks to View Sample Files The specimen name is hyperlinked to the Specimen View. You can view the data for a failed sample file by clicking its hyperlink to open the Sample View for the selected sample. Using the hyperlink to open the Sample View 73

82 4 View the Library Search Report Analyst: Set Up a Project Evaluating Results: Library Search Report The next step in the evaluation process is to check the library matches against each specimen consensus sequence. View the Library Search Report to: Identify the unknown organism. Assess the accuracy of the identification. Examine results for all matches. View phylogenetic relationships between the unknown and the top matches in a Neighbor Joining Tree. Determine if a match was made against a validated (proprietary) library or a custom library. View the Library Search Report To view the Library Search Report: 1. In the Project Navigator pane of the Project window, select the project name (Tutorial500KitProject). Project name 2. Select Analysis Report Manager to open the Report Manager for the Tutorial500KitProject. 3. In the Reports navigation pane, select Library Search Report. You can display the Project View beneath the Library Search Report. From the toolbar, select Window Tile. If you cannot see all the text in the Reports pane, click Report Settings, then select Wrap Text. 74

83 Analyst: Set Up a Project 4 1. Review the data in the Hit List section. See Interpreting the Hit List Section on page Review the data in the Concise Alignment section. See Interpreting the Concise Alignment Section on page Review the data in the Phylogenetic Tree section. See Interpreting the Phylogenetic Tree Section on page Continue with Editing Data in the Analyzed Project on page 82. Interpreting the Hit List Section In the Hit List section, the report lists for each specimen: Column Description Specimen Library Library Entry Name The name of the specimen The name of the library searched for possible matches An entry found in the library that possibly matches the consensus sequence % Match Indicates how closely the unknown isolate matches the top library sequence (Top Match). See % Match on page 72. Consensus Length The number of bases of the consensus sequence. 75

84 4 Interpreting the Concise Alignment Section Analyst: Set Up a Project Column Library Entry Length Total Mismatches Description The number of bases of the library sequence entry. The number of bases in the consensus sequence that do not match the library sequence. Interpreting the Concise Alignment Section In the Concise Alignment section, the report lists for each specimen with a % Match <100: The position at which each mismatch occurred. The position numbers displayed are the internal index numbers assigned to the consensus sequence bases in the MicroSEQ ID Analysis software. Position 0 is the first base in the consensus sequence. The base called in the consensus sequence for that position. The bases letter colors corresponds to their Quality Value colors as set in the Display Settings dialog box. The base present in the library sequence for that position. Note: Your Administrator can configure the number of top matches to display. The Concise Alignment table shown below displays several positions highlighting low quality values. To display both the Project view and the Library Search, select Window Tile. Customize the Quality Values colors by selecting Analysis Display Settings Bases tab and clicking on the color bar to choose different colors. Click on the base of interest; it turns grey, then the Assembly tab opens. For this example, the Assembly tab displays position 337, W s quality value location. 76

85 Analyst: Set Up a Project 4 Click the base to hyperlink to the assembly tab. View the QV and electropherogram data. The Concise Alignment table tiled with the Assembly tab Interpreting the Phylogenetic Tree Section In the Phylogenetic Tree section, the report lists for each specimen in the Hit List section: Specimen The name of the specimen N.Join % A percentage representing how closely the unknown is related to its closest library matches. It is calculated based on the percentage of the bases that differ between the unknown and the library. A smaller percentage indicates a closer relationship. The % value is the sum of the horizontal branch lines between two different species. Phylogenetic Tree diagram A graph composed of nodes and branches, where the nodes represent the Genus or Species. The Neighbor Joining Tree used in the MicroSEQ ID Analysis software represents the evolutionary history of organisms based on the sequence of the 16S rrna Gene. Entries with synonyms are denoted with *. Note: There are examples where two different species have the same rrna gene sequence and where two different rrna gene sequences belong to two different strains of the same species. 77

86 4 Interpreting the Phylogenetic Tree Section Analyst: Set Up a Project For example, in the Phylogenetic Tree shown below, the specimen Unknown_1 is the same species as its closest library match, but the specimen may be a different serotype, biovar, strain, and so on. Unknown_1 Phylogenetic Tree Creating a Phylogenetic Tree This tutorial procedure is to familiarize you with the Phylogenetic Tree Viewer, shown below, as you search for specimens and create a specific type of phylogenetic tree, called a neighboring joining tree. Neighboring Joining Tree diagram pane Status field Search Results pane Phylogenetic Tree Viewer window 78

87 Analyst: Set Up a Project 4 Searching Libraries To search for specimens using library search criteria: 1. Select Tools Create Phylogenetic Tree to open the Phylogenetic Tree Viewer. 2. Click Search Libraries to open the Search Specimens dialog box with library search criteria. a. Select Library Name from the Library entry name drop-down list. b. Select Contains in the contains drop-down list, then in the right-side field, enter Tutorial. c. Select AND in the no more criteria drop-down list. d. In the second line of criteria, select: Genus Contains Enter Aceto Select Case Sensitive e. Click Search to view the criteria used and the results. Your search should look like below: 79

88 4 Searching Projects to Add to Tree Analyst: Set Up a Project f. Select all four results and click Add Selected Specimens to Tree. 3. Click Search Projects to open the Search Specimens dialog box with project search criteria. See Searching Projects to Add to Tree below and go to step 2. Searching Projects to Add to Tree To search for specimens using project search criteria: 1. Select Tools Create Phylogenetic Tree to open the Search Specimens dialog box. 2. Search for a specimen using project search criteria: a. In the Specimens name drop-down list, select Project Name. b. Select Begins With in the Contains drop-down list, then enter Tutorial500 in the right-side field. c. Select Case Sensitive. d. In the No More Criteria drop-down list, select AND to add more criteria fields. e. In the second line of criteria, select: Top Match Name Contains Enter Ac f. Click Search to view the criteria used and the results. Your criteria and results search should look like below: 3. Select your result and click Add Selected Specimens to Tree. Creating a Neighboring Joining Tree Create a neighboring joining tree or phylogenetic tree by continuing the following tutorial procedure. To create a neighboring joining tree: 1. Click Compute Tree. The Tree Computation dialog box opens, then click OK. 80

89 Analyst: Set Up a Project 4 Your screen should look similar to the one below: 2. Click Clear to clear the tree and the search results pane. Clicking Save opens the Save Phylogenetic Tree dialog box. You can name the file, save it as an.xml file, and browse to a folder. Clicking Load opens the Load Phylogenetic Tree dialog box where you browse to your saved.xml file, select it, then click Load and the neighbor tree displays in the Phylogenetic Tree Viewer. Searching for Specimens to Create New Project Open the Search Specimens dialog box to select specimens and create a new project. 1. Select Tools Search and follow Searching Projects to Add to Tree on page 80 from step a through step f. 2. Select your result and click Create New Project with Selected Specimens to view the New Project Wizard. See Creating a Project on page 61 for more details. Click Cancel to return to the Search Specimens dialog box. 3. Click Clear Results to clear the results pane then click Close to end this tutorial section. 81

90 4 When to Edit Data Analyst: Set Up a Project Editing Data in the Analyzed Project When to Edit Data After analyzing and evaluating your own projects, edit the data if: An analysis step (basecalling, filtering, or assembling) fails. See Analysis Quality on page 71. A Specimen Score value is below 30. See Specimen Score on page 71. Consensus sequence needs to be edited. For example, if a base is incorrectly called or there are missing bases. How to Edit Data You can edit data in your analyzed projects by making changes to the: Consensus sequence Sample file Note: The screen captures in this section are examples only of failed data. They do not appear in the tutorial data for this Getting Started Guide. To perform the tasks in this section, use one of your own projects that contains failed data. Editing a Consensus Sequence The consensus sequence is the sequence assembled by the MicroSEQ ID Analysis software from all sample sequences in the specimen that meet the clear range and filtering thresholds. The consensus sequence is compared with a library of sequences to generate the closest matches. You can edit the consensus sequence by inserting, deleting, or changing a base. When you edit the consensus sequence, the sample sequences change to reflect the consensus edits. To edit a consensus sequence: 1. Select File Open Project and select one of your projects that contains failed data. The Project window opens. 2. In the Project Navigator pane, select the project name. 3. Select Analysis Report Manager. 4. In the Reports navigation pane, select Analysis QC Report. 5. In the Specimen Analysis section, double-click a specimen name (left-most column) to display the Specimen View for that specimen. 82

91 Analyst: Set Up a Project 4 Click a specimen name to display the Specimen View. 6. Click the Assembly tab in the Specimen View. The Assembly tab displays the nucleotide sequence of the consensus and sample, sample electropherogram data, and view position. 7. Check for medium (default color yellow) or low (default color red) QV bars. Medium or low values can decrease the Specimen Score. Note: If QV bars are not displayed, click (Show/Hide consensus QV) and (Show/Hide sample QV) in the Project window toolbar. Low and medium QV bars 83

92 4 Viewing the Assigned Analysis Protocol Analyst: Set Up a Project 8. Check for incorrectly called bases (for example, a noise peak called as a base or a true base peak not labeled). Note: If electropherograms are not displayed, click Electropherograms) in the Project window toolbar. (Show/Hide All 9. Make base changes following the information in the table below. To... Click... Then... Change a base The base to edit Type a new base letter. Delete a base The base to edit Press the Delete key. Insert a base Between two bases Type a new base letter. 10. Click OK. The edit is applied to the consensus sequence and all sample sequences in the specimen. Note: If the Audit Trail state is On, the Audit Reason Editor is displayed. Select a reason, type a comment, then click OK. 11. Reanalyze your project. Viewing the Assigned Analysis Protocol To view the assigned analysis protocol, open a project to get to the Project Navigator tree pane, then: Level Right-click on the... Project Specimen Sample Project name. Select Display Analysis Protocol to view the analysis protocol settings. Specimen name. You will see either: A message that the Specimen is using the project level analysis protocol or Select Display Analysis Protocol to view the analysis protocol settings Sample name. Select Display Last Successful Analysis Protocol. 84

93 Analyst: Set Up a Project 4 Selecting Another Analysis Protocol Select another analysis protocol at the project level or at the specimen level. To select another analysis protocol at the project level: Only the Administrator and Scientist user groups can edit analysis protocols. MicroSeqID Manager s Analysis Protocols tab is greyed out for the Analyst user group. 1. In the Project Navigator pane of the Project window, select Tutorial500KitProject as the project name, then right-click to select Properties and Settings. 2. In the Project Analysis Protocol pane, select an analysis protocol from the dropdown list. To select another analysis protocol at the specimen level: 1. In the Project Navigator pane of the Project window, select Unknown_1 the specimen name, then right-click to select Properties and Settings. 2. In the Project Analysis Protocol pane, select an analysis protocol from the dropdown list. 85

94 4 Reanalyzing and Saving the Project Analyst: Set Up a Project 3. Click Apply. Reanalyzing and Saving the Project 1. In the Project window, click (Analyze samples) to reanalyze the project. 2. Re-evaluate the Analysis QC Report (see page 70). 3. Re-evaluate the Library Search Report (see page 74). 4. When you finish editing the project, click File Close Project. 5. Click Yes at the prompt to save your changes and close the Project window. Exporting Your Project You may want to back up your projects by exporting them to a designated folder. 1. Close the project. 2. Select Tools MicroSeqID Manager, then select the Projects tab. 3. Select the project(s) to be exported and click Export. 4. Browse to your designated folder. 5. Click Export, then Close. 86

95 Analyst: Set Up a Project 4 Exporting or Printing Reports After editing data and reanalyzing the project, you can export or print the following reports: Analysis QC Report Library Search Report Audit Trail Report Electronic Signature History Report A custom report designed by a Scientist For this Getting Started Guide, follow the tasks below to export and print the Analysis QC Report for the Tutorial500KitProject. Exporting the Report To export the Analysis QC Report for the Tutorial500Kit Project: 1. In the Analysis QC Report, select File Export Report to open the Export Report browser. 2. Navigate to a destination for the exported file. 3. Type a name for the file or keep the default name (Tutorial500KitProject_Analysis_QC_Report). 4. Select a file type from the drop-down menu: PDF HTML XML TXT 5. Click Export. Printing the Report To print the Analysis QC Report for the Tutorial500Kit Project: 1. In the Analysis QC Report, select File Print to open the Print Report dialog box. 87

96 4 Analyst: Set Up a Project 2. Select the paper orientation and size. 3. Optional: Click Header/Footer to customize the header and footer. 4. Optional: Click Print preview to see the report before printing. 5. Click Print. 6. Select a printer, specify the page range, then click OK. For More Information Access the Online Help by opening the MicroSEQ ID Analysis software and doing one of the following: Select Help Contents and Index Press F1 Examples of information in the Online Help include: Troubleshooting topics Using a sample sheet template in the autoanalysis feature KB Basecaller Frequently Asked Questions (FAQs) To access context-sensitive help in the software application, click selected windows or dialog boxes., located on 88

97 A Appendix A Using Autoanalysis with Applied Biosystems 3130 Series Genetic Analyzers This chapter covers: Integrating MicroSEQ and Data Collection Software Automatically Exporting Projects After Autoanalysis Creating Required Files in the Data Collection Software Scheduling and Starting a Run Launching Autoanalysis Manager

98 A Overview Using Autoanalysis with Applied Biosystems 3130 Series Genetic Analyzers Integrating MicroSEQ and Data Collection Software Overview You can automatically analyze sequencing data generated on Applied Biosystems 3130/3130xl Genetic Analyzers using the MicroSEQ ID Analysis software without requiring user interaction. Autoanalysis uses three software packages automatically installed by the installer program: 3130/3130xl Analyzer Data Collection Software v3.0 Autoanalysis Manager A hidden version of MicroSEQ ID Analysis software that the Autoanalysis Manager uses to analyze the data in the projects Note: Autoanalysis can be performed only on the same instrument computer that collected the sample files. IMPORTANT! The data collection software must be running when installing MicroSEQ ID Analysis Software Version 2.2 on a computer that is connected to a 3130 Series Genetic Analyzer. If data collection software is not running, MicroSEQ ID Analysis software does not register with the Data Service. Requirements Autoanalysis requires that: Data Collection is running when MicroSEQ ID Analysis software is installed. You have launched MicroSEQ ID Analysis software at least once, registered the software, and created the appropriate user names. You have created a project in the MicroSEQ ID Analysis software. Autoanalysis Manager software is running. For More Information For information on setting up and using the 3130/3130xl Genetic Analyzer and the 3130 Data Collection software, see the Applied Biosystems 3130/3130xl Getting Started Guide (PN ). 90

99 Using Autoanalysis with Applied Biosystems 3130 Series Genetic Analyzers A Automatically Exporting Projects After Autoanalysis To automatically export projects after autoanalysis: 1. Select Tools Options to open the Options dialog box. 2. In the General tab, select Export Project after Auto-Analysis. 3. Type or browse to a destination folder, then click OK. 91

100 A Create an Instrument Protocol Using Autoanalysis with Applied Biosystems 3130 Series Genetic Analyzers Creating Required Files in the Data Collection Software Create an Instrument Protocol To create an instrument protocol: 1. In the navigation pane, click the Protocol Manager icon to display the Protocol Manager. 2. In the Instruments Protocols section, click New or select an existing instrument protocol file, then click Edit to open the Protocol Editor. 92

101 Using Autoanalysis with Applied Biosystems 3130 Series Genetic Analyzers A 3. Complete the Protocol Editor: a. Type a name for the protocol. b. Optional: Type a description for the protocol. c. Select Regular in the Type drop-down list. d. Select the correct run module for your run. (See the appropriate instrument user guide for information on selecting the correct run module.) e. Select the correct Dye Set for your run. (See the appropriate instrument user guide for information on selecting the correct Dye Set.) f. Click OK. Creating an Analysis Protocol Note: If you created an appropriate analysis protocol in MicroSeq ID Analysis Software v2.0, you can use it in data collection software. You must have Administrator or Scientist privileges. To create an analysis protocol: 1. In the Analysis Protocols section of the Protocol Manager, click New. Note: If more than one analysis application is installed on the data collection computer, the Analysis Applications dialog box opens. Select MicroSeqID, then click OK. The Analysis Protocol Editor dialog box opens. 93

102 A Creating an Analysis Protocol Using Autoanalysis with Applied Biosystems 3130 Series Genetic Analyzers 2. In the General tab, enter a unique name and comments for the new protocol. 3. Select the Basecalling tab, then: a. Select the appropriate basecaller and dye set/primer file. IMPORTANT! Make sure that the basecaller and the DyeSet/Primer file types match. Do not rename the files. Refer to your instrument user guide for basecaller and dye set/primer files. b. If desired, select one or more stop points for data analysis. Base your selection on the basecaller being used, as indicated below. Basecaller KB KB Stop Point At PCR Stop check box After Bases check box c. For the KB basecallers only, select True Profile or Flat Profile for processed data, then select a quality threshold. 4. For the KB Basecaller only, select the Mixed Bases tab, then: a. For mixed bases only, select Use Mixed Base Identification. b. Use the default setting of 25% or change the detection level by entering a new value or dragging the % line up or down. 94

103 Using Autoanalysis with Applied Biosystems 3130 Series Genetic Analyzers A 5. Select the Clear Range tab, then, if desired, select one or more stop points for data analysis. Note: The clear range is the region of the sequence that remains after excluding the low-quality or error-prone sequence at both the 5 and 3 ends. 6. Select the Filter tab, then, if desired, change one or more of the settings. (See Specifying Filter Settings on page 49 for more details.) 7. Select OK to save the protocol and close the Analysis Protocol Editor. Creating a Results Group To create a results group: 1. Click the Results Group icon in the navigation pane to open the Results Group Editor. 2. Click New, or select an existing group, then click Edit. 3. Make the following entries in the General tab: a. Type a Results Group Name. The name can be used in naming and sorting sample files and must be unique. b. Optional: Type a Results Group Owner. The owner name can be used in naming and sorting sample files. c. Optional: Type a Results Group Comment. 4. Select the Analysis tab. 95

104 A Creating a Results Group Using Autoanalysis with Applied Biosystems 3130 Series Genetic Analyzers 5. Make the following entries in the Analysis tab: a. In the Analysis Type drop-down list, select MicroSeq_ YourInstrumentName. b. In the Analysis Actions section, select Do Autoanalysis. c. In the text boxes, type a valid MicroSEQ Login ID and Password. IMPORTANT! Failure to use the proper login and password causes your project to fail being transferred into the MicroSEQ ID Analysis software or being analyzed. Use reextraction and try again. 6. Select the Destination tab to use the default location or define a new location. 96

105 Using Autoanalysis with Applied Biosystems 3130 Series Genetic Analyzers A 7. To define a new location: a. Select Use Custom Location, then click Browse to navigate to a different save location. b. Click Test to test the Location path name connection. If the path name is valid, a message box displays Path Name test successful. If the path name is not valid, a message box displays Could not make the connection. Please check that the Path Name is correct. Click and retry to establish a connection. c. Click OK. 8. Select the Naming tab, then define custom names for the sample file and the run folder, if desired.as you select from the drop-down list in the format fields in either the Sample File Name Format or Run Folder Name Format panes, another item field appears. 9. Click OK to save the Results Group and close the Results Group Editor. Creating a Plate Record To create a new plate record: 1. Click the Plate Manager icon in the navigation pane. 97

106 A Creating a Plate Record Using Autoanalysis with Applied Biosystems 3130 Series Genetic Analyzers 2. Click New to open the New Plate Dialog dialog box. 3. Complete the information in the New Plate Dialog box: a. Type a name for the plate. b. Optional: Type a description for the plate. c. Select MicroSeq_YourInstrumentName in the Application drop-down list. d. Select 96-well or 384-well in the Plate Type drop-down list. e. Type a name for the owner and operator. f. Click OK to open the MicroSeq Plate Editor. 98

107 Using Autoanalysis with Applied Biosystems 3130 Series Genetic Analyzers A Completing a Plate Record The MicroSeq Plate Editor dialog box below, allows you to fill out a plate record with the run conditions, sample information, and analysis conditions. To use the same specimen name for the entire plate, enter the name then click Ctrl+D to fill down the column Blank Plate Editor To complete a plate record: 1. Type sample names. Note: You should not use duplicate sample names in the same specimen. 2. Optional: Add comments. 3. Optional: Change the priority of the sample. (The default value 100 automatically displays.) 4. Select a project or create a new one by clicking in the project column cell and selecting from the drop-down list. The project s analysis protocol will be copied into the analysis protocol column. 5. Select a specimen or create one by clicking in the specimen column cell and selecting from the drop-down list. Note: If the specimen has an assigned analysis protocol, that analysis protocol will be copied into the analysis protocol column, replacing the project analysis protocol. 6. Select a results group, duplicate one, or create one by clicking in the Results Group 1 column cell and selecting from the drop-down list. 7. Select an instrument protocol, edit one, or create one by clicking in the Instrument Protocol 1 column cell and selecting from the drop-down list. 8. To add additional runs to the plate record: a. Select Edit Add Sample Run. b. Select a results group, and instrument protocol. 9. Click OK. 99

108 A Completing a Plate Record Using Autoanalysis with Applied Biosystems 3130 Series Genetic Analyzers Scheduling and Starting a Run 1. Click the Run Scheduler icon in the navigation pane. 2. Click Find All in the Plate View to find your plate record. You can also select Barcode or Advanced in the Type of Search drop-down menu to find your plate record, enter matching criteria, and click Search. 100

109 Using Autoanalysis with Applied Biosystems 3130 Series Genetic Analyzers A 3. Link the plate record to a plate layout: a. Select your plate record. b. Click the appropriate plate layout, A or B. The selected plate turns green. 4. Click (Run) to start the run. 101

110 A Launch Autoanalysis Manager Using Autoanalysis with Applied Biosystems 3130 Series Genetic Analyzers Launching Autoanalysis Manager Launch Autoanalysis Manager To launch the Autoanalysis Manager, select Start Applied Biosystems Autoanalysis Manager Autoanalysis Manager. Autoanalysis Manager General tab IMPORTANT! Do not run other applications when the Autoanalysis Manager and the data collection software are running. Never open a project while it is in use by the Autoanalysis Manager. Otherwise, the Autoanalysis Manager will not analyze the project. Sample Sheet Template By creating a sample sheet template you can save time because you can create a plate editor or use a text editor. For more information on using a sample sheet template in the autoanalysis feature, see the MicroSEQ ID Online Help. Press F1 Contents Autoanalysis topic Creating a sample sheet template. General Tab The General tab shows the jobs that have been submitted and their statuses. Note: Each Job in a row represents one run. 102

111 Using Autoanalysis with Applied Biosystems 3130 Series Genetic Analyzers A General tab with jobs in the queue General tab command buttons Button Name Delete Job Delete Completed Jobs Move Job Up Move Job Down Function Deletes a pending job Deletes all completed jobs Moves a pending job higher in the queue Moves a pending job lower in the queue MicroSeq ID Tab The MicroSeq ID tab shows the jobs, project, and status information. Note: Each Job in a row represents one project. Autoanalysis Manager MicroSeq ID tab job queue 103

112 A MicroSeq ID Tab Using Autoanalysis with Applied Biosystems 3130 Series Genetic Analyzers MicroSeq ID tab command buttons Button Name Details Function Displays the project in the navigation pane. Resubmit Edit Properties Stop Processing Auto-delete Jobs Delete Resubmits a failed job for analysis. Edits the name and password (active only if analysis failed). Interrupts the processing of the current job. The job is marked failed and the Autoanalysis Manager goes to the next job. Automatically deletes the completed jobs from the Autoanalysis Manager. This option allows you to select when to delete (in number of days) the completed jobs. This option is a convenient way to save disk space. Deletes a job from the Autoanalysis Manager. 104

113 B Appendix B Using Autoanalysis with Applied Biosystems 3500 Series Genetic Analyzers This chapter covers: Integrating MicroSEQ and Data Collection Software Preparing the MicroSEQ ID Analysis Software Preparing the 3500 Series Data Collection Software Creating a Plate for Autoanalysis Starting an Autoanalysis Run

114 B Overview Using Autoanalysis with Applied Biosystems 3500 Series Genetic Analyzers Integrating MicroSEQ and Data Collection Software Overview You can automatically analyze sequencing data generated on Applied Biosystems 3500 Series Genetic Analyzers using the MicroSEQ ID Analysis software without requiring user interaction. Autoanalysis uses three software packages automatically installed by the installer program: 3500 Series Data Collection Software v1.0 or later Autoanalysis Manager A hidden version of MicroSEQ ID Analysis software that the Autoanalysis Manager uses to analyze the data in the projects. Note: Autoanalysis can be performed only on the same instrument computer that collected the sample files. IMPORTANT! The data collection software must be running when installing MicroSEQ ID Analysis Software Version 2.2 on a computer that is connected to a 3500 Series Genetic Analyzer. If data collection software is not running, MicroSEQ ID Analysis software does not register with the Data Collection Software. Overview of Steps To prepare the 3500 Series Data Collection Software to run autoanalysis with the MicroSEQ ID Analysis software (one time): 1. Create a MicroSEQ ID secondary analysis protocol. This protocol identifies the location of the MicroSEQ ID Analysis software (page 110). 2. Create a plate template for autoanalysis (page 111). To run a plate with autoanalysis: 1. MicroSEQ ID Analysis software: Create a project for the autoanalysis run, then close the software. 2. In 3500 Series Data Collection software: Create a plate for autoanalysis (page 116). 3. Start the autoanalysis run (page 121). 106

115 Using Autoanalysis with Applied Biosystems 3500 Series Genetic Analyzers B During Autoanalysis When you start a run that is set up for autoanalysis, the following occurs: After each injection, the Autoanalysis Manager copies the base-called.ab1 files from the 3500 directory to the MicroSeq ID directory. The MicroSEQ ID Analysis Software uses the settings in the MicroSeq ID Analysis protocol to basecall and analyze the.ab1 files in the MicroSeq ID directory. Note: The KB basecaller v1.4.1 is used in the 3500 Series Data Collection Software and the MicroSEQ ID Analysis Software Series Genetic Analyzer features Shorter run times than the 3130 Series Genetic Analyzer New MicroSEQ ID-specific run module Requirements Autoanalysis requires that: The 3500 Series Data Collection Software is running when MicroSEQ ID Analysis software is installed. You have launched MicroSEQ ID Analysis software at least once, registered the software, and created the appropriate user names. You have created a project in the MicroSEQ ID Analysis software. The project is not open in the MicroSEQ ID Analysis software. Autoanalysis Manager software is running. For More Information For information on setting up and using the3500 Series Genetic Analyzer and Data Collection software, see the Applied Biosystems 3500 Series User Guide (PN ). 107

116 B Using Autoanalysis with Applied Biosystems 3500 Series Genetic Analyzers Preparing the MicroSEQ ID Analysis Software Automatically Exporting Projects After Autoanalysis 1. Select Tools Options to open the Options dialog box. 2. In the General tab, select Export Project after Auto-Analysis. 3. Type or browse to a destination folder, then click OK. 108

117 Using Autoanalysis with Applied Biosystems 3500 Series Genetic Analyzers B Preparing the 3500 Series Data Collection Software New concepts for 3130 Series Genetic Analyzer Users In the 3130 Series Data Collection Software, each injection was referred to as a run. In the 3500 Series Data Collection Software, a run refers to all injections in an injection list. An injection is an instance of 8 or 24 samples (depending on instrument configuration) processed simultaneously under the same conditions. The 3500 Series Data Collection Software uses new elements to specify settings for data collection. Note that you no longer need to create or select an instrument protocol; it is part of an assay (described below). New 3500 Series Data Collection Software Element Specifies settings for Settings previously Specified in 3130 Series Data Collection Software Element Primary Analysis (basecalling) protocol and templates Secondary Analysis (MicroSeqID) protocol File Name Convention and templates Results Group and templates Assay and assay templates Plate template Basecalling Location of the MicroSEQ ID Analysis Software for autoanalysis File naming Naming, sorting, and customizing the folders in which sample data files are stored. Data collection and processing. It contains: Instrument protocol (dye set and run configuration) Primary analysis (basecalling) protocol Secondary analysis (MSID) protocol Plate parameters and autoanalysis. Can also contain: Assays File name conventions Results groups Analysis protocol Results Group Results Group Results Group N/A N/A 109

118 B Using Autoanalysis with Applied Biosystems 3500 Series Genetic Analyzers Creating a MicroSEQ ID secondary analysis protocol for autoanalysis configuration 1. Access the MicroSeq ID Protocols library. 2. Click Create. 3. In the Create New MicroSeq ID Protocol dialog box (see figure below), specify settings (see Table 3 on page 111). 4. Select the remaining secondary analysis items, then click Save. IMPORTANT! The auto-analysis settings you specify for the plate to run with this protocol must contain the same secondary software and location settings. For more information, see Creating a Plate for Autoanalysis on page 116. Create New MicroSeq ID Protocol 110

119 Using Autoanalysis with Applied Biosystems 3500 Series Genetic Analyzers B Table 3 MicroSeq ID Protocol Settings Setting Description Protocol Name Lock Application Type Secondary Analysis Software Secondary Analysis Software Instance Name of the protocol, for example, Autoanalysis. Names must be unique. When enabled, allows the entry to be unlocked and modified only by the user who created it, the administrator, or another user with unlock permissions. Useful when your system includes the SAE module. For more information on this function, see the 3500 Series Data Collection Software User Guide. Automatically set to Sequencing. The secondary analysis software must be installed and properly configured with the 3500 Series Data Collection Software before it is listed as a selection in this screen. For information on setting up the MicroSEQ ID Analysis Software for auto-analysis, see Installing the Software on page 19. Computer on which the secondary analysis software is running (must be the 3500 Series Genetic Analyzer computer). Project Select a factory-provided MicroSEQ ID project, for example, AB_Bacterial500_Project_V2.0. You can select any project here; an entry is required but the project is not used by autoanalysis. You specify the MicroSEQ ID software project in the Assign Plate Contents screen (see Assigning plate contents on page 117). Specimen Automatically set to Specimen 1. You specify the MicroSEQ ID software specimen in the Assign Plate Contents screen (see Assigning plate contents on page 117). Create a plate template for autoanalysis from the MSID plate template provided Modifying the Plate Settings 1. In the Dashboard, click Create Plate From Template to display the Open Plate Template from Library dialog box. 111

120 B Specifying Autoanalysis Settings Using Autoanalysis with Applied Biosystems 3500 Series Genetic Analyzers 2. In the Filter field, select Sequencing. MicroSEQ ID templates 3. Select a MicroSEQ ID plate template, then click Open. 4. In the Define Plate Properties screen, type a name for the modified plate template. 5. Select the plate type: 96 Supports 96-well standard reaction plate. 8-strip standard tubes are also supported with appropriate retainers. 96-Fast Tube Supports 96-well Fast reaction plate. 8-strip fast tubes are also supported with appropriate retainers. Specifying Autoanalysis Settings 1. In the bottom of the Define Plate Properties screen, select Perform Auto- Analysis. 112

121 Using Autoanalysis with Applied Biosystems 3500 Series Genetic Analyzers B 2. Select MicroSeq ID for Software Type (automatically selected if you do not have any other secondary analysis software installed on this computer). The software automatically lists the current computer name for software location. Note: If MicroSeq ID is not listed under Software Type, the MicroSEQ ID Analysis Software is not correctly installed on the current computer. See Installing the Software on page 19. IMPORTANT! Software Type and Software Location settings must match the settings you specified in the secondary analysis protocol you created on page Enter your MicroSEQ ID Analysis Software Username and Password to allow the Autoanalysis Manager to access the MicroSEQ ID Analysis Software. 4. Click Assign Plate Contents. Note: Do not click Save yet. If you do, a plate error is displayed. Click Details to see the reason for the error. Editing the Assay The factory-defined assay included in the MicroSeq ID plate templates contains an instrument protocol/run module (POP-6 polymer) and a basecalling protocol optimized for MicroSeq ID applications. You cannot use the factory-defined assay directly in an autoanalysis run, you must save under a new name and add a secondary analysis protocol. Note: If you use POP-7 polymer, create a plate template that specifies the MicroSeqID POP-7 assay. IMPORTANT! Applied Biosystems validation studies were run with POP-6 polymer. Before running POP-7 polymer, perform POP-7 polymer validation studies. Edit the assay associated with the plate template to add the MicroSeq ID protocol you created on page 110: 1. In the Assign Plate Contents screen, click to display the Edit Assay screen. 113

122 B Editing the Assay Using Autoanalysis with Applied Biosystems 3500 Series Genetic Analyzers Assign Plate Contents Screen Edit Assay Edit Assay Screen 2. In the Assay Name field, add for AA to the end of the name. 3. In the SeqScape/MicroSeqID Protocol field, select the MSID protocol you created on page Click Apply to Plate, click Save to Library, then click Close. 114

123 Using Autoanalysis with Applied Biosystems 3500 Series Genetic Analyzers B Reviewing the File Name Convention and Results Group 1. Click to display the Edit File Name Conventions screen. 2. Review file name convention settings, edit as needed. If you make changes, specify a new name for the file name convention. 3. If you will specify Forward and Reverse sample attributes in User Defined Field 1, add User Defined Field 1 to the file name convention. Note: The Specimen Name attribute is not functional. Even when selected, specimen name is not included in the file name. 4. Click Apply to Plate, click Save to Library, then click Close. 5. Click to display the Edit Results Group screen. 6. Review results group settings, edit as needed. If you make changes, specify a new name for the results group. 7. Click Apply to Plate, click Save to Library, then click Close. Saving the plate Template Select Save As Template and save the template to use when you create a plate for autoanalysis. 115

124 B Saving the plate Template Using Autoanalysis with Applied Biosystems 3500 Series Genetic Analyzers Creating a Plate for Autoanalysis Creating a Plate From a Template 1. In the Dashboard, click Create Plate From Template to display the Open Plate Template from Library dialog box. 2. In the Filter field, select Sequencing. Note: You can set Sequencing as the default for this dialog box by selecting Preferences Plate Setup. You can filter by name by typing a name in the Search field, selecting Name, then clicking Go. Plate template from page

125 Using Autoanalysis with Applied Biosystems 3500 Series Genetic Analyzers B 3. Select the MicroSEQ ID plate template you created on page 111, then click Open. 4. Enter a name. 5. Select Save, then click Assign Plate Contents. Assigning plate contents Accessing the Assign Plate Contents Screen 1. After you create a plate, access the Assign Plate Contents screen (see figure on next page) from: The Define Plate Properties screen by clicking Assign Plate Contents (described above). The navigation pane by selecting Assign Plate Contents in the navigation pane. The Dashboard by clicking the Main workflow arrow, then selecting Assign Plate Contents in the navigation pane. 2. Click Show In Wells to specify the attributes to display in wells. Show well attributes Name samples Assign assays, file name conventions, and results groups Assign sample type, project name, and specimen name Link the plate Assign Plate View of the Assign Plate Contents screen 117

126 B Assigning Sample Name, Sample Type, Project Name, Specimen Name, Assay, File Name Convention, and Results Group Assigning Sample Name, Sample Type, Project Name, Specimen Name, Assay, File Name Convention, and Results Group 1. In the plate view, do any of the following to assign names to samples: Click a well, then type a sample name directly into the well, then press Enter. Click-drag multiple wells, then right-click and select Fill or Fill Series to populate the selected fields. (To use Fill Series, type a number as the last character of the named well). Copy and paste sample names instead of using fill commands. Number required for Fill Series 2. At the bottom right of the Assign Plate Contents screen, expand the Customize Sample Info pane. 3. Specify the Sample Type for the selected wells, then press Enter. 4. To specify the project name for autoanalysis: In User Defined Field 4, enter the name of the MicroSeq ID project in which to store the data. The name you enter must exactly match the name of an existing project in the MicroSEQ ID Analysis software. Do not include spaces. You can specify different project names on the same plate. IMPORTANT! If you do not enter a project name for any samples, or if the project name you enter does not exactly match the name of an existing project, the autoanalysis run fails. Note: You can select the entire plate, then specify a project name for all wells. 118

127 Using Autoanalysis with Applied Biosystems 3500 Series Genetic Analyzers B 5. To specify specimen names for each forward-reverse sample pair, you have two options: Option Use sample name for specimen name Set specimen name for each sample Procedure 1. Make sure sample names specified in step 1 for forward and reverse sample are identical (that is, do not indicate Forward or Reverse in the sample name. 2. If desired, specify this information in User Defined Field 1. Make sure to include User Defined Field 1 in the file name convention for the assay (see page 115). 3. Leave User Defined Field 3 blank. 1. In the plate view, select the forward-reverse sample pair. 2. In User Defined Field 3, enter the name of the: Existing MicroSEQ ID specimen to add the files to (existing samples in the specimen are not deleted or overwritten) or New MicroSEQ ID specimen to create Do not include spaces. The name you enter must follow the specimen naming conventions in MicroSEQ ID Analysis software. For information, see the Creating a New Project topic in the MicroSEQ ID Analysis software help. IMPORTANT! Amplicon and Specimen fields are not used. 6. Assign an assay, file name convention, and a results group for each sample. Note: If you specify different assays for samples within the same injection, one injection is made for each assay. For example, on an 8-capillary instrument, samples A01 through H01 are part of injection 1. If you specify Assay 1 for A01 and Assay 2 for samples B01 through H01, two injections are made. 119

128 B Assigning Sample Name, Sample Type, Project Name, Specimen Name, Assay, File Name Convention, and Results Group 120

129 Using Autoanalysis with Applied Biosystems 3500 Series Genetic Analyzers B Starting an Autoanalysis Run Starting the Autoanalysis Manager 1. Close the MicroSEQ ID Analysis Software. 2. Click Applied Biosystems 3500 Autoanalysis Manager Autoanalysis Manager. 3. Minimize the Autoanaylsis Manager. IMPORTANT! Do not run other applications when the Autoanalysis Manager and the data collection software are running. Never open a MicroSeq ID Analysis project while it is in use by the Autoanalysis Manager otherwise, the Autoanalysis Manager will not analyze the project. Parts of the Autoanalysis Manager General Tab The General tab shows the jobs that have been submitted and their statuses. Note: Each Job in a row represents one injection. General tab command buttons Button Name Delete Job Delete Completed Jobs Move Job Up Move Job Down Function Deletes a pending job Deletes all completed jobs Moves a pending job higher in the queue Moves a pending job lower in the queue 121

130 B Parts of the Autoanalysis Manager Using Autoanalysis with Applied Biosystems 3500 Series Genetic Analyzers MicroSeq ID Tab The MicroSeq ID tab shows the jobs, project, and status information. Note: Each Job in a row represents one injection. Samples are added to the project after each completed injection. Therefore, a 96-well plate that specifies one MicroSEQ ID project will generate twelve jobs (8-capillary instrument) or four jobs (24-capillary instrument). MicroSeq ID tab command buttons Details Button Name Displays the project in the navigation pane. Function Resubmit Edit Properties Stop Processing Auto-delete Jobs Delete Resubmits a failed job for analysis. Edits the name and password (active only if analysis failed). Interrupts the processing of the current job. The job is marked failed and the Autoanalysis Manager goes to the next job. Automatically deletes the completed jobs from the Autoanalysis Manager. This option allows you to select when to delete (in number of days) the completed jobs. This option is a convenient way to save disk space. Deletes a job from the Autoanalysis Manager. 122

131 Using Autoanalysis with Applied Biosystems 3500 Series Genetic Analyzers B Linking the Plate and Starting the Run 1. Prepare the plate and load it in the instrument. 2. In the Assign Plates for Run screen, click Link Plate for Run. 123

132 B Using Autoanalysis with Applied Biosystems 3500 Series Genetic Analyzers 3b 3a 3c Load Plates for Run 3. In the Load Plates for Run screen: a. Review the consumables information and the calibration information and ensure the status is acceptable for a run. b. Enter a Run Name or use the default run name: <Start Instrument Run Date/Time Stamp> YYYY-MM-DD-hh-mm-ss-SSS (milliseconds), for example, Run where the run start date is February and the run start time is 15:03:42:096. c. Click Create Injection List. 4. In the Preview Run screen: a. Configure the injection list. b. Click Start Run. The Monitor Run screen is automatically displayed. IMPORTANT! It takes, approximately, 10 seconds for the instrument to initialize after the instrument door is closed. Do not start a run until the instrument status light is green. IMPORTANT! You must specify re-injections before the run completes. 124

133 Appendix C C Electronic Signatures This chapter covers: About Electronic Signatures for Users Electronically Signing Your Work Viewing and Verifying Electronic Signatures

134 C Electronic Signatures About Electronic Signatures for Users Use the MicroSeq ID Analysis Software Electronic Signature (esig) system to regulate the creation, deletion, and modification of data stored in the database. Note: The electronic signature feature is turned off by default. Your administrator can turn on the electronic signature feature and configure the esig system by: Selecting which users can sign for their own actions. Selecting the events that will prompt an Electronic Signature Verification dialog box. Your administrator can select all or any of the following events: Save/Save As Project Print Export Report Export Project Creating any new action item. Review and Approve are the two default actions. For details on how your administrator sets up electronic signatures, see Setting Up Electronic Signatures on page 31. You, the user, authenticate or sign for your action by typing your user name (optional) and password. If you perform an action, such as saving a project that requires an electronic signature, the following dialog box opens: 126

135 Electronic Signatures C Drop-down list of actions Optional: Enter comments User types his or her user name User types his or her password Optional: Have two signatures to verify the Electronic Signature Verification dialog box If you enter the correct username and password, the MicroSeq ID Analysis Software v2.2 performs the requested action and records the electronic signature transaction. If you enter an invalid userid or passwords in three attempts within a minute, the software locks you out for 3 minutes. Note: The Electronic Signature Software tracks the number of failed login attempts. 127

136 C Electronic Signatures Electronically Signing Your Work You can activate the electronic signature system two ways: Perform an event that prompts an Electronic Signature Verification dialog box. See About Electronic Signatures for Users on page 126. Select a project, specimen or sample in the Project navigator pane, then select Tools Electronic Signature Sign to open the Electronic Signature Verification dialog box. Signing Your Work To complete the Electronic Signature Verification dialog box: 1. Select an action from the Action drop-down list. 2. Enter any comments, your user ID, and your password. 3. Optional: Select 2 signatures for you (or another valid electronic signature user) and another person to sign with each of your user ID and password. The Electronic Signature Verification dialog box adds another User ID and Password field for the second user. 4. Click OK. A message is displayed either stating you do not have electronic signature privileges or your verification was successful. See Invalid Project on page 130 if the verification is invalid. 5. Click OK to close the message. If you do not have electronic signature privileges, another user who does have them may sign with his/her user ID and password, then click OK. 6. Click OK to close the Electronic Signature Verification dialog box. 128

137 Electronic Signatures C Viewing and Verifying Electronic Signatures Viewing the Electronic Signature History To view the electronic signature history for a project: 1. In the main tool bar, click to open the Report Manager. 2. Select the Electronic Signature History Report in the Reports navigation pane. 3. Optional: Export the report In the main MicroSeq ID window, select File Export Report. Verifying a Project If you have electronically signed a project, you can verify if the project has changed since you last signed. To determine if a project has changed since the last signature: 1. Select File Open project. 2. Click Tools Electronic Signature Verify. A message states that the current electronic signature is VALID or NOT VALID. 3. Click OK. 129