MicroSeq ID Analysis Software Version 1.0

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1 MicroSeq ID Analysis Software Version 1.0 Getting Started Guide

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3 Copyright 2003, Applied Biosystems. All rights reserved. For Research Use Only. Not for use in diagnostic procedures. Information in this document is subject to change without notice. Applied Biosystems assumes no responsibility for any errors that may appear in this document. This document is believed to be complete and accurate at the time of publication. In no event shall Applied Biosystems be liable for incidental, special, multiple, or consequential damages in connection with or arising from the use of this document. NOTICE TO PURCHASER: 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 Applera Corporation, either expressly or by estoppel. NOTICE TO PURCHASER: PLEASE REFER TO THE MICROSEQ IDENTIFICATION KIT PROTOCOL FOR LIMITED LABEL LICENSE OR DISCLAIMER INFORMATION TRADEMARKS: ABI PRISM, Applied Biosystems, BigDye, and MicroSeq are registered trademarks of Applera Corporation or its subsidiaries in the U.S. and/or certain other countries. AB (Design), Applera, and POP-6 are trademarks of Applera Corporation or its subsidiaries in the U.S. and/or certain other countries. All other trademarks are the sole property of their respective owners. Part Number Rev. A 08/2003 August 14, :07 pm, MSID_GSG_TitlePage.fm

4 Contents Preface How to Use This Guide vii Purpose of This Guide vii Audience vii Assumptions vii Text Conventions vii User Attention Words viii How to Obtain More Information viii Related Documentation viii Send Us Your Comments ix How to Obtain Services and Support ix Chapter 1 Software Basics Start Here: Getting the Most out of This Guide Step Step Step What is the MicroSeq ID Analysis Software? User Groups and Privileges User Workflow Tutorial Data Software Structure Options Window MicroSeqID Manager Window Project Window Software Toolbars General Toolbar Analysis Toolbar Display Toolbar Terms Used in This Guide MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, MSID_GSG_Book_TOC.fm iii

5 File-Naming Conventions For More Information Online Help Context-Sensitive Help Chapter 2 Administrator: Set Up the Software Review Requirements and Workflow User Privileges Administrator Workflow Register the Software License and Warranty Register the Software Check Hardware and Software Requirements Minimum System Requirements Hard Drive Partitions Install the Software Prepare for Installation Install the Software Start the Software for the First Time Set Up General Options Set Up User Accounts Create New Users Change User Information Import/Export User Accounts Set Up Authentication and Auditing Set Up Authentication Set Up the Audit Trail Import/Export Audit Settings Chapter 3 Scientist: Set Up Analysis Information Review Requirements and Workflow User Privileges User Accounts Scientist Workflow Log In iv MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, MSID_GSG_Book_TOC.fm

6 Create a Custom Library Create a Master Analysis Protocol Use the MicroSeqID Manager Open a New Master Analysis Protocol Specify Basecalling Settings Specify Mixed Bases Settings Specify Clear Range Settings Specify Filter Settings Chapter 4 Analyst: Set Up a Project Review Requirements and Workflow User Privileges User Accounts Analysis Information Analyst Workflow Log In Customize the Display Settings Create a Project Select Project Settings Create a New Specimen and Import Sample Files View the Project Setup Summary Analyze the Project Data Options for Analyzing Finish and Analyze the Project Data Evaluate Results: Analysis QC Report View the Analysis QC Report Interpret the Specimen Analysis Section Interpret the Sample Analysis Section Evaluate Results: Library Search Report View the Library Search Report Interpret the Hit List Section Interpret the Concise Alignment Section Interpret the Phylogenetic Tree Section Edit Data in the Analyzed Project When to Edit Data How to Edit Data Edit a Consensus Sequence Edit the Sample File MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, MSID_GSG_Book_TOC.fm v

7 Reanalyze and Save the Project Export and Print Reports Customize the Report Export the Report Print the Report Appendix A Software Warranty Information Computer Configuration A-2 Limited Product Warranty A-2 Limited Warranty A-2 Warranty Period Effective Date A-3 Warranty Claims A-3 Warranty Exceptions A-3 Warranty Limitations A-3 vi MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, MSID_GSG_Book_TOC.fm

8 Preface How to Use This Guide Purpose of This Guide Audience Assumptions Text Conventions The Applied Biosystems MicroSeq ID Analysis Software Version 1.0 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 reference tutorial data that is supplied on the software CD-ROM. This guide is intended for new users of the MicroSeq ID Analysis software. There are three user groups: Administrator, Scientist, and Analyst. (For more information on user groups, see page 1-3.) This guide assumes that you have: A working knowledge of the Microsoft Windows operating system Access to the MicroSeq ID Analysis software Online Help This guide uses the following conventions: Bold indicates user action. For example: Type 0, then press Enter for each of the remaining fields. Italic text indicates variables and new or important words. Italic text is also used for emphasis. For example: Before analyzing, always prepare fresh matrix. A right arrow bracket (>) separates successive commands you select from a drop-down or shortcut menu. For example: Select File > Open > Spot Set. Right-click the sample row, then select View Filter > View All Runs. MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, 00_Preface.fm vii

9 Preface 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. Examples of the user attention words appear below: Note: The size of the column affects the run time. Note: The Calibrate function is also available in the Control Console. IMPORTANT! To verify your client connection to the database, you need a valid Oracle user ID and password. IMPORTANT! You must create a separate Sample Entry Spreadsheet for each 96-well microtiter plate. How to Obtain More Information Related Documentation The following related documents are available: MicroSeq ID Analysis Software Version 1.0 Online Help Describes the MicroSeq ID Analysis software and provides procedures for common tasks. The Online Help is supplied as part of the MicroSeq ID Analysis software. When the software is installed and running, press F1, or select Help > Contents & Index. MicroSeq ID Analysis Software Version 1.0 Quick Reference Card Provides the Analyst with a brief description of the tasks required to set up a project in the MicroSeq ID Analysis software. A portable document format (PDF) version of the Quick Reference Card is provided on the software CD-ROM. Note: For additional documentation, see How to Obtain Services and Support on page ix. viii MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, 00_Preface.fm

10 How to Obtain Services and Support Send Us Your Comments Applied Biosystems welcomes your comments and suggestions for improving its user documents. You can your comments to: How to Obtain Services and Support For the latest services and support information for all locations, go to then click the link for Services and Support. At the Services and Support page, you can: Download PDF documents Obtain information about customer training Download software updates and patches In addition, the Services and Support page provides access to worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities. MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, 00_Preface.fm ix

11 Preface x MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, 00_Preface.fm

12 Software Basics 1 1 This chapter covers: Start Here: Getting the Most out of This Guide What is the MicroSeq ID Analysis Software? User Groups and Privileges User Workflow Tutorial Data Software Structure Software Toolbars Terms Used in This Guide File-Naming Conventions For More Information MicroSeq ID Analysis Software Version 1.0 Getting Started Guide 1-1 August 14, :07 pm, 01_SWBasics.fm

13 Chapter 1 Software Basics Start Here: Getting the Most out of This Guide The Applied Biosystems MicroSeq ID Analysis Software Version 1.0 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 reference tutorial data that is supplied on the software CD-ROM. To get the most out of this Getting Started Guide, we recommend proceeding as follows: Step 1 Step 2 Step 3 Read this Software Basics chapter. Determine which user group you belong to. See User Groups and Privileges on page 1-3. Work through the tutorial tasks appropriate for your user group. See User Workflow on page 1-4. What is the MicroSeq ID Analysis Software? MicroSeq ID Analysis software version 1.0 is a tool for microbial identification of bacteria and fungi. The software analyzes data generated using a MicroSeq chemistry kit and an ABI PRISM 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. 1-2 MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, 01_SWBasics.fm

14 User Groups and Privileges User Groups and Privileges There are three user groups defined in the software: Administrator, Scientist, and Analyst. Each of these user groups has the privileges listed in the table below. User Group Privileges Administrator Install and start the software Set up defaults Set up users Set up auditing All Scientist privileges All Analyst privileges Scientist Create libraries Create analysis protocols All Analyst privileges Analyst Create a project Analyze the project Evaluate the results Edit data in the analyzed project Export or print reports MicroSeq ID Analysis Software Version 1.0 Getting Started Guide 1-3 August 14, :07 pm, 01_SWBasics.fm

15 Chapter 1 Software Basics User Workflow The workflow below provides an overview of the tasks presented in this Getting Started Guide. The tasks reference tutorial data (see page 1-5) 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. Administrator: Set up the software Set up defaults. Set up users. Set up auditing. See Ch. 2 Scientist: Set up reference data Create libraries. Create analysis protocols. See Ch. 3 Create a project. Analyst: Set up a project Analyze the project. Evaluate the results. Edit data in the analyzed project. Export or print reports. See Ch MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, 01_SWBasics.fm

16 Tutorial Data 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 1-1 defines the two type of files provided and their folder names, as well as the specific file names and extensions. Table 1-1 Tutorial data referenced in this guide File Type Folder Name File Name and Extension Library Bacterial500CustomLibrary 500KitCustomLibrary.fsta Sample file 500EColi MicroSeq041703_C03_H_5F_05.ab1 MicroSeq041703_D03_H_535R_07.ab1 Note: The tutorial data was electrophoresed on an ABI PRISM 3100 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. MicroSeq ID Analysis Software Version 1.0 Getting Started Guide 1-5 August 14, :07 pm, 01_SWBasics.fm

17 Chapter 1 Software Basics 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 View Report Manager Sample Manager User accounts Libraries Specimen View Analysis QC Report Sample analysis protocol Authentication and audit trails Master analysis protocols Sample View Library Search Report Audit Trail Report Create and edit settings Manage analysis information View project data View, export, or print results Edit and apply to project Options Window Access the Options window by clicking Tools > Options. Use the Options window to create and/or edit: General defaults, which include Displaying reports after analysis Exporting reports after analysis Specifying a directory to which reports are automatically exported User accounts Authentication and audit trails 1-6 MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, 01_SWBasics.fm

18 Software Structure MicroSeqID Manager Window Access the MicroSeqID Manager window by clicking Tools > MicroSeqID Manager. Project Window Access the Project window by clicking File > Open Project and selecting an existing project. Use the MicroSeqID Manager window to enter and manage the information necessary to perform analyses on your projects: Edit, delete, import, and export projects Create, edit, delete, import, and export libraries Create, edit, delete, import, and export master analysis protocols Use the Project window to: View, export, or print project data (Project View, Specimen View, or Sample View) Access the Sample Manager to edit sample file information Access the Report Manager to view, export, or print project analysis results MicroSeq ID Analysis Software Version 1.0 Getting Started Guide 1-7 August 14, :07 pm, 01_SWBasics.fm

19 Chapter 1 Software Basics 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 1-8 to 1-10) 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 Save Project Ctrl+S Saves a modified project Import Samples To Project Ctrl+M 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 New Specimen Opens a new specimen Find Ctrl+F Finds a selected string/iub code in a sequence 1-8 MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, 01_SWBasics.fm

20 Software Toolbars Analysis Toolbar The Analysis toolbar contains analysis tools for the projects you create. Analyze Samples Ctrl+R Analysis Settings Ctrl+B When a sample file is selected, displays the Analysis Protocol settings When a project is selected, displays the Project Analysis Settings MyScience Opens your Applied Biosystems MyScience web page. Sample Manager Opens the Sample Manager window Report Manager Ctrl+1 Opens the Report Manager window MicroSeq ID Analysis Software Version 1.0 Getting Started Guide 1-9 August 14, :07 pm, 01_SWBasics.fm

21 Chapter 1 Software Basics 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 1-10 MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, 01_SWBasics.fm

22 Terms Used in This Guide Terms Used in This Guide Term Aligned consensus sequence Analysis protocol Clear range Consensus quality value (consensus QV) Consensus sequence Kit Project Sample file Sample quality value (sample QV) Definition The consensus sequence after pairwise alignment with the library sequence. May contain gaps. 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. MicroSeq ID Analysis Software Version 1.0 Getting Started Guide 1-11 August 14, :07 pm, 01_SWBasics.fm

23 Chapter 1 Software Basics Term Specimen Specimen score Start and End delimiters Unknown isolate A container that holds the sample data (sample files) from a single bacterial or fungal unknown isolate. Average consensus QV. Definition 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). (Number of bases that match / Length of alignment) 100 Where: Number of bases that match = Number of matching bases in the pairwise alignment between consensus sequence and the library search hit. Length of alignment = Length of pairwise alignment without considering leading gaps and trailing gaps MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, 01_SWBasics.fm

24 File-Naming Conventions 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 Context-Sensitive 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. The GPS Explorer 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. MicroSeq ID Analysis Software Version 1.0 Getting Started Guide 1-13 August 14, :07 pm, 01_SWBasics.fm

25 Chapter 1 Software Basics 1-14 MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, 01_SWBasics.fm

26 Administrator: Set Up the Software 2 2 This chapter describes tasks an Administrator performs with the MicroSeq ID Analysis software. Instructions are provided for installing the software. A brief tutorial follows for setting up defaults, user accounts, and authentication and auditing. This chapter covers: Review Requirements and Workflow Register the Software Check Hardware and Software Requirements Install the Software Start the Software for the First Time Set Up General Options Set Up User Accounts Set Up Authentication and Auditing MicroSeq ID Analysis Software Version 1.0 Getting Started Guide 2-1 August 14, :07 pm, 02_SettingUpSW.fm

27 Chapter 2 Administrator: Set Up the Software Review Requirements and Workflow User Privileges Administrator Workflow 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. 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 2-3 to 2-9). Administrator: Set up the software Set up defaults (page 2-10). Set up users (page 2-12). Set up auditing (page 2-15). 2-2 MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, 02_SettingUpSW.fm

28 Register the Software Register the Software License and Warranty Register the Software Before you begin, read Appendix A, Software Warranty Information. Appendix A 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. 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. 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: MicroSeq ID Analysis Software Version 1.0 Getting Started Guide 2-3 August 14, :07 pm, 02_SettingUpSW.fm

29 Chapter 2 Administrator: Set Up the Software Check Hardware and Software Requirements Minimum System Requirements Table 2-1 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. Table 2-1 Minimum system requirements System Component CPU CD-ROM drive 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 Operating system Microsoft Windows 2000 OS with Service Pack 2 or Service Pack 3 Microsoft Windows XP OS with Service Pack 1 RAM Disk space Monitor At least 256 MB Applied Biosystems recommends 512 MB. 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. 2-4 MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, 02_SettingUpSW.fm

30 Check Hardware and Software Requirements Table 2-1 Minimum system requirements (continued) System Component Minimum Requirements Printer An HP 4500, 8100, 990cxi or an Epson 980 printer is recommended. Hard Drive Partitions The installer uses the following location for the MicroSeq ID Analysis software files: drive letter:\applied Biosystems\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 2-2 Drive letter conditions If the computer Is not connected to a genetic analyzer Has Data Collection software that is connected to the Applied Biosystems 3730/3730xl DNA Analyzers The installer selects drive D (default) OR C (if D drive is not available) E MicroSeq ID Analysis Software Version 1.0 Getting Started Guide 2-5 August 14, :07 pm, 02_SettingUpSW.fm

31 Chapter 2 Administrator: Set Up the Software Install the Software Prepare for Installation Install the Software To prepare for the installation: 1. Ensure that your system meets the minimum requirements (see Check Hardware and Software Requirements on page 2-4). 2. 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. To install the MicroSeq ID Analysis software: 1. Insert the MicroSeq ID Analysis Software v1.0 CD into the computer CD-ROM drive. 2. If the installer does not start automatically, navigate to the CD-ROM drive and double-click setup.exe. 3. Follow the instructions to install the software. 2-6 MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, 02_SettingUpSW.fm

32 Install the Software 4. When the InstallShield Wizard Complete window opens, 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. MicroSeq ID Analysis Software Version 1.0 Getting Started Guide 2-7 August 14, :07 pm, 02_SettingUpSW.fm

33 Chapter 2 Administrator: Set Up the Software Start the Software for the First Time The MicroSeq ID Analysis software is designed with a user login process. Logging in with a user name allows MicroSeq ID Analysis software to track each user s interactions with each project. When you start the software for the first time, you are prompted with a registration dialog box that automatically creates an Administrator (Admin) account. 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 and password must be 6 to 15 characters long. 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. 2-8 MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, 02_SettingUpSW.fm

34 Start the Software for the First Time 6. Click OK. The main MicroSeq ID Analysis window opens. MicroSeq ID Analysis Software Version 1.0 Getting Started Guide 2-9 August 14, :07 pm, 02_SettingUpSW.fm

35 Chapter 2 Administrator: Set Up the Software Set Up General Options Set up the general options 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 options: 1. In the MicroSeq ID Analysis main window, select Tools > Options to open the Options dialog box. 2. Select the General tab. 3. If desired, select the Display Reports after Analysis check box. 4. If desired, select the Export Reports after Analysis check box, then select the format in which to export them from the Format drop-down list. 5. If desired, 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 MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, 02_SettingUpSW.fm

36 Set Up General Options b. Navigate to the desired directory. c. Click Set Directory. 6. Click OK to save your defaults and close the Options dialog box. MicroSeq ID Analysis Software Version 1.0 Getting Started Guide 2-11 August 14, :07 pm, 02_SettingUpSW.fm

37 Chapter 2 Administrator: Set Up the Software Set 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 who uses MicroSeq ID Analysis software on the computer. The Users tab allows exporting of user names and access privileges for these users. Create 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 MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, 02_SettingUpSW.fm

38 Set Up User Accounts 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 Select the user group 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. Change 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. MicroSeq ID Analysis Software Version 1.0 Getting Started Guide 2-13 August 14, :07 pm, 02_SettingUpSW.fm

39 Chapter 2 Administrator: Set Up the Software 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 three 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. 6. Click OK to close the Options dialog box. Import/Export User Accounts To set up a default directory for importing/exporting, see page 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 MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, 02_SettingUpSW.fm

40 Set Up Authentication and Auditing Set Up Authentication and Auditing The MicroSeq ID Analysis software includes authentication and auditing features to support 21 CFR part 11 for electronic records requirements. Authentication helps ensure user integrity. Auditing tracks user edits to consensus sequences. Set Up Authentication 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 & Audit tab. Note: The settings shown below are the defaults for authentication and auditing. 3. Complete the fields in the Authentication Settings section: Field Lockout user after invalid login attempts Description Indicates the number of times a user can enter an incorrect password or user name before being locked out of the software. Enter a number or accept the default. MicroSeq ID Analysis Software Version 1.0 Getting Started Guide 2-15 August 14, :07 pm, 02_SettingUpSW.fm

41 Chapter 2 Administrator: Set Up the Software Field Within minutes Maintain lockout for minutes Automatic timeout after minutes Change password every days Description Indicates the amount of time a user has to enter the maximum number of login attempts before being locked out the software. Enter a number or accept the default. Indicates 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. Indicates the number of minutes that must elapse before the software times out. Enter a number or accept the default. Indicates the number of days before the user must enter a new password. Enter a number or accept the default. 4. Continue with Set Up the Audit Trail below. Set Up the Audit Trail To set up the audit trail: 1. In the Audit Trail section of the Authentication & Audit tab, select one of the following: Audit Trail On When a user makes a change to a project, the software displays a dialog box, which requires the user to select the reason for the change. The software logs the change, the user making the change, and the time/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/date the change was made in the Audit Trail. 2. Click New to open the Audit Reason Editor MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, 02_SettingUpSW.fm

42 Set Up Authentication and Auditing 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. 6. Click OK to save the authentication and audit settings. Import/Export Audit Settings To set up a default directory for importing/exporting, see page You can import or export the Authentication & Audit settings from one computer to another. For example, an Administrator may want to set up authentication and audit 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 & Audit 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 and Audit settings, then select export or import. MicroSeq ID Analysis Software Version 1.0 Getting Started Guide 2-17 August 14, :07 pm, 02_SettingUpSW.fm

43 Chapter 2 Administrator: Set Up the Software 2-18 MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, 02_SettingUpSW.fm

44 Scientist: Set Up Analysis Information 3 3 This chapter describes tasks a Scientist performs with the MicroSeq ID Analysis software. A brief tutorial is provided for creating a custom library and a master analysis protocol. This chapter covers: Review Requirements and Workflow Log In Create a Custom Library Create a Master Analysis Protocol MicroSeq ID Analysis Software Version 1.0 Getting Started Guide 3-1 August 14, :07 pm, 03_SettingUpRefData.fm

45 Chapter 3 Scientist: Set Up Analysis Information Review Requirements and Workflow User Privileges User Accounts Scientist Workflow 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. Before you can log in to the software for the first time, an Administrator must create a user account for you (Chapter 2). The workflow below provides an overview of the Scientist tasks presented in this Getting Started Guide. IMPORTANT! Perform each task in the order given. Scientist: Set up reference data Create libraries (page 3-4). Create analysis protocols (page 3-8). 3-2 MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, 03_SettingUpRefData.fm

46 Log In Log 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. 2. Enter your user name and password, then click OK. MicroSeq ID Analysis Software Version 1.0 Getting Started Guide 3-3 August 14, :07 pm, 03_SettingUpRefData.fm

47 Chapter 3 Scientist: Set Up Analysis Information Create 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. This section provides a procedure for creating your own custom library. 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: a. In the Name field, type AB_Bacterial500CustomLibrary (spaces aren t allowed; use the underscore character). b. From the Kit drop-down list, select Bacterial500Kit. c. In the Comments field, type Custom library for the Getting Started Guide (Bacterial 500 Kit). 3-4 MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, 03_SettingUpRefData.fm

48 Create a Custom Library 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) 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. MicroSeq ID Analysis Software Version 1.0 Getting Started Guide 3-5 August 14, :07 pm, 03_SettingUpRefData.fm

49 Chapter 3 Scientist: Set Up Analysis Information c. Select 500KitCustomLibrary.fsta. d. Click Import. 7. Click OK to acknowledge the import. 8. The sequences in the AB_Bacterial500CustomLibrary 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. 3-6 MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, 03_SettingUpRefData.fm

50 Create a Custom Library 9. 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. MicroSeq ID Analysis Software Version 1.0 Getting Started Guide 3-7 August 14, :07 pm, 03_SettingUpRefData.fm

51 Chapter 3 Scientist: Set Up Analysis Information Create 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 Getting Started Guide, follow the specifications given in the tasks 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. Use the MicroSeqID Manager Open a New Master Analysis Protocol IMPORTANT! To create a master analysis protocol, you must access the Analysis Protocol Editor from the MicroSeqID Manager. This ensures that the protocol will be available for selection in other projects. The master analysis protocol is not applied any projects that are reanalyzed. To open a new master analysis protocol: 1. In the MicroSeq ID Analysis main window, open the MicroSeqID Manager window: Select Tools > MicroSeqID Manager, or click (MicroSeqID Manager). 2. Select the Analysis Protocols tab. 3-8 MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, 03_SettingUpRefData.fm

52 Create a Master Analysis Protocol 3. Click New to open the Analysis Protocol Editor, then select the General tab. 4. In the Name field, type GSG_AnalysisProtocol (spaces aren t allowed; use the underscore character). 5. In the Comments field, type New master analysis protocol for the Getting Started Guide. MicroSeq ID Analysis Software Version 1.0 Getting Started Guide 3-9 August 14, :07 pm, 03_SettingUpRefData.fm

53 Chapter 3 Scientist: Set Up Analysis Information 6. Continue with Specify Basecalling Settings below. IMPORTANT! Do not click OK until you complete all the tabs in the Analysis Protocol Editor. Specify 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: a. In the Basecaller drop-down list, select Basecaller-3100POP6SR.bcp. b. In the DyeSet/Primer drop-down list, select DT3100POP6{BD}v2.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 Specify Mixed Bases Settings below. IMPORTANT! Do not click OK until you complete all the tabs in the Analysis Protocol Editor MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, 03_SettingUpRefData.fm

54 Create a Master Analysis Protocol Specify 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. You 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. 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 Specify Clear Range Settings on page IMPORTANT! Do not click OK until you complete all the tabs in the Analysis Protocol Editor. MicroSeq ID Analysis Software Version 1.0 Getting Started Guide 3-11 August 14, :07 pm, 03_SettingUpRefData.fm

55 Chapter 3 Scientist: Set Up Analysis Information Specify Clear Range Settings 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. 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. 4. Check that the completed Clear Range tab looks like the one shown below. 5. Continue with Specify Filter Settings below. IMPORTANT! Do not click OK until you complete all the tabs in the Analysis Protocol Editor MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, 03_SettingUpRefData.fm

56 Create a Master Analysis Protocol Specify 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. 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. MicroSeq ID Analysis Software Version 1.0 Getting Started Guide 3-13 August 14, :07 pm, 03_SettingUpRefData.fm

57 Chapter 3 Scientist: Set Up Analysis Information 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 MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, 03_SettingUpRefData.fm

58 Analyst: Set Up a Project 4 4 This chapter describes tasks an Analyst performs with the MicroSeq ID Analysis software. A brief tutorial is provided for setting up a project. This chapter covers: Review Requirements and Workflow Log In Customize the Display Settings Create a Project Analyze the Project Data Evaluate Results: Analysis QC Report Evaluate Results: Library Search Report Edit Data in the Analyzed Project Export and Print Reports MicroSeq ID Analysis Software Version 1.0 Getting Started Guide 4-1 August 14, :07 pm, 04_SettingUpProjects.fm

59 Chapter 4 Analyst: Set Up a Project Review Requirements and Workflow User Privileges User Accounts Analysis Information Analyst Workflow 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. Before you can log in to the software for the first time, an Administrator must create a user account for you (Chapter 2). 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 (GSG_AnalysisProtocol, page 3-8). 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 4-6). Analyst: Set up a project Analyze the project (page 4-12). Evaluate the results (pages 4-15 and 4-20). Edit data in the analyzed project (page 4-24). Export or print reports (page 4-28). 4-2 MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, 04_SettingUpProjects.fm

60 Log In Log 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. 2. Enter your user name and password, then click OK. Customize 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. In the MicroSeq ID Analysis main window, select Analysis > Display Settings to open the Display Settings window. 2. Select the General tab. 3. Note that your user name automatically appears in the Display Settings For User field. You cannot edit this field. Your settings are saved under your name and applied each time you log in. 4. In the Comments field, type My display settings. MicroSeq ID Analysis Software Version 1.0 Getting Started Guide 4-3 August 14, :07 pm, 04_SettingUpProjects.fm

61 Chapter 4 Analyst: Set Up a Project 5. Select the Bases tab and choose the font, scale, and colors as desired. 6. Select the Electropherogram tab and choose the scaling and axes as desired. 4-4 MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, 04_SettingUpProjects.fm

62 Customize the Display Settings 7. Select the Views tab and choose the General, Sample, and Specimen Views as desired. 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. MicroSeq ID Analysis Software Version 1.0 Getting Started Guide 4-5 August 14, :07 pm, 04_SettingUpProjects.fm

63 Chapter 4 Analyst: Set Up a Project Create a Project Creating a project requires that you: Select project settings Create new specimens and import sample files View the project setup summary Select 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 500KitProject. 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. 4-6 MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, 04_SettingUpProjects.fm

64 Create a Project 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 Libraries Available field, select Bacterial500Lib. 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. 7. Check that the completed Select Project Settings dialog box looks like the one shown below. 8. Continue with Create a New Specimen and Import Sample Files on page 4-8. MicroSeq ID Analysis Software Version 1.0 Getting Started Guide 4-7 August 14, :07 pm, 04_SettingUpProjects.fm

65 Chapter 4 Analyst: Set Up a Project Create 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. The next step in creating a project is to 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: MicroSeq041703_C03_H_5F_05.ab1 MicroSeq041703_D03_H_535R_07.ab1 Options for Naming Specimens You can assign a specimen name: Manually, by clicking New Specimen, or Automatically, by entering the desired Start and/or End delimiter to extract a specimen name from the sample name or the sample file name. Assign a specimen name manually Assign a specimen name automatically For this Getting Started Guide, follow the procedure on page 4-9 to assign the specimen name manually. For information on assigning 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. 4-8 MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, 04_SettingUpProjects.fm

66 Create a Project Creating Specimens and Importing Sample Files To create specimens and import sample files: 1. In the Select Project Settings dialog box of the New Project Wizard, click Next. The Create Specimens dialog box appears. 2. Click New Specimen. A new specimen (Specimen1) is added to the Samples to Add section. Note: The default specimen name (Specimen1) can only be changed in the Project Navigator pane after the project has been created. You will perform this step after project analysis; see step 2 on page In the Files tab, navigate to and select the 500Ecoli sample folder: drive letter\appliedbiosystems\microseqid\tutorial Data\ Bacterial500Samples\500EColi MicroSeq ID Analysis Software Version 1.0 Getting Started Guide 4-9 August 14, :07 pm, 04_SettingUpProjects.fm

67 Chapter 4 Analyst: Set Up a Project Sample files for this Getting Started Guide 4. Click Add Sample. The sample files in the 500Ecoli sample folder are added to Specimen1 in the Samples To Add section. 5. Continue with View the Project Setup Summary on page MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, 04_SettingUpProjects.fm

68 Create a Project View 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 Analyze the Project Data on page MicroSeq ID Analysis Software Version 1.0 Getting Started Guide 4-11 August 14, :07 pm, 04_SettingUpProjects.fm

69 Chapter 4 Analyst: Set Up a Project Analyze the Project Data After specifying the project settings and creating a new specimen (pages 4-6 to 4-11), analyze the project data. 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 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 MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, 04_SettingUpProjects.fm

70 Analyze the Project Data 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. 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 appears. 2. In the Project Navigator pane, change the default specimen name: a. Double-click the default specimen name (Specimen1). b. Type Uknown_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 (500KitProject) to view the Library Search Results (that is, the % Match results for all the specimens). MicroSeq ID Analysis Software Version 1.0 Getting Started Guide 4-13 August 14, :07 pm, 04_SettingUpProjects.fm

71 Chapter 4 Analyst: Set Up a Project b. The specimen name (Unknown_1) to view the Layout and Assembly Views for that specimen. 4. Continue with Evaluate Results: Analysis QC Report on page MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, 04_SettingUpProjects.fm

72 Evaluate Results: Analysis QC Report Evaluate Results: Analysis QC Report To verify that the sample files passed each step of the analysis process, you need to evaluate the information provided in the Analysis QC Report. View the Analysis QC Report To view the Analysis QC Report: 1. In the Project Navigator pane of the Project window, select the project name (500KitProject). Select project name 2. Select Analysis > Report Manager to open the Report Manager for the 500KitProject. 3. In the Reports navigation pane, select Analysis QC Report. If you cannot see all the text in the Reports pane, click Report Settings, then select Wrap Text. If desired, you can display the Project View beneath the Analysis QC Report. From the toolbar, select Window > Tile. 4. Review the data in the Specimen Analysis section. See Interpret the Specimen Analysis Section on page MicroSeq ID Analysis Software Version 1.0 Getting Started Guide 4-15 August 14, :07 pm, 04_SettingUpProjects.fm

73 Chapter 4 Analyst: Set Up a Project 5. Review the data (if any) in the Sample Analysis section. See Interpret the Sample Analysis Section on page Continue with Evaluate Results: Library Search Report on page Interpret the Specimen Analysis Section In the Specimen Analysis section displays a table that lists for each specimen: Specimen - The name of the specimen. Note: The specimen name is hyperlinked to the Specimen View. # Samples - The number of sample files in the specimen Basecalling, Filter, and Assembly columns - The status of these analysis steps. See Analysis Quality below. Specimen Score - Average consensus quality value (QV). See Specimen Score on page 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 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. Table 4-1 Analysis quality icons Icon Green square Description Complete: All of the sample files in the specimen successfully completed the analysis step. Symbol Linked to 4-16 MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, 04_SettingUpProjects.fm

74 Evaluate Results: Analysis QC Report Table 4-1 Analysis quality icons Icon Yellow triangle Red octagon Description Partial output: At least one of the sample files in the specimen did not successfully complete the analysis step. No output: 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 Partial or No output icon, clicking the icon links you to the affected sample files in the Sample Analysis section. See page 4-18 for more information. Specimen Score 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 (Pe) Where: Pe is the probability of error of the basecall The range of a QV is 1 (low confidence) to 50 (high confidence). Values above 30 indicate high-quality results. Note: Mixed basecalls yield lower sample QVs than pure basecalls. Table 4-2 on page 4-18 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. MicroSeq ID Analysis Software Version 1.0 Getting Started Guide 4-17 August 14, :07 pm, 04_SettingUpProjects.fm

75 Chapter 4 Analyst: Set Up a Project Table 4-2 Probability of basecall errors QV Pe QV Pe 10 a 10% % 20 a 1% 40 a 0.01% % % 30 a 0.1% 50 a 0.001% a. 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: (Number of bases that match / Length of alignment) 100 Where: Number of bases that match = Number of matching bases in the pairwise alignment between consensus sequence and the library search hit. Length of alignment = Length of pairwise alignment without considering leading gaps and trailing gaps. Interpret the Sample Analysis Section If the Specimen Analysis section displays the Partial or No output icon (see Table 4-1 on page 4-16) 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: Sample - The name of the failed sample file Step - The analysis step at which the failure occurred: basecalling, filtering, or assembling Description - A detailed description of the particular analysis failure 4-18 MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, 04_SettingUpProjects.fm

76 Evaluate Results: Analysis QC Report In Figure 4-1 on page 4-19 for example, clicking (Partial output icon) under the Filter column for Specimen1 links you to the Sample Analysis section. The Sample Analysis section displays the failed sample file (MicroSeq041703_C03_H_5F_05) and a description of the failure. Hyperlink Figure 4-1 Description of failure for Specimen1 Hyperlinks to Viewing 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 (Figure 4-2) to open the Sample View for the selected sample. Figure 4-2 Using the hyperlink to open the Sample View MicroSeq ID Analysis Software Version 1.0 Getting Started Guide 4-19 August 14, :07 pm, 04_SettingUpProjects.fm

77 Chapter 4 Analyst: Set Up a Project Evaluate 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: Examine results for all matches. Determine if a match was made against a validated (proprietary) library or a custom library. View phylogenetic relationships between the unknown and the top matches in a Neighbor Joining Tree. 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 (500KitProject). Select project name 2. Select Analysis > Report Manager to open the Report Manager for the 500KitProject. 3. In the Reports navigation pane, select Library Search Report MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, 04_SettingUpProjects.fm

78 Evaluate Results: Library Search Report If you cannot see all the text in the Reports pane, click Report Settings, then select Wrap Text. You can display the Project View beneath the Library Search Report. From the toolbar, select Window > Tile. 4. Review the data in the Hit List section. See Interpret the Hit List Section on page Review the data in the Concise Alignment section. See Interpret the Concise Alignment Section on page Review the data in the Phylogenetic Tree section. See Interpret the Phylogenetic Tree Section on page Continue with Edit Data in the Analyzed Project on page Interpret the Hit List Section In the Hit List section, the report lists for each specimen: Specimen - The name of the specimen Library - The name of the library searched for possible matches Sequence Name - A sequence 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 # of Bases Searched - The number of bases searched (clear range) MicroSeq ID Analysis Software Version 1.0 Getting Started Guide 4-21 August 14, :07 pm, 04_SettingUpProjects.fm

79 Chapter 4 Analyst: Set Up a Project Total Mismatches - The number of bases in the consensus sequence that did not match the library sequence Interpret 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 base present in the library sequence for that position For example, in the Concise Alignment section shown below, the specimen Unknown_3 was matched to the library sequence Brevibacterium casei. The mismatches occurred at several positions (49,?, 50, etc.). The bases called in the consensus sequence for those positions are C, -, C, etc. The bases present in the library sequence for those position are T, G, G, etc. Note: A? indicates that there is a gap in the aligned consensus sequence. Interpret 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 MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, 04_SettingUpProjects.fm

80 Evaluate Results: Library Search Report 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. For example, in the Phylogenetic Tree shown below, the specimen Unknown_1 is the same species as its closest library match, but may be a different serotype, biovar, strain, and so on. MicroSeq ID Analysis Software Version 1.0 Getting Started Guide 4-23 August 14, :07 pm, 04_SettingUpProjects.fm

81 Chapter 4 Analyst: Set Up a Project Edit Data in the Analyzed Project When to Edit Data How to Edit Data Edit a Consensus Sequence After analyzing and evaluating your own projects, edit the data if: An analysis step (basecalling, filtering, or assembling) fails. See Analysis Quality on page A Specimen Score value is below 30. See Specimen Score on page 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. 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 to 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 MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, 04_SettingUpProjects.fm

82 Edit Data in the Analyzed Project Click a specimen name to display the Specimen View. 5. In the Specimen Analysis section, double-click a specimen name (left-most column) to display the Specimen View for that specimen. 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. Low (red) and medium (yellow) QV bars MicroSeq ID Analysis Software Version 1.0 Getting Started Guide 4-25 August 14, :07 pm, 04_SettingUpProjects.fm

83 Chapter 4 Analyst: Set Up a Project 7. Check for medium (yellow) or low (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. 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 (Show/Hide All Electropherograms) in the Project window toolbar. 9. Make bases changes per 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. See Reanalyze and Save the Project on page Edit the Sample File To edit the Sample File: 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 > Sample Manager to open the Sample Manager for your project MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, 04_SettingUpProjects.fm

84 Edit Data in the Analyzed Project Only the Administrator and Scientist user groups can change analysis protocols. The Edit Analysis Protocol and Apply Analysis Protocol buttons are greyed out for the Analyst user group. 4. For the sample file you want to edit, you may modify the following fields: Sample Name BaseCaller DyeSet/Primer Spacing Peak1 Start Stop Note: For a description of each of these fields, refer to the MicroSeq ID Analysis software Online Help. Press F1, or select Help > Contents & Index. Go to the Index tab and type sample manager, then select parameters. 5. Click OK. The changes are applied to the selected sample file. Note: If the Audit Trail state is On, the Audit Reason Editor is displayed. Select a reason, type a comment, then click OK. 6. Reanalyze your project. See Reanalyze and Save the Project on page MicroSeq ID Analysis Software Version 1.0 Getting Started Guide 4-27 August 14, :07 pm, 04_SettingUpProjects.fm

85 Chapter 4 Analyst: Set Up a Project Reanalyze and Save the Project 1. In the Project window, click (Analyze samples) to reanalyze the project. 2. Re-evaluate the Analysis QC Report (see page 4-15). 3. Re-evaluate the Library Search Report (see page 4-20). 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. Export and Print Reports After editing data and reanalyzing the project, you can customize, then export or print the following reports: Analysis QC Report Library Search Report Audit Trail Report For this Getting Started Guide, follow the tasks below to customize, export, and print the Analysis QC Report for the 500KitProject. Customize the Report To customize the Analysis QC Report for the 500Kit Project: 1. Select File > Open Project and select the 500KitProject. 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 MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, 04_SettingUpProjects.fm

86 Export and Print Reports Click 5. Click Report Settings to display the Report Settings dialog box. 6. Set the options 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. 7. Click okay to enable the options and return to the Analysis QC Report. MicroSeq ID Analysis Software Version 1.0 Getting Started Guide 4-29 August 14, :07 pm, 04_SettingUpProjects.fm

87 Chapter 4 Analyst: Set Up a Project Export the Report To export the Analysis QC Report for the 500Kit 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 (500KitProject_Analysis_QC_Report). 4. Select a file type from the drop-down menu: PDF HTML XML TXT 5. Click Export. Print the Report To Print the Analysis QC Report for the 500Kit Project: 1. In the Analysis QC Report, select File > Print to open the Print Report dialog box. 2. Select the paper orientation and size MicroSeq ID Analysis Software Version 1.0 Getting Started Guide August 14, :07 pm, 04_SettingUpProjects.fm

88 Export and Print Reports 3. Optionally, click Header/Footer to customize the header and footer. 4. Optionally, click Print preview. to see the report before printing. 5. Click Print. 6. Select a printer, specify the page range, then click OK. MicroSeq ID Analysis Software Version 1.0 Getting Started Guide 4-31 August 14, :07 pm, 04_SettingUpProjects.fm