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ViroSeq®

HIV-1 Genotyping
Software v3.0
For use with the ViroSeq
HIV-1 Genotyping System v2.0

Software Manual

4J94-28

5003698 Rev. A

Celera Corporation
1401 Harbor Bay Parkway
Alameda, CA 94502
USA
ABBOTT
1300 E. Touhy Avenue
Des Plaines, IL 60018
USA

FPO
Robert Lue and Alain Viel, Biovisions at Harvard University
Contents

Preface...................................................................................................................................1
Introduction.......................................................................................................................... 1
Use of the ViroSeq HIV-1 Genotyping Software.................................................................. 1
How to Use This Manual ..................................................................................................... 1
Safety .................................................................................................................................. 1
Text Conventions................................................................................................................. 2
Key to Symbols.................................................................................................................... 2
How to Obtain Services and Support .................................................................................. 2

Section 1: ViroSeq Software Requirements...........................................................................3


Materials Required .............................................................................................................. 3
Computer Requirements for ViroSeq Software v3.0 ........................................................... 3
External Software Requirements......................................................................................... 3

Section 2: Installation, Activation, and Settings .....................................................................6


Installation and Uninstallation.............................................................................................. 6
Registration and Activation.................................................................................................. 6
Initial Set up......................................................................................................................... 7

Section 3: Overview ............................................................................................................. 12


ViroSeq HIV-1 Genotyping Software Window ................................................................... 12
Sample Trace View Window.............................................................................................. 15
Sample Report Window..................................................................................................... 19

Section 4: Sample Data Analysis Procedure ....................................................................... 21


Recommended Workflow .................................................................................................. 21

Section 5: Bibliography ........................................................................................................30

Appendix A: User Account Management ............................................................................. 31


Software License Renewal ................................................................................................ 31
Manage Users ................................................................................................................... 31

Appendix B: Insertions and Deletions .................................................................................. 33


Indel Symbol...................................................................................................................... 33
In-frame Insertions and Deletions...................................................................................... 33
Out-of-frame Insertions and Deletions............................................................................... 33
Three-base Deletion Example ........................................................................................... 33
Insertion Adjustment for Multiples of Three ....................................................................... 34
Auto-correction .................................................................................................................. 36

ViroSeq HIV-1 Genotyping Software i


Appendix C: Data Management .......................................................................................... 37
Sample Data File Management ......................................................................................... 37
Archiving Analyzed Data.................................................................................................... 38
Deleting Samples, Projects, and Repositories................................................................... 38
Export to File Formats........................................................................................................ 39

Appendix D: Example Data Set........................................................................................... 40


Drug-Resistance ................................................................................................................40
Edit .................................................................................................................................... 40
INDEL ............................................................................................................................... 41
HIV-3.................................................................................................................................. 42
Example of a Sample Report ............................................................................................. 43

Appendix E: Troubleshooting .............................................................................................. 49


Software Messages and Warnings .................................................................................... 50

Appendix F: Glossary .......................................................................................................... 53

ii ViroSeq HIV-1 Genotyping Software


Preface •

Bibliography (Section 5)
ViroSeq software user account management (Appendix A)
• Information about insertion and deletion mutations (Appendix B)
• Data management (Appendix C)
• Information about the Example Data Set (Appendix D)
• Troubleshooting and software messages (Appendix E)
• A glossary of terminology used within this manual (Appendix F)
Introduction Example Data Set
The ViroSeq® HIV-1 Genotyping Software v3.0 is designed for use with the ViroSeq
HIV-1 Genotyping System v2.0, and may be used in place of the ViroSeq HIV-1 The ViroSeq HIV-1 Genotyping Software is pre-loaded with a data set composed of
Genotyping Software v2.8. Refer to the applicable operator’s manual or instructions common examples. Refer to Example Data Set on page 40.
for use for additional information regarding the assay and instrumentation:
• ViroSeq HIV-1 Genotyping System v2.0 Operator’s Manual, For Use With the
ABI PRISM® 3100/3100-Avant Genetic Analyzers and ViroSeq HIV-1
Safety
Genotyping System Software v2.8 (REF 4J94-12)
• ViroSeq HIV-1 Genotyping System v2.0 Operator’s Manual, For Use With the User Attention Words
Applied Biosystems 3130/3130xl Genetic Analyzers and ViroSeq HIV-1 User attention words used within this manual imply a particular level of
Genotyping System Software v2.8 (REF 4J94-57) recommended user observation or action.

Note: Calls attention to useful information.


Additional Reference
IMPORTANT! Indicates information that is necessary for proper
The ViroSeq Algorithm Advisor™ is packaged with the ViroSeq HIV-1 Genotyping
operation and accurate or safe usage.
Software v3.0. The ViroSeq Algorithm Advisor includes the proprietary algorithm
that determines the relative impact of mutations in the protease and reverse Indicates a potentially hazardous situation, which, if not
transcriptase genes and the development of resistance to antiretroviral drugs. It also avoided, may result in minor or moderate injury. This
informs users of any changes to the software or to the algorithm used in the software. symbol may also be used to alert against unsafe
practices.

Use of the ViroSeq HIV-1 Genotyping Computer Workstation Safety


Software Correct ergonomic configuration of your computer workstation can prevent stress
• The ViroSeq software is for use only by trained operators. induced effects such as fatigue, pain, and strain. Minimize or eliminate these effects
• Accurate and reliable results are dependent on good sequence quality. High on your body by designing your workstation to promote neutral or relaxed working
background and noisy data can interfere with precise basecalling. positions.

• Understanding and using the editing recommendations found within this MUSCULOSKELETAL AND REPETITIVE
document will assist in making more accurate base calls, and may help identify MOTION HAZARD. These hazards are caused by
mutations that otherwise may be missed. potential risk factors that include, but are not limited to,
• Refer to the applicable operator’s manual or instructions for use for additional repetitive motion, awkward posture, forceful exertion,
product limitations. holding static unhealthy positions, contact pressure, and
other workstation environmental factors.

How to Use This Manual Use equipment that comfortably supports a neutral working position and maintains
adequate accessibility to the keyboard, monitor, and mouse.
Position keyboard, mouse, and monitor to promote a relaxed body and head posture.
Purpose of This Manual
The ViroSeq HIV-1 Genotyping Software v3.0, Software Manual provides
instructional information on the use of the ViroSeq HIV-1 Genotyping Software and
is designed to be used in conjunction with the applicable ViroSeq HIV-1 Genotyping
System v2.0 operator’s manual or instructions for use.
For In Vitro Diagnostic Use.

How to Navigate This Manual


This manual is designed as a guide for both new and experienced users on the use of
the ViroSeq HIV-1 Genotyping Software. The information is organized as described
below:
• ViroSeq software requirements (Section 1)
• ViroSeq software installation, activation, and settings (Section 2)
• An overview of the ViroSeq software (Section 3)
• The recommended sample data analysis workflow and procedure (Section 4)

ViroSeq HIV-1 Genotyping Software 1 of 54


Text Conventions
This manual uses the following conventions:
• Bold indicates user action or is also used to clarify a software element or user
entry. 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:
The dialog box will close without saving any information.
• A right arrow bracket (>) separates successive commands that denote a software
path or a drop-down menu. For example:
Select Programs > Celera > ViroSeq HIV v3.0.
From the drop-down menu, select Tools > Import... > Browse.

Key to Symbols

For In Vitro
= Diagnostic Use = Catalog Number

Celera Product Consult Instructions


= Part Number = For Use

= Manufacturer = Distributor

How to Obtain Services and Support

US Sales and Support


Toll Free: +1 800-553-7042

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Section 1: ViroSeq External Software Requirements
These are required steps that must be performed prior to using the ViroSeq HIV-1
Software Requirements Genotyping Software v3.0 to analyze sample data. Follow the appropriate
procedures below, depending upon the genetic analyzer instrument platform used for
sequencing.

Applied Biosystems 3130 Series Genetic Analyzers


The ViroSeq HIV-1 Genotyping Software v3.0 supports data files (.ab1) generated
by Applied Biosystems 3130 series Genetic Analyzers with Data Collection v3.0
and Sequencing Analysis Software v5.2 or above, with KB Basecaller v1.2 or above.
Materials Required A change to the ViroSeq 3130/3130xl Data Collection Analysis Protocol
Refer to the applicable operator’s manual or instructions for use for a complete list requirements in the ViroSeq HIV-1 Genotyping System v2.0 Operator's Manual
of required materials. (REF 4J94-57) under “To Create a ViroSeq Analysis Protocol” or Instructions For
Use (REF 4J94-58), “To Create a ViroSeq Analysis Protocol” is required to enable
Description mixed base calls.
IMPORTANT! Enabling mixed base identification is required to ensure that
ViroSeq® HIV-1 Genotyping System Software v.3.0, with 4J94-27 mixed bases are properly identified by the ViroSeq HIV-1 Genotyping Software
• ViroSeq® Algorithm Advisor™ Protease and Reverse v3.0.
Transcriptase, For use with the ViroSeq HIV-1 Genotyping
System v2.0, First Edition Create a ViroSeq Analysis Protocol in Data Collection Software
ViroSeq HIV-1 Genotyping System Software v.3.0 4J94-28 1. Select GA Instruments > ga3130 or ga3130xl > Protocol Manager.

Software Manual, CD 2. In the Analysis Protocol section, click New. The Sequencing Analysis
Protocol Editor opens. Complete the Sequencing Analysis Protocol
Editor as described in the following steps.
For 3100/3100-Avant Users
3. Under the General tab:
Sequencing Analysis Software v5.2 or above, with KB™ Basecaller v1.2 or above.
a. Name: ViroSeqAnalysisProtocol_MixedBases
b. Description: optional
Computer Requirements for ViroSeq c. Sequence File Formats: uncheck all check boxes
4. Basecalling tab:
Software v3.0 a. Basecaller: KB.bcp
The following minimum requirements were validated for the installation and
b. DyeSet / Primer: KB_3130_POP6_BDTv1.mob
operation of the ViroSeq HIV-1 Genotyping Software v3.0.
c. Processed Data: True Profile
d. Ending Base: At PCR Stop
Configuration 1 Configuration 2
e. Quality Threshold: Do not assign N’s to Basecalls
Operating system requirements: Operating system requirements: 5. Mixed Bases tab:
® ®
• Microsoft Windows 7 • Microsoft Windows XP • Select the Use Mixed Base Identification check box with the default
Professional, service pack 1 (SP1), Professional, service pack 3, US- setting of ≥ 30%.
for 32-bit or 64-bit processor, US- English, French (France), and Dutch
English, French (France), and Dutch Version
Version
Computer Platform: Computer Platform:
• RAM – 1 GB for 32-bit processor or • 1 GB RAM or above
2 GB for 64-bit processor • Hard drive – 5 GB or more of free
• Hard drive – 5 GB or more of free space
space • Keyboard
• Keyboard • 2-button mouse with scroll wheel
• 2-button mouse with scroll wheel • Monitor supporting screen
• Monitor supporting screen resolution of 1024 x 768
resolution of 1024 x 768

ViroSeq HIV-1 Genotyping Software 3 of 54


6. Clear Range tab: 5. Mixed Bases tab:
• Uncheck all check boxes. • Select the Use Mixed Base Identification check box with the default
7. Click OK to return to the Analysis Protocol Manager -- DataStore setting of ≥ 30%.
window.
8. Click Done.
9. Analyze data using ViroSeq HIV-1 Genotyping Software v3.0

ABI PRISM 3100 Series Genetic Analyzers


Data files (.ab1) generated by the ABI PRISM 3100 Genetic Analyzer with Data
Collection v1.1 or v1.0.1 or the ABI PRISM 3100-Avant Genetic Analyzer with Data
Collection v1.0, must first be re-analyzed using the Sequencing Analysis Software
v5.2 or above, with KB Basecaller v1.2 or above, before being imported into the
ViroSeq HIV-1 Genotyping Software.
To re-analyze data files, create a ViroSeq analysis protocol with KB Basecaller and
enable mixed base calls .
IMPORTANT! Enabling mixed base identification is required to ensure that
mixed bases are properly identified by the ViroSeq HIV-1 Genotyping Software
v3.0.

Create a ViroSeq Analysis Protocol in Sequencing Analysis


Software
1. From the Main menu in the Sequencing Analysis Software, select 6. Clear Range tab:
Analysis > Analysis Protocol Manager. The Analysis Protocol Manager • Uncheck all check boxes.
- DataStore window opens. 7. Click OK to return to the Analysis Protocol Manager -- DataStore
2. Click New in the Analysis Protocol Manager - DataStore window and window.
create the ViroSeq Analysis Protocol by clicking on the appropriate tabs as 8. Click Done.
described in the following steps.
3. Under the General tab: Add Sample Files to the Sample Manager in Sequencing Analysis
a. Name: 3100_Reanalysis Software
b. Description: optional After the new protocol has been created, the 3100/3100-Avant collected sample files
c. Sequence File Formats: uncheck all check boxes. must be added.
4. Basecalling tab: 1. Click on the Add Samples button in the toolbar or select File > Add
Samples(s).
a. Basecaller: KB.bcp
2. Navigate to the location of the sample folder. The individual sample files are
b. DyeSet / Primer: KB_3100_POP6_BDTv1.mob
located within the sample folder.
c. Processed Data: True Profile
3. Select the desired sample files from the sample folder and click on the Add
d. Ending Base: At PCR Stop
Selected Samples button to add the sample files.
e. Quality Threshold: Do not assign N’s to Basecalls
4. Once the desired sample files have been added, click on the OK button in
the Add Samples dialog box to close the dialog box.

Re-analyze the Sample Files in Sequencing Analysis Software


After the sample files have been loaded into the Sample Manager, the files are
ready to be re-analyzed with the ViroSeq Analysis Protocol.
1. From the Main menu, select Analysis > Analysis Protocol Manager.
2. Select 3100_Reanalysis and click the Apply to All Samples button.
3. Click on the Done button.

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4. Verify that the following settings are applied to each sample:
• BC Check Box – box is checked
• PP Check Box – box is not checked
• P Check Box – box is not checked
• Basecaller – KB.bcp
• DyeSet/Primer – KB_3100_POP6_BDTv1.mob
• Matrix File – None
• Spacing – 0
• Peak 1 Location – 0
• Start Point – 0
• Stop Point – 0
5. Click on the Start Analysis button or select Analysis > Start Analysis.
6. Save the re-analyzed data by selecting File > Save All Samples or clicking
on the Save All Samples button from the toolbar.
7. After analysis, visually check:
• that the Spacing, Peak 1 Location, Start Point and Stop Point have
been defined by the software, and
• the color of the BC check box. The color indicates the analysis status.
8. Analyze data using ViroSeq HIV-1 Genotyping Software v3.0.

ViroSeq HIV-1 Genotyping Software 5 of 54


Section 2: Installation, Registration and Activation
The ViroSeq HIV-1 Genotyping Software must be registered and activated before

Activation, and Settings


use. After inputting and saving the registration information, the registration key file
is created. The registration key file is emailed to Celera with a request to issue a
license key file; refer to Request a License Key File on page 7. The license key is
required to activate the software.
IMPORTANT! Each license key is issued for use only with the computer system
that originated the registration information and generated the registration key file.
1. Launch the ViroSeq HIV-1 Genotyping Software by double-clicking on
the Desktop shortcut or from the Start > Programs > Celera > ViroSeq
HIV 3.0 menu to open the License & Registration dialog box.
Installation and Uninstallation 2. From the License & Registration dialog box, click on the Enter
Before proceeding with software installation, review Section 1: ViroSeq Software Registration Information... button to open the Registration Information
Requirements. If the computer system does not meet the minimum system dialog box.
requirements, the installation process will abort and an error message will display.
Ensure that a user with Administrator privileges is logged in to the system to enable
installation of the ViroSeq HIV-1 Genotyping Software.
Only one copy of the ViroSeq HIV-1 Genotyping Software v3.0 may be installed on
a given Windows 7 computer. For Windows XP users, the ViroSeq HIV-1
Genotyping Software v3.0 can be installed on the same system as the v2.8 software.
Note: Refer to Computer Requirements for ViroSeq Software v3.0 on page 3.

Install the ViroSeq Software


1. Close all desktop applications.
2. Insert the ViroSeq HIV-1 Genotyping Software CD into the CD drive.
Note: During the installation process, if a Destination Folder other than 3. Complete the registration information as indicated. Form fields marked with
the default folder (C:\Programs\Celera\ViroSeq HIV 3.0\) is selected, an asterisk (*) indicate fields in which information must be provided.
make note of the folder location. Navigation instructions within this
software manual refer to the default folder location, therefore, users using a
custom folder location should defer to their folder location.
3. The installer should start automatically. If it does not, perform the following
actions:
a. Double-click My Computer on the desktop (Windows XP) or click
Start, right-click on Computer, then select a drive for software
installation (Windows 7).
b. Right-click on the drive that contains the CD.
c. Click on the Open option from the pop-up window.
d. Double-click on SetupHIV.
4. Accept the terms of the End-user License Agreement and Third Party
Licenses to continue with software installation. Not accepting the terms will
terminate the installation process.
Note: The End-user License and Third Party Licenses are presented to the
end-user during the installation of the ViroSeq HIV-1 Genotyping Software.
When the installation process is complete, users may find the license
contents as text (.txt) files in the C:\Program Files\Celera\ViroSeq HIV
a. Institution – The name of the institution or company to which the
3.0 folder.
software is registered.
5. Follow the instructions on the installer screen to complete the software
b. Department (optional) – The name of the department that will use the
installation.
software.
c. Address 1 – The street address of the institution or company.
Uninstall the Software d. Address 2 (optional) – Additional street address information.
From the Windows Control Panel, select the Add or Remove Programs utility in e. City – The city in which the institution or company resides.
Windows XP or Uninstall a Program in Windows 7 to remove the ViroSeq HIV-1 f. State/Province – The state or province in which the institution or
Genotyping Software. company resides.
Workspaces and user-generated data are not removed when the program is g. Postal Code – The Postal or ZIP Code in which the institution or
uninstalled. User-generated folders and files may be managed using Windows file company resides.
management functions. h. Country – The country in which the institution or company resides.
i. First Name – The first name of the person to whom the software is
registered.

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j. Last Name – The last name of the person to whom the software is Workspace Launcher
registered.
The Workspace Launcher window opens after entering the license key (refer to
k. Title (optional) – The title/position of the person to whom the software Software Activation on page 7) or when launching the program for the first time.
is registered.
To launch the workspace every time the program is started, refer to General Settings
l. Phone Number – A contact phone number of the person to whom the
on page 9.
software is registered.
m. E-mail Address – The e-mail address of the person to whom the Workspace features and characteristics
software is registered.
• A workspace can be defined by an Administrator or Lab Technician.
4. After entering the registration information, click on the Save registration
• One workspace can be utilized by a single user (e.g., private) or multiple
file... button to open the Save As dialog box.
users, based upon the Windows access rights to a given folder location.
Note: The Save registration file... button is enabled once all of the
• Users accounts (e.g., Administrator or Lab Technician) are work-space
required fields are populated.
specific; a user has access to the workspace in which their account was
Note: If the OK button is selected prior to saving the registration file, the created.
Registration Information dialog box will close without saving any • A workspace cannot be deleted in the ViroSeq software (refer to Appendix C:
information.
Data Management for additional information).
5. Navigate to a location or folder to store the Registration.key file, then click • A workspace can be archived (refer to Appendix C: Data Management for
on the Save button. additional information).
Note: Clicking on the Save button more than once will generate an error
message. If this occurs, click on the OK button to close the dialog box. Create a Workspace
6. Click OK to close the Registration Information dialog box. 1. Double-click on the Desktop shortcut or select the program
from the Start > Programs menu. The first time the software is
started following software activation, the Workspace
Request a License Key File Launcher window opens.
1. Send the Registration.key file (see above) as an email attachment to
softwareregistration@celera.com.
2. A license key file will be sent via e-mail. Save the file to a location for use
in the activation step.
3. Proceed to Software Activation on page 7.

Software Activation
A license key file is required to activate the ViroSeq HIV-1 Genotyping Software.
Refer to Request a License Key File on page 7 for information on how to obtain a
license key.
1. Launch the software by double-clicking on the Desktop shortcut
or from Start > Programs > Celera > ViroSeq HIV 3.0 to 2. Click on the Browse button to open the Select Workspace Directory dialog
open the License & Registration dialog box. box. The default folder is the user’s Windows directory.
Navigate to a destination on a local drive or a shared network drive in which
the workspace will be located.

2. Click on the Browse... button to access the Open dialog box.


3. Navigate to the location that contains the license key file. Double-click on
the license key file, or click on the file and then click on the OK button.
4. The Workspace Launcher dialog box is then displayed. Proceed to Initial
Set up on page 7.

Initial Set up
The first step in setting up the ViroSeq software is to define a Workspace. A
workspace is a user-defined location that stores user information, user-preferences,
and data.

ViroSeq HIV-1 Genotyping Software 7 of 54


3. Click on the location in which the workspace will be located, then click on Create Multiple Workspaces
the Make New Folder button.
Follow the default user login instructions every time a new workspace is created as
the User ID and Password are workspace-specific (refer to Default User Login on
page 8).
Only one workspace may be open at a time, but there is no limit to the number of
workspaces that can be created as long the computer hard drive has adequate space.
When multiple workspaces are set up, the user can be prompted to select a
workspace when the software is started, based upon the selections under General
Settings (refer to General Settings on page 9).
To create additional workspaces, enable the Prompt for workspace at startup
option under General Settings. Exit from the program and then follow the steps in
Create a Workspace on page 7 to create a new workspace. Repeat steps to create
additional workspaces.

Default User Login


Note: Only one session of ViroSeq HIV-1 Genotyping Software v3.0 can be in
operation at any given time.
1. Following a new workspace setup, the ViroSeq Login dialog box is
4. In the highlighted area, input the folder name, then click on the OK button displayed. In the User ID and Password fields, input the default login
to return to the Workspace Launcher window or click on Cancel to exit information for an Administrator account as shown below:
without further changes.
• User ID: Administrator
• Password: ChangeMeNow
Note: The User ID and Password fields are case-sensitive.

2. The Change Password screen is displayed. Input a new password in the


5. Verify the pathway shown in the Workspace text box, then click on the OK Password and Confirm Password text fields. Click on the OK button to
button to proceed to the next step. proceed.
IMPORTANT! Do not rename the folder in the Windows environment as Note: The case-sensitive password must consist of at least 6 alphanumeric
the ViroSeq software will not recognize changes made to the folder name. characters. The underscore (_) symbol may be included in the password; no
other symbols are allowed.
3. To create an account for a laboratory technician or additional administrator
users, refer to Manage Users on page 31.

Settings
The next step in set up is to customize the settings:
• Analysis Settings
• General Settings
• Naming Configuration
• Report Settings
• Site Information

8 of 54 Software Manual
To begin, click the Change Settings button from the main window to open the The minimum requirement for a segment is 100 bases. If this minimum requirement
Settings dialog box. is not met after autotrimming has been performed, then a Status Message is
displayed for the sample (refer to Appendix E: Troubleshooting for additional
information).
To modify the Analysis Settings:
1. Click in the text field adjacent to Left Trim Threshold, Right Trim
Threshold, or Sliding Window Length.
2. Enter the new quality value that meets the settings described in the above
parameter section.
3. Press the Enter key or click the OK button.
Analysis Settings
Reference Sequence
The Analysis Settings are unique to each workspace. Sample files must be re-
analyzed after the Analysis Settings have been changed. The Reference Sequences section displays a text file representation of the reference
sequence (HXB-2, GenBank K03455).
The Analysis Settings dialog box is composed of the:
• Automatic Trimming Parameters General Settings
• Reference Sequences
The General Settings dialog box is used to enable or disable user prompts at the
• Restore Defaults button – restores the Analysis settings to their default application level, affecting all workspaces. To enable a selection, click the check
settings. box, then click on the OK button. To disable, deselect the check box, then click on
• OK button – applies the current settings. the OK button.
• Cancel button – closes the dialog box without applying any changes to Note: Only users with Administrator credentials are allowed to change these
settings. settings.
• Prompt for workspace at startup – when enabled, users are prompted to select
a workspace when the software is launched.
• Prompt to enter comments when saving changes – when enabled, a comments
field is displayed after selecting the Save button in the Sample Trace View.
Note: The comments entered when saving changes are displayed only in the
Sample History comments field.
• OK button – applies the current selections.
• Cancel button – closes the dialog box without applying any changes to settings.

Automatic Trimming Parameters


The Automatic Trimming Parameters section is used to specify the quality value
thresholds used for automatic trimming of segments. It contains the following fields:
• Left Trim Threshold – The quality value that must be met by the peaks on the
left end of a segment in order to be considered for assembly. The default setting
is 20. The quality value setting must be an integer ≥ 0 and ≤ 40.
• Right Trim Threshold – The quality value that must be met by the peaks on the
right end of a segment in order to be considered for assembly. The default setting
is 20. The quality value setting must be an integer ≥ 0 and ≤ 40.
• Sliding Window Length – Indicates the number of bases used to calculate the
average quality value. The default setting is 15. The sliding window length must
be an integer ≥ 5 and ≤ 20.
If the average quality value is less than the predefined threshold quality value, then
the first base in the window is trimmed and the window slides one more base. This
step is repeated until the average quality value is greater than or equal to the
threshold number.

ViroSeq HIV-1 Genotyping Software 9 of 54


Naming Configuration The sample name is identical in all seven file names, but the primer name is unique
for each primer segment as designated by the letter A, B, C, D, F, G, or H.
The ViroSeq HIV-1 Genotyping System Software v3.0 allows the flexibility to
analyze sample data files with varying file naming conventions. The Naming sample name
Configuration settings are used to identify the sample name and primer name
within the file names of imported sample data files.(.ab1). The Naming
Drug-Resistance_A.ab1
Configuration settings are defined for each workspace. Before importing a data set,
make sure that the Naming Configuration settings are appropriately constructed.
primer name
A data set for a single sample is composed of several sample files (.ab1) with unique
sequence primers. • For the sample name “Drug-Resistance”, the settings are shown below:
Note: The ViroSeq HIV-1 Genotyping Software v3.0 allows for one or more
underscores or other delimiters to separate the sample name and primer name.

Start Location (sample name and/or primer name)


1. Click on the Beginning of the file name radio button to indicate that the
software should start to parse the sample file name from the beginning of
the file name.
2. Click on the Delimiters radio button if the beginning of the sample file
name is followed by a delimiter. The four default options are:
• Period – Start Location, select Beginning of the file name.
• Dash The sample name appears at the very beginning of the file name and is not
• Underscore preceded by any delimiters.
• Comma – End Location, select Delimiters, _Underscore, and 1 Occurrence
Custom delimiters may be added by clicking in the Delimiters text field and The end of the sample name is identified by 1 occurrence of an underscore
inputting a new character. The new delimiter will be included in the drop- (_).
down menu for future settings. • For the primer name of “A”, the settings are shown below:
3. Enter the total number of delimiters that must be detected before starting the
sample file name.

End Location (sample name and/or primer name)


1. Click on the End of the file name radio button to indicate that the software
should stop parsing at the end of the sample file name.
2. Click on the Delimiters radio button if the end of the sample file name
immediately precedes a delimiter. The four default options are:
– Start Location, select Delimiters, _Underscore, and 1 Occurrence
• Comma
The primer name begins after 1 occurrence of an underscore (_) is
• Dash identified. Note that this is the same underscore that marks the end location
• Period of the sample name.
• Underscore – End of the file name, select End Location
Custom delimiters may be added by clicking in the Delimiters text field and The end of the primer name is also the end of the file name, without the
inputting a new character. The new delimiter will be included in the drop- occurrence of any delimiters.
down menu for future use.
Example 2
3. Enter the number of delimiters that must be detected before ending the
sample file name. A sample named “SAMPLE12_6” was analyzed using seven primers:
4. Click on the Apply button to apply the changes. – 2012-08-16_SAMPLE12_6__A_A05..ab1
5. Click on the Restore Defaults button to reapply the original settings. – 2012-08-16_SAMPLE12_6__B_A05.ab1
– 2012-08-16_SAMPLE12_6__C_A05.ab1
6. Click on the OK button to confirm changes or on the Cancel button to exit
without changes. – 2012-08-16_SAMPLE12_6__D_A05.ab1
– 2012-08-16_SAMPLE12_6__F_A05.ab1
Example 1
– 2012-08-16_SAMPLE12_6__G_A05.ab1
For example, a sample named “Drug-Resistance” was analyzed using seven primers: – 2012-08-16_SAMPLE12_6__H_A05.ab1
– Drug-Resistance_A.ab1 The sample name is identical in all seven file names, but the primer name is unique
– Drug-Resistance_B.ab1 for each primer segment as designated by the letter A, B, C, D, F, G, or H.
– Drug-Resistance_C.ab1
sample name primer name
– Drug-Resistance_D.ab1
– Drug-Resistance_F.ab1
– Drug-Resistance_G.ab1 2012-08-16_SAMPLE12_6__A_A05.ab1
– Drug-Resistance_H.ab1

delimiter occurrence Œ  Ž+ 

10 of 54 Software Manual
• For the sample name “SAMPLE12_6”, the settings are shown below: To change a logo:
1. Click on the Browse button to navigate to an acceptable graphic file (i.e.,
GIF, JPEG, JPG, PNG). The file size must be at least 16 x 16 pixels or not
greater than 48 x 48 pixels. Select the file and click on Open.
Note: A warning is displayed if the image size is invalid; the OK button is
disabled until an acceptable graphic file is selected.
2. Click on the Apply button.
3. Click on OK to close the Report Settings dialog box.

To restore default logo settings:


– Start Location, select Delimiters, _Underscore, and 1 Occurrence,
1. Click on the Restore Defaults button and then click on the Apply button.
The sample name is preceded by the date of analysis and separated by an
2. Click on OK to close the Settings dialog box.
underscore. The sample name begins after one occurrence of a delimiter,
_Underscore.
Site Information
– End Location, select Delimiters, _Underscore, and 3 Occurrence,
Site Information is used for input of the site-specific information to be incorporated
The end of the sample name is identified by the occurrence of the third
into sample reports. Site information is applied at the application level, affecting all
underscore (_) in the file name: the delimiter count is cumulative from the
workspaces. Refer to Report header on page 26.
beginning of the file name.
• For the primer name of “A”, the settings are shown below: When site information is revised, any new report will contain the updated
information; older reports must be regenerated before the updated information is
included.
Note: Only users with an Administrator credential can edit these settings.

– Start Location, select Delimiters, _Underscore, and 4 Occurrence,


The primer name begins after the fourth occurrence of an underscore (_) is
identified.
– End of the file name, select Delimiters, _Underscore, and 5 Occurrence
The end of the primer name is marked by the occurrence of the fifth
delimiter. The file name information following the fifth and final underscore
(e.g., plate position) is not relevant to the Naming Configuration settings.

Report Settings
Report Settings allows the user to select a custom logo for use in the sample report.
Changes made to the Report Settings are made at the application level, affecting all
workspaces. A change is effective for new reports generated after the change was
made.
Note: English is the only language currently available for sample reports.

ViroSeq HIV-1 Genotyping Software 11 of 54


Section 3: Overview Menu Bar
The menu bar provides the following menu and drop-down
selections:
• File – the drop-down menu contains Exit. Select Exit to close the ViroSeq
HIV-1 Genotyping Software.
• Tools – the drop-down menu contains Export Analyzed Sample, Import
Analyzed Sample, Import... and Change Password.

The ViroSeq® HIV-1 Genotyping Software v3.0 consists of three primary user – Select Export Analyzed Sample to export an analyzed sample file (.VAS)
interfaces: that may be used for assessment by a secondary reviewer or to archive the
analyzed data for a sample. The exported file (.VAS) can only be opened in
• the ViroSeq HIV-1 Genotyping Software window, or main window,
the ViroSeq HIV-1 Genotyping Software v3.0.
• the Sample Trace View window, and
– Select Import Analyzed Sample to import a sample file (.VAS) that has
• the Sample Report window. been previously analyzed and exported. Sample files can only be imported
For additional instructions on the use of the software features, refer to Appendix D: into a different project from which they were exported.
Example Data Set. – Select Import... to upload files (.ab1) into the software for analysis (note
that this provides the same function as the Import Source Files button).
– Select Change Password to replace the current password with a new
ViroSeq HIV-1 Genotyping Software password.
Window • Help – the drop-down menu contains About, License & Registration, and
Show Keyboard Shortcuts.
The ViroSeq HIV-1 Genotyping Software window, or main window, consists of
the: – Select About to display the information about the software (i.e., version,
copyright).
• menu bar
– Select License & Registration to access Registration Information. The
• main toolbar
license expiration date can also be viewed within 30 days of the expiration
• Explorer panel date.
• sample section – Select Show Keyboard Shortcuts to display a list describing functions for
• Audit Trail and Sample History use of the mouse, keyboard, and editing palette.

ViroSeq HIV-1 Genotyping Software Window


main toolbar
menu bar

sample section
Explorer panel

Audit Trail /
Sample History

12 of 54 Software Manual
Main Toolbar Explorer Panel
The main toolbar displays across the top of the window. The Explorer panel is located along the
left side of the main window and
displays the user-accessible repositories
and projects within the current
Note: The appearance of the toolbar buttons indicates when an item is currently workspace. The current Windows user
available (full color) for selection or not available (shades of grey). name and workspace file path is
displayed at the bottom of the panel
Main Toolbar Icons
when the Explorer panel or sample
Icon Name Description section is selected.

Delete Deletes a sample, project, or repository. Refresh button: located in the


upper right corner of the
Explorer panel, the Refresh
Add New Repository Creates a new repository. button is used to update the
view.
Add New Project Creates a new project.
Sample Section
Import Samples Imports data files into a project or a The sample section contains information about the imported sample files:
repository.
Analyze Samples Analyzes one or more selected sample
file(s) (.ab1).
Show Trace View Opens the Sample Trace View for one
or more selected sample file(s). For
unanalyzed sample files, performs an
analysis before opening the Sample
Trace View.
Enter Patient Information Allows for the entry of patient
information related to a selected • Project name – the name of the current project folder and the quantity of sample
sample file. Patient information is files within that folder are displayed at the top of the sample section.
incorporated into the sample report. • Sample Name – the imported sample name and the primer file names (.ab1)
associated with that sample.
Show Report Launches the Sample Report window
and creates a sample report for the • Path – the location of the imported sample within the workspace.
selected sample. • Date modified – the date that the sample file was last edited.
Export to file Exports file(s) in alternate file formats. • Review – following sample analysis, a colored box indicates a sample’s review
status. Refer to Table 1. Sample Review Status on page 23.
Show Consensus Sequence Displays the nucleotide and amino acid • Status – indicates whether a sample is not processed (e.g., not yet analyzed) or
sequences for a sample. analyzed.
Change Settings Contains the settings for the software. • Status Message – indicates the current sample status.

Manage Users Allows for the creation and


modification of the users in the current
workspace.

ViroSeq HIV-1 Genotyping Software 13 of 54


Audit Trail and Sample History Sample History
The Audit Trail and Sample History are displayed as two tabbed panels in the lower
right half of the main window.

Audit Trail

The Sample History tab displays recorded actions performed on the currently
selected sample. The information includes sample- and segment-level actions, such
as when a sample is imported, analyzed, and edited, when a segment is added or
The Audit Trail records application-level actions, such as login activity and Change deleted, when a sample-specific analysis settings change is made, and when patient
Settings activity. The Audit Trail is not workspace-specific. information is edited.
The Audit Trail records the Date, User, Action, Target, Old Value, and New Note: Actions that are not saved are not recorded in the Sample History.
Value fields. The left side of the Sample History tab displays the sample name and a summary of
• Date – the date the action was performed. when an action was performed (e.g., date, time) and the user who performed the
action. The summary can be expanded or hidden by clicking on the Windows
• User – the login name of the user who performed the action.
symbol adjacent to the sample name.
• Action – the type of action performed (e.g., login).
The right side of the tab displays the sample history detail table:
• Target – the target is dependent upon the type of action performed. For example,
• Description – the type of action performed (e.g., Imported).
the target for a login displays the login path, whereas the target for a Change
Settings action is displayed as the type of action performed (e.g., logo change, • Target – the sample or segment name on which the action was performed.
prompt for workspace). • Property/Position – the location of the sample (e.g., base number) affected by
• Old Value – the value before the action was performed. the action.
• New Value – the value after the action was performed. • Old Value – the value before the action was performed.
• New Value – the value after the action was performed.
Export Audit Trail and Archive Audit Trail
• Comments – displays comments for the selected sample. Comments are entered
The Audit Trail may be exported and/or archived as an un-
when changes are saved to a sample in the Sample Trace View window. Refer to
editable portable document format (PDF) file using the buttons
General Settings on page 9 for additional information.
or drop-down menu in the upper right corner of the panel.
• Export current audit trail to PDF: click on the white arrow button to open the Export Sample History
Export Audit Trail dialog box. Navigate to a folder of choice and click on the The Sample History may be exported as an un-editable portable
Save button. The exported file name consists of the software version, audit trail document format (PDF) file using the buttons or drop-down menu in
description, and the date range in the format of year, month, and day (e.g., the upper right corner of the panel.
VS3_Audit_Trail_20121101-20121105.pdf). Exporting an audit trail does not
• Export Sample History to pdf: click on the green arrow button to open the
affect the information presented in the Audit Trail panel.
Export Sample History dialog box. Select the data to be exported, then navigate
• Archive specified records and delete from the application: click on the red to a folder of choice and click on the Save button. The exported file name
arrow button to open the Archive Audit Trail dialog box. The audit trail history consists of the software version, sample history description, and the sample
can be archived by calendar weeks. Select a week to be archived and then click name. Exporting the sample history does not affect the information presented in
the Save button. The audit trail history from the selected week and any prior the Sample History tab.
weeks is archived. Audit trail entries occurring after the selected week(s) will
• Drop-down arrow: clicking on the drop-down
not be archived.
arrow accesses the Export Sample History
The exported file name consists of the software version and the calendar year option. Click on the option to access the export
week(s) (e.g., VS3_Audit_Trail_2012-43-2012-44.pdf). Archiving an audit trail dialog box.
deletes information presented in the Audit Trail panel, with the exception of the
current day’s activity and any entries made after the week(s) selected for
archival.
Note: If the audit trail entries are less than 7 days old, a message is displayed
to alert the user that “No messages are old enough to archive.”
• Drop-down arrow: clicking on the drop-down arrow
accesses the Export Audit Trail and Archive Audit
Trail menu. Click on a menu option to access the
export or archive dialog box.

14 of 54 Software Manual
Sample Trace View Window

 

 

Sample Trace View Window Icon Name Description


Next POI Jumps to the POI to the immediate right
The Sample Trace View window is displayed by selecting a sample and of the current position.
clicking the Show Trace View button from the main window or by double-clicking
on a sample. The Sample Trace View window is used to review and edit analyzed Last POI Jumps to the last (rightmost) POI within
data. The elements within the Sample Trace View window are integrated, working the sequence.
together to assist in the review and editing process:
Show Base Call Line Displays vertical reference lines in the
 Trace View toolbar Sample Trace View electropherograms.
 Navigation panel
Show Quality Values Displays the quality value (QV) bars for
 Editing palette and Position of Interest (POI) filters the individual bases in the Sample
 Mutations or Variants tables Trace View electropherograms.
 Reference and Consensus Panel Fill traces with color Displays the electropherogram peaks
 Sample Traces (Electropherograms) filled with color.

Selects one or more bases for display as


Show Traces A (Adenine)
Trace View Toolbar an electropherogram. All of the base
icons are selected in the default setting.
The Trace View toolbar displays across the top of the Sample Trace View window.
Show Traces C (Cytosine)
Each color-coded base has a
Note: Active icons are displayed in color; inactive icons are displayed in shades of
corresponding electropherogram trace
grey. Show Traces G (Guanine) color:
Icon Name Description • Adenine – Green
Save Edits Saves changes made by the user. Show Traces T (Thymine)
• Cytosine – Blue
• Guanine – Black
Show Report Launches the Sample Report window • Thymine – Red
and creates a sample report for the
Show POI Table Displays the position of interest tables.
selected sample.
First POI Jumps to the first (leftmost) position of
interest (POI) within the sequence. Show Keyboard Opens the Keyboard Shortcuts
Shortcuts window containing a list of key
Previous POI Jumps to the POI to the immediate left functions associated with the mouse,
of the current position. keyboard, and the editing palette.

ViroSeq HIV-1 Genotyping Software 15 of 54


Navigation Panel Area of No Coverage Indicator
If the Consensus Sequence has an area in which no data is available from the
navigation assembled segments, this is considered an area of no coverage. The area of no
bar coverage is indicated in the navigation bar by a dark grey shaded area. In the
example below, an area of no coverage occurs in the Protease gene as indicated by
assembled the dark grey shaded area.
segments
area of no coverage
guide line

The navigation panel provides a visual representation of the analyzed pol gene,
highlighting POIs for review in the electropherograms. The navigation panel is
displayed below the Trace View toolbar and consists of the navigation bar and the
assembled segments. The guide line provides a point of reference between the
navigation bar and the assembled segments, and corresponds to the highlighted base
position within the sample traces.

Navigation Bar

bar
guide POI

Assembled Segments
The primer segments are assembled to generate a consensus sequence. The
assembled segments are displayed in the lower portion of the navigation panel,
base position horizontal scale depicting segment overlap and the direction of primer extension. Each segment is
(codon position)
labeled with the source file (.ab1) name.
The navigation bar guides the user through the Sample Trace View window. Up to five rows of segments are displayed within the navigation panel. When
• Vertical yellow lines within the navigation bar indicate the location of POIs; the segments are deleted or added, the segments are adjusted and shifted vertically,
corresponding POIs in the consensus and reference sequences are highlighted in which may result in a segment being placed into a different row than shown in the
yellow. figure below.

• The horizontal scale indicates the codon position. An area of known single-coverage is present in the reverse transcriptase codon
region of 315 to 335 (RT315 - RT335).
• The bar guide points to the location of the base position and codon currently
selected in the sample traces. The guide can be moved in the following ways: There are four primers in the forward direction (i.e., A, B, C, D) and three primers in
the reverse direction (i.e., F, G, H).
– by clicking and dragging it along the bar,
– by clicking on the toolbar POI buttons;
– by clicking on a base within a segment or an electropherogram; and
– by clicking on a variant or mutation within the variant and mutation tables.
Note: A pink-highlighted segment provides a visual link to its corresponding pink-
Area of Single Coverage Indicator highlighted sample trace.
If the Consensus Sequence has an area in which data is available from only one IMPORTANT! A minimum of six valid segments is required for an accurate
segment, this is considered an area of single coverage. The area of single coverage is assessment of the patient sample. One of the missing segments can be A or D.
indicated in the navigation bar by a light grey shaded area. In the example below, an
area of single coverage occurs in the RT gene as indicated by the light grey shaded Remove and Add Segments
area. Segments can be removed and added from an assembled sample with the following
area of single coverage restrictions:
• The option to add segments is disabled if the sample already has seven
segments.
• A segment can be added only if the sample name is identical to the other
segments and the primer ID has not already been added.
• If only one segment remains, the option to remove segments is disabled.
Remove a Segment: Removed segments are not considered when the consensus
sequence is created.
1. Determine which segment is to be removed.
2. Right-click on the segment in the navigation panel to display the pop-up
menu.
3. Click on the Remove Segment option from the drop-down menu. The
remaining segments in the Sample Trace View may be vertically adjusted
to optimize the segment display.

16 of 54 Software Manual
Add a Segment: A maximum of seven segments per sample can be loaded at one Editing Base Calls
time.
Editing base calls includes inserting, deleting, and changing a base. When editing a
1. Right-click on one of the current segments in the navigation panel to display base in the segment sequence, the consensus sequence is automatically recalculated.
the pop-up menu. If a change is made to a base in the majority of the segments, the consensus sequence
2. Click on the Add Segment option to display the Import dialog box. is updated to reflect the majority base call.
Note: Adding or removing segments causes the sample to be re-analyzed Inserting a Base Into the Segment Sequence
and all previous edits are lost.
1. Identify and select the base in the segment sequence where a new base is to
3. From the Import dialog box, the segments that can be added appear in a be inserted.
black-colored font; non-viable options appear greyed-out. Double-click on a 2. While holding down the Alt key, click on the base to be inserted from the
segment to add it, or click on the segment and then select the Import button. editing palette. The selected base is inserted into the segment and all other
bases are shifted one space to the right.
3. Click on the Save button.
Editing Palette and POI Filters
Deleting a Base From the Segment Sequence
Editing Palette 1. Select a base call, then click on the DELETE button in the editing palette or
The editing palette is designed for the user to edit base calls at the segment level. click the Delete key on the keyboard.
The palette is composed of IUB letter codes for the four single nucleotides (i.e., A, 2. Repeat this process to delete additional base calls.
C, G, T) and mixtures of nucleotides.
3. Click on the Save button.

Changing a Base in the Segment Sequence


1. Select a base call.
2. Using the editing palette or the corresponding letter key in the keyboard,
click on the new base.
3. Repeat this process to change additional base calls.
4. Click on the Save button.

Undoing and Redoing Edits


IUB Codes The UNDO and REDO buttons allow a user to reverse the most recent user-made
edits.
A = adenosine U = uracil S = G or C D = A, G, or T
Undo edits
C = cytidine K = G or T W = A or T H = A, C, or T
• Select the UNDO button to reverse the most recent
G = guanosine M = A or C Y = C or T V = A, C, or G
change made in the Sample Trace View window.
T = thymidine R = A or G B = C, G, or T N = aNy base
Redo edits
Amino Acid Abbreviations • Select the REDO button to reverse the most recent
change made using the UNDO button.
Amino Acid Three Letters One Letter Note: If a change is reverted using the UNDO button and then
Alanine Ala A another change is made to a sequence, the REDO button will not
Arginine Arg R implement the reverted change.
Asparagine Asn N
Aspartic Acid Asp D Position of Interest (POI) Filters
Cysteine Cys C The POI filters are located in the upper right
Glutamic Acid Glu E corner of the Sample Trace View window.
Glutamine Gln Q Select one or more filters of a similar type (e.g.,
Glycine Gly G mutation, variant, edit) to customize the
contents of the Sample Trace View window
Histidine His H
and the Mutations or Variants tables. The
Isoleucine Ile I
number to the right of the POI filter indicates
Leucine Leu L
the total quantity of a given POI.
Lysine Lys K
Methionine Met M Mutation POI Filters
Phenylalanine Phe F • Drug Resistance Mutations – mutations
Proline Pro P identified within the consensus sequence that are used in the calculation of drug
Serine Ser S resistance.
Threonine Thr T • Additional Mutations – mutations identified that are not used in the calculation
Tryptophan Trp W of drug resistance.
Tyrosine Tyr Y
Valine Val V
Any Amino Acid Xaa X

ViroSeq HIV-1 Genotyping Software 17 of 54


Variant POI Filters • Un-confirm All – the stacked check box icon in the upper right corner can
• Mismatch – bases identified that differ between the consensus and reference be selected to unconfirm all of the previously confirmed nucleotide base
sequences. variants.
• Mixture – mixed bases (more than one nucleotide) identified at a single base Clicking on the column heading above the check boxes will sort the content of
position. the column.
• Insertion – identified base insertions.
• Deletion – identified base deletions. Reference & Consensus Panel
Edited POI Filter a
• Edited – user-edited bases.
b

Mutations or Variants Table c


The mutations or variants tables are displayed in the left-side of the Sample Trace d
View window. The type of tables displayed correspond to the POI filter selections.
e
Mutations Tables
f
The mutations tables are displayed when Drug
Resistance Mutations and/or Additional
Mutations are selected as POI filters. The a The codon position in the reference and consensus sequence.
tables display a summary of the Protease and
RT mutations identified within a sample. The
b Reference Translation – the amino acid sequence translated from the
total count for each is displayed in the table
heading. reference nucleotide base sequence.

• AA – Describes the amino acid mutation. c Consensus Translation – the amino acid sequence translated from the
• (R) – The presence of a blue letter “R” consensus nucleotide base sequence.
adjacent to the amino acid variant, indicates
a drug resistant mutation. d Reference Sequence – the reference nucleotide base sequence to which the
consensus nucleotide sequence is compared.

e Consensus Sequence – the consensus nucleotide base sequence is


determined by assembling the individual segment sequences and is aligned
to the reference nucleotide base sequence.
Yellow highlighted bases indicate the presence of a POI and correspond to
the vertical yellow POI lines displayed in the navigation bar. The check box
below a POI indicates when a base call has been confirmed (checked) or has
not yet been confirmed (unchecked).

f The average quality value (QV) for all of the bases at a given position.
Variants Tables
Legend
The variants tables are displayed when any of
the Variant and/or the Edited POI filter(s) are – Provides information for each adjacent row within
selected. The tables display a summary of the the Reference & Consensus panel.
Protease and RT variants identified within a – Base Count – the total number of bases detected
sample. The total count for each is displayed in within the Consensus Sequence.
the table heading. – QV below 20 – the number of bases within the
• Base – Describes the nucleotide base Consensus Sequence with a QV below 20
variants. – The histogram represents a tally of the QV, providing a visual indication of
• (E) – The presence of a blue letter “E” the overall QV distribution
adjacent to the nucleotide base variant is an • x-axis (horizontal), starting from left, QV of 1 to 60
edit marker and indicates that a user-edit
• y-axis (vertical), the base count for each QV
has been performed.
• Check boxes – a checked box indicates that
a nucleotide base variant has been
confirmed.

18 of 54 Software Manual
Sample Traces Maximize a segment panel
1. Double-click on a segment panel to maximize the segment panel view in the
Segment Sequence Panels Sample Trace View window.
Each primer segment sequence panel consists Note: Maximizing one panel will hide other panels from view.
of: 2. Double-click on the panel segment again to return to the previous view.
– the nucleotide bases within the
segment
– the direction of the segment (forward Sample Report Window
or reverse) The Sample Report window is displayed by right-clicking on an
– the name of the segment source file (.ab1) analyzed sample from the main window and selecting Show Report
– the segment sequence electropherogram or trace from the drop down menu, or by clicking on the Show Report button from either the
main window toolbar or the Sample Trace View toolbar. The Sample Report
Trimming a Segment window is used to review, print, and save reports. Reports are saved in PDF
(portable document format) files. The Sample Report window consists of the
Automatic Trimming: Automatic trimming of a segment is based upon predefined Sample Report toolbar and a sample report. For additional information on the
quality values (QV) selected in the Analysis Settings section. Automatic trimming
sample report, refer to Reports on page 26.
is performed on both ends of a segment.
The automatic trimming mechanism employs a sliding window to evaluate each base
for trimming. The average QV for the sliding window is calculated and, if the Sample Report Toolbar
average QV is less than the predefined threshold QV, the first base in the window is The Sample Report toolbar is displayed in the Sample Report window and is used
trimmed and the window slides down one base. This process is repeated until the to navigate, resize, save, and print the displayed report.
average QV becomes greater than or equal to the threshold value. For information on
how to set the automatic trimming parameters see Analysis Settings on page 9.
Manual Trimming: Manual trimming can be performed at the beginning or end of a
segment in the electropherogram view.

1. Identify a segment end to be trimmed. Button Name Description

• If the segment to be trimmed is on the left-end of the segment, right-click Print Opens the print dialog to produce a
on a base to the right of the area to be trimmed and select Trim to the hard copy printout of the report.
Left of selection.
Save as PDF Saves the report as a PDF (portable
• If the segment to be trimmed is on the right-end of the segment, right- document format).
click on a base to the left of the area to be trimmed and select Trim to the
Right of selection. First page Jumps to the first page of the report.
2. Click on the Save button.

Adjust the Electropherogram Display Previous page Accesses the page immediately
before the currently displayed page.
The mouse scroll wheel may be combined with key strokes to adjust the visual
representation of the sample traces.
Page Number Field Displays the page number of the
Adjust the horizontal display (shifting view, left and right) current page.
Use of this feature shifts all of the segment panels in unison.
• Click on any segment pane: Next page Accesses the page immediately
following the currently displayed
– Move the mouse wheel forward to cause the trace to scroll to the right.
page.
– Move the mouse wheel backward to cause the trace to scroll to the left.
Last page Jumps to the final page of the
Adjust the peak height report.
Use of this feature adjusts the peak heights within a selected panel.
Actual size Adjusts the report to display at
1. Click on a segment panel to select it. The selected panel displays a
100% scale.
highlighted border.
2. Hold down the Ctrl key and move the mouse wheel forward or backward. Fit page Adjusts the report to display one
• A forward motion decreases peak height. complete page in the Sample Report
window.
• A backward motion increases peak height.
Fit width Adjusts the report to fit the width of
Adjust the peak width the Sample Report window.
Use of this feature adjusts all peaks widths within all panels.
Zoom in Increases the displayed report in
1. Click on any segment panel. 25% increments, up to a maximum
2. Hold down the Alt key and move the mouse wheel forward or backward. of 200%.
• Move the mouse wheel forward to compress the horizontal trace view.
• Move the mouse wheel backward to expand the horizontal trace view.

ViroSeq HIV-1 Genotyping Software 19 of 54


Button Name Description
Zoom out Decreases the displayed report in
25% increments, down to a
minimum of 50%.
Zoom drop-down Displays the current zoom level
with a drop-down selection, from
50% to 200%.

20 of 54 Software Manual
Section 4: Sample Data Review Step Description

Analysis Procedure Basecaller Verify that the Basecaller file selected in the ViroSeq
Analysis Protocol was KB.bcp.

Sufficient Review the signal strength numbers for each base.


Signal Strength If the total A, C, G, and T signal strength value is below
400, inspect the raw and analyzed data and discard any
noisy data.

The recommended workflow and procedures for sample data analysis and output are Sequence IMPORTANT! Six of the seven segments must
described in this section Segments be successfully sequenced in order to proceed with
the analysis in the ViroSeq software. One of the
missing segments can be A or D.
Recommended Workflow
B. Repository
A data repository is used to store one or more projects. Multiple
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contain one or more projects.
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Create a New Repository
1. In the ViroSeq software, click on the Add New Repository button in the
main toolbar, or right-click in the Explorer panel and select Add New
Repository from the drop-down menu.
2. In the Data Repository dialog box, input a name for the repository in the
'&RQILUPILOH
1DPLQJ (,PSRUW'DWD Display Name text field.
)$QDO\]H'DWD
&RQILJXUDWLRQ )LOHV
VHWWLQJV
3. Click on the OK button.

Access an Existing Repository


In the ViroSeq software, select a previously created repository from the Explorer
panel.

*$VVHVVWKH +(GLW &RQILUP ,&UHDWHD


C. Project
$QDO\]HG'DWD $QDO\]HG'DWD 6DPSOH5HSRUW Project folders are created within a repository and are used to assist with
data management. Projects have the same access restrictions as the
repository in which they are located. Each project can contain one or more sets of
sample data.
A. Check the DNA Sequencing Analysis Data Files Create a New Project
Before analyzing the DNA Sequencing Analysis data with the ViroSeq HIV-1
1. Select a repository and then click on the Add New Project button in the
Genotyping Software v3.0, check the files for the following:
main toolbar, or right-click on the repository name and select Add New
Project option from the pop-up menu.
Review Step Description
2. In the New Project dialog box, input a name for the project in the Name
text field and click on the OK button.
File Names Verify that the sequence segment file names meet the
following requirements:
Access an Existing Project
• Sample names are identical across all seven segment file
names to ensure proper assembly of the sample file by Click on the Windows symbol ( + or >) adjacent to the repository name to expand
the ViroSeq software. the view and display project folders. Select a project from the existing folders.

• Each segment file has a unique primer name.


D. Confirm File Naming Configuration Settings
• One or more delimiters separate the sample name and
the sequence primer name. The Naming Configuration settings allow users to set the parsing elements that are
applied to imported data. Settings can be specified for both the sample file name and
DyeSet/Primer For 3100 genetic analyzers: verify that the DyeSet/Primer the primer name. Confirm that the Naming Configuration settings are appropriately
File file set in the Sequencing Analysis software was constructed for the sample data set before importing sample data. Refer to Naming
KB_3100_POP6_BDTv1.mob Configuration on page 10.
For 3130 genetic analyzers: verify that the DyeSet/Primer
file selected in the ViroSeq Analysis Protocol was
KB_3130_POP6_BDTv1.mob.

ViroSeq HIV-1 Genotyping Software 21 of 54


E. Import Data Files 6. Select all of the .ab1 files for each sample to be analyzed and click on the
Import button. Select files using one of the following methods:
Data consists of a set of imported DNA Sequencing Analysis files (.ab1);
data files are imported into a project folder. Set up the repository and • Import select files – Hold down the Ctrl key and click on individual file
project before importing a data set. After import of data files, enter patient names.
information. Up to 1,000 samples may be imported at one time. • Import a range of files – Hold down the Shift key and click on the first
IMPORTANT! Refer to External Software Requirements on page 3 for file in the list and then click on the last file to be imported.
instructions on how to prepare data files for use with the ViroSeq HIV-1 Genotyping • Import all files – Press Ctrl + A to select all sample files (.ab1) in the list.
Software v3.0.
1. Select a project and click on the Import Samples button or right-click and
select the Import Samples option in the pop-up menu to launch the Import
Samples dialog box.
2. From the Import Samples dialog box:
• Select the Include Subdirectories check box to enable the software to
search and retrieve .ab1 files from all subdirectories.
• Click on the Browse button to display the New source file(s) Location
dialog box.

3. Select the sample folder to be imported, then click on the OK button.


4. The .ab1 files located within the folder and subfolders are displayed in the
IMPORTANT! File names that do not meet the Naming Configuration
File Name column.
settings are displayed in a red-colored font within the Import Samples
dialog box. The settings within the Naming Configuration dialog box must
then be changed to match the file names. When the file names match the
Naming Configuration settings, the file names are displayed in a black-
colored font.
7. Imported files are displayed in the sample section of the main window. The
sample files can be expanded or hidden by clicking on the Windows symbol
adjacent to the sample name.

File Import Failure


An error message is displayed if one or more files fail to import. A file will not
import if any of the following conditions occur:
• The project contains a sample name that is the same as the file being
imported.
5. Click on the Naming Configuration button in the lower right side of the • The file is corrupt.
Import Samples dialog box. To facilitate data import, review the Naming • The Basecaller verification fails.
Configuration settings to confirm that they accurately describe the source
Refer to Troubleshooting on page 49 for additional information.
data file names. Refer to Naming Configuration on page 10 for more
information.
Patient Information
After importing data, input the patient information for each sample.
1. Access patient information:
• Select the sample and click the Enter Patient Information button, or
• Right-click on the sample and select the Enter Patient Information
option from the pop-up menu.
2. Input patient information as indicated in the dialog box:
Note: The information for items (a) to (j) below is limited to 32 characters
in length.
a. Accession Number – The identification number of the record.
b. Assay Operator Name – The name of the person who performed the
assay.
c. Institution Name – The name of the institution where the assay was
performed.
d. Physician – The name of the physician requesting the assay.
e. Field 1 – Additional information as required, up to 32 characters in
length.
f. Field 2 – Additional information as required, up to 32 characters in
length.

22 of 54 Software Manual
g. ID – The identification number for the patient. Re-analyze a Sample
h. First Name – The patient’s first name. A previously analyzed sample can be reanalyzed with different automatic trimming
i. Middle Name – The patient’s middle name. parameters. The revised automatic trimming parameters are applied to the selected
j. Last Name – The patient’s last name. sample and are not applied to all samples.
k. Gender – The patient’s gender. Note: Re-analyzed samples will not retain user-edits made prior to sample re-
l. Date of Birth and Date Drawn – The patient’s date of birth and the analysis.
sample collection date, respectively. The date field can be populated by Note: Ensure that the Sample Trace View window is closed before attempting to
manually inputting the date in the mm/dd/yyyy format or by clicking re-analyze a sample.
the Browse button next to the date field to access the pop-up Date 1. Right-click on the sample name from the sample section to display the pop-
Selection dialog box.
up menu.
1. Click on the Browse button to access the Date Selection dialog
2. Select Analysis Setup to edit the automatic trimming parameters, then
box.
select Re-analyze.
2. In the calendar header, use the scroll buttons to navigate to the
desired month and year. Sample Review Status
3. In the body of the calendar, click on the desired day.
Once analysis is complete, the Review, Status, and Status Message columns are
4. Click on the OK button to accept the date or click on Cancel to updated. The Review column displays a color-coded flag that indicates the
close the box without changes. importance of the user review:
m. Comment – Any additional comments about the patient, with a
Table 1. Sample Review Status
maximum comment length of 500 characters.

Review Status Message Description


F. Analyze Data
The ViroSeq software data analysis can be performed on a single sample or on At least 6 valid segments are The sample has less than 6
multiple samples. The ViroSeq software data analysis verifies that: required by this assay valid segments.

• A sufficient number of sequencing files are assembled


This sample has region(s) of Part of the Protease and/or RT
• Sample names are correct no coverage region has an area of no
• Sequencing files are aligned in the correct order and orientation coverage.

Invalid Segment(s) Out of frame indel(s) are Out of frame insertion or


detected deletion at the codon level.
IMPORTANT! An error message is displayed as a pop-up message or in the
sample section if analysis is performed on an invalid segment.
Missing required segment X There are 6 valid segments for
A segment is invalid when one or more of the following conditions is met: (X = primer name) a sample; One of the missing
• The segment is shorter than 100 bases segments is B, C, F, G, or H.
• The average QV is less than 20
Missing segment X There are 6 valid segments for
• No signal
(X = primer name) a sample; One of the missing
Refer to Appendix E: Troubleshooting for additional information on invalid segments is A or D.
segments.
This sample has no There were no mutations
Analyze a Single Sample mutations found in the analyzed sample
• Use the Analyze Samples button in the tool bar. Select a sample for analysis prior to user review.
and click on the Analyze Samples button.
This sample has region(s) of Part of the Protease and/or RT
• Use the Show Trace View button in the tool bar. Select a sample for analysis insufficient coverage region has single coverage
and click on the Show Trace View button. outside of the known single-
• Use the Analyze option from the pop-up menu. Right-click on a sample and coverage area (RT315-
select the Analyze button. RT335).
• Double-click on the sample name. Double-click on the sample name to
analyze the data and automatically open the Sample Trace View window. Ready for review POIs have not yet been
confirmed by the user.
Analyze Multiple Samples
All positions of interest All POls are confirmed and
Up to 1,000 samples can be selected for analysis at one time. (POI) have been confirmed reviewed by the user. No
• Select a group of samples and click the Analyze Samples button from the mutations were detected by
tool bar or right-click on the group of samples and select the Analyze option the ViroSeq software after
from the pop-up menu. user review and editing.

ViroSeq HIV-1 Genotyping Software 23 of 54


G. Assess Analyzed Data Edit Workflow
The following steps are the recommended workflow for editing:
Launch the Sample Trace View Window
1. Enable the editing palette by clicking on a segment in the assembled
The Sample Trace View window can be launched in one of two ways: segments or on an electropherogram.
• With a sample selected, click on the Show Trace View button, or 2. Remove a segment of poor quality sequence (refer to Remove and Add
• double-click on a sample name. Segments on page 16) and add the new re-injected segment (refer to Data
Assessment on page 24).
Data Assessment IMPORTANT! Removal or addition of a segment after editing base calls
1. Assess the assembled sample data to verify that the sequences are will cause a reanalysis and any previously confirmed POIs will revert to an
positioned as expected and that the peaks in the electropherograms are well- unconfirmed status.
resolved. 3. Manually trim segment ends to remove poor quality sequence segments
2. Identify areas of poor segment quality that may need to be removed, (refer to Trimming a Segment on page 19).
replaced, or trimmed. If necessary, re-inject the primer segment to generate 4. Select the POI filters for Mismatch, Mixture, Insertion, and Deletion.
a new sequence. The new segment may be added to the existing sample files Beginning at the leftmost POI, verify and reconcile mixed and mismatched
(refer to Edit Workflow on page 24). base calls, and inserted and deleted bases.
3. Review all POIs, paying particular attention to areas of interest as indicated Note: If mixed bases are not identified by ViroSeq software, refer to
by the analysis status message and red or yellow flags. External Software Requirements on page 3 for instructions on how to enable
mixed base identification. Follow the instructions and re-analyze the sample
To Distinguish Peaks from Background Signal (Noise) data in ViroSeq software.
IMPORTANT! Carefully review areas with insertions. Refer to the
A peak is an electrophoretic signal that has a well-defined maximum point with
Appendix B: Insertions and Deletions on page 33 for additional information.
clearly resolved slopes on either side. Peaks represent the signal strength of the
fluorescent dye bound to each nucleotide base. Peaks are read and analyzed by the 5. Select the POI filters for Drug Resistance Mutations and Additional
Data Collection and Sequencing Analysis programs. Mutations. Beginning at the leftmost POI, review every position identified
as a mutation.
6. Save the file.

Identify Misaligned or Mismatched Base Calls


When the ViroSeq software determines that a misalignment was caused by a
mobility issue, the software autocorrects the misalignment by deleting one or more
base calls in the segments.
Segments with mismatches are represented by the white vertical bars within the blue
Well-resolved Peaks Poorly-resolved Peaks assembled segment (navigation panel) and also by the yellow highlighted bases with
the corresponding electropherograms.
Note: Shoulders on peaks are defined as a peak only if they also have a well-defined
maximum point.
Noise is the background signal present in an electropherogram. It can be caused by
the instrument or by the quality of the capillary array, polymer, sample, buffer, or
Hi-Di formamide. The quality of the sequence data determines the accuracy of base
calls: fewer user edits are required with lower background noise levels.
Mixed basecalled peaks should be distinct from noise peaks. However, when high
levels of continuous noise are present in a sequence, real data can be obscured and
secondary peaks cannot be distinguished from noise.
The electropherogram display can be resized by the user to facilitate the review
process. Refer to Adjust the Electropherogram Display on page 19 for additional
information.
segments with mismatches are
H. Edit and Confirm Analyzed Data represented by white vertical bars
This section provides guidance on how to edit an analyzed sample. There are several
reasons why a sample may need to be edited, from trimming segments to manually
adding, deleting, or changing bases. After editing a base in the segment sequence,
the consensus sequence is automatically recalculated. If a change is made to a base
in the majority of the segments, the consensus sequence is updated to reflect the
majority base call.

24 of 54 Software Manual
Confirm Variant POIs and Review Drug Resistant
Mutations
Confirm Variant POIs
Refer to Position of Interest (POI) Filters on page 17
for additional information.
1. Check the highlighted filter(s) of the variant
POI(s) to be confirmed.
Note: Mutation POIs do not require
confirmation.
2. Click on the First POI button in the Sample Trace View toolbar to position
the cursor at the first POI in the navigation panel and the electropherograms.

mismatches indicated by
yellow highlighted bases

If mismatch(es) are edited by the user, such that the Consensus Sequence agrees with
the Reference Sequence, the white bars within the segment are removed as are the
yellow highlights within the electropherogram. For an example of a sample with a
mismatch, refer to the Edit sample in Appendix D: Example Data Set.

Guidelines for Manually Identifying Mixed Base Calls


Note: If mixed bases are not identified by ViroSeq software, refer to External
Software Requirements on page 3 for instructions on how to enable mixed base
identification. Follow the instructions and re-analyze the sample data in ViroSeq
software.
In most cases, the ViroSeq software accurately identifies mixed base calls (mixture).
If a user-made determination is required to identify a mixture, follow the guidelines
below. A mixture can be called when the following circumstances occur:
• Guideline 1 – Confirming peak is present; one sequence segment contains a 3. To edit a POI before confirming, refer to Editing Base Calls on page 17.
secondary peak at least 30% of the primary peak and the segment in the 4. To confirm a POI, press the spacebar on the keyboard. Confirmation of the
opposite direction confirms the mixture. POI is indicated by a check marked box in both the consensus panel and the
• Guideline 2 – Two opposite sense sequence segments contain secondary Variants table. Press the spacebar a second time to revert to an
peaks clearly above the local noise (approximately double the noise level). unconfirmed state.
• Guideline 3 – Confirming peak is not present; only one sequence segment 5. Navigate to the next POI by selecting the right arrow key on the keyboard,
contains a secondary peak at least 30% of the primary peak and three times clicking on the Next POI button, or clicking on a variant within the
the background noise. Variants table. Press the spacebar to confirm a selected POI. Press the
spacebar on a confirmed POI to revert to an unconfirmed state.
Take note of mixed base calls identified within regions of single coverage – only
Guideline 3 can be used to identify a mixed base call. 6. All confirmed changes may be returned to an unconfirmed state by clicking
on the Unconfirm All button in the upper right corner of the Variants table.

7. After all of the POIs have been confirmed, click on the Save button to
preserve changes.

ViroSeq HIV-1 Genotyping Software 25 of 54


I. Reports Interpret a Report
Reports contain a report header and a report body. Refer to Example of a Sample
Generate a Report Report on page 43 for a sample report example.
After sample analysis has been performed and user-edits have been
Report header
saved, a report can be generated from the main window or from the
Sample Trace View window.
ViroSeq® HIV-1 Antiretroviral Drug Resistance Report
• From the main window, right click on an analyzed sample and select Show
Report. Patient ID:
Accession Number:
-----

-----
Institution Name:
Report Generated by:
Cooper Medical Suites
Administrator

Sample Name: Report Date & Time:


• From the Sample Trace View toolbar, click on the Show Report button.
Drug-Resistance Apr 25, 2013 9:55:32 AM

D R i t
The Sample Report window opens and displays the multipage sample report. Refer The report header on pages 1 and 2 of the report contain general report information:
to Sample Report Window on page 19 for a description of the toolbar buttons.
• Patient ID – the patient ID input in the Patient Information dialog.
• Accession Number – the accession number input in the Patient
Information dialog.
• Sample Name – the sample name as imported into the ViroSeq software.
• Institution Name – the institution name as input during the software
registration process.
• Report Generated by – the ID of the current user.
• Report Date & Time – the date and time of report generation.

Report body
Drug Resistance:
NRTI Class Evidence of Resistance
EMTRIVA® (emtricitabine, FTC) Resistance***

EPIVIR® (lamivudine, 3TC) Resistance***

RETROVIR® (zidovudine, ZDV) Resistance***

VIDEX® (didanosine, ddI) Resistance***


VIREAD® (tenofovir, TDF) Resistance***

ZERIT® (stavudine, d4T) Resistance***

ZIAGEN® (abacavir, ABC) Resistance***

NNRTI Class Evidence of Resistance

EDURANT® (rilpivirine, RPV) Possible Resistance***


INTELENCE® (etravirine, ETR) Possible Resistance***
SUSTIVA® (efavirenz, EFV) Resistance***

VIRAMUNE® (nevirapine, NVP) Resistance***

PI+ Class Evidence of Resistance

APTIVUS® (tipranavir, TPV) Possible Resistance***


CRIXIVAN® (indinavir, IDV) Resistance***

FORTOVASE® / INVIRASE® (saquinavir, SQV) Resistance***

KALETRA® (lopinavir + ritonavir, LPV) Resistance***

LEXIVA® (fosamprenavir, FPV) Resistance***

PREZISTA® (darunavir, DRV) Possible Resistance***


REYATAZ® (atazanavir, ATV) Resistance***

VIRACEPT® (nelfinavir, NFV) Resistance***

* NOTE: At least one mutation used to determine Evidence of Resistance for this drug has not been fully validated.
** NOTE: At least one mutation used to determine Evidence of Resistance for this drug has not been clinically verified.
*** NOTE: For at least one mutation used to evaluate Evidence of Resistance for this drug, both notes above apply.
+ Evidence of Resistance for Protease Inhibitors estimates response to ritonavir-boosted regimens. Refer to section titled "Notes on Evidence of Resistance"

Notes on Evidence of Resistance:


Antiretroviral resistance is reported for three classes of drugs: Resistance Mutations present constitute a high level of genetic evidence for viral resistance
Possible Resistance Mutations present suggest the possibility of viral resistance

• Nucleoside Reverse Transcriptase Inhibitors (NRTIs)


None There is insufficient evidence for viral resistance

The protease inhibitor (PI) evidence of resistance interpretations were developed to estimate the expected virological response to standard doses of protease inhibitors with
pharmacokinetic boosting by ritonavir. This has become the most common method of administering each of the protease inhibitors, except nelfinavir (ref. 1), to ensure adequate drug
levels in all patients. Boosted PIs are more active in the presence of resistance than non-boosted PIs. (ref. 2,3)

• Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs)


• Protease Inhibitors (PIs) • Drug Resistance – reports information about possible drug resistance for the
patient sample.
– Information is presented by drug class [NRTI, Nucleoside Reverse
Transcriptase Inhibitors; NNRTI, Non-Nucleoside Reverse Transcriptase
Inhibitors; and PI, Protease Inhibitors] and by drug.
– Evidence of Resistance, reported for each drug, is based upon the
mutations found within the sample. The presence of a specific mutation or
combination of mutations results in one of the following calls:
• Resistance indicates a high level of genetic evidence for viral resistance.
• Possible Resistance indicates possible evidence of viral resistance;
mutations or combinations of mutations that do not meet the criteria
required for Resistance, but are suggestive of resistance.
• None indicates insufficient evidence for resistance.
Note: Refer to the ViroSeq® Algorithm Advisor™, Protease and Reverse
Transcriptase, for individual and combination mutation scores.

26 of 54 Software Manual
– Disclaimer Levels: The Evidence of Resistance levels can be labeled with • Drug Resistance Mutations Identified – reports mutations present in the
zero to three asterisks (*) or a plus symbol (+). The meanings of the patient sample that may cause drug resistance.
symbols are described in the following table: • Additional Mutations – identifies amino acids that differ from the reference
sequence (HXB-2, accession number K03455) at the indicated codon positions
Meaning in Terms of and may be useful as a baseline determination of virus genotype. The
Disclaimer
Level Disclaimer Analytical and Clinical
Studies performance characteristics of the additional mutations have not been
established. Additional mutations are specified either for the Protease gene or for
the Reverse Transcriptase (RT) gene.
(No symbol) No disclaimer All mutations present
have been verified in Drug Resistance Mutations Identified:
clinical and analytical NRTI Class:
studies. M41L, D67N, L74V, M184V, L210W, T215Y, K219R

NNRTI Class:
* Note: At least one All mutations present K101E, K103R, V108I, V179D, H221Y
mutation used to have been validated in PI Class:
determine Evidence of clinical studies but may L10I, M46I, I47V, A71V, L76V, I84A
Resistance for this drug not have been verified in
Additional Mutations:
has not been fully analytical studies. Additional Mutations: The following amino acids differing from the reference sequence (HXB-2, accession number K03455) at the indicated

validated.
codon positions were identified and may be useful as a baseline determination of virus genotype.

Protease

V3I, G16A, K20R, M36I, S37D, R41K, Q61N, I62V, L63P, C67Y, I93L
** Note: At least one All mutations present
RT
mutation used to have been verified in
K20R, V35M, T39A, K43E, E44A, V118I, I202M, H208Y, R211K, L214F, R277K, T286A, I326V
determine Evidence of analytical studies but may
Resistance for this drug not have been verified in • Patient Information– information about the patient based upon the input from
has not been clinically clinical studies. the Patient Information dialog.
verified. • Site Information– information about the site input from the Site Information
window..
*** Note: For at least one At least one mutation was
mutation used to not verified in both • Comments – comments input by the user in the Patient Information dialog.
evaluate Evidence of clinical and analytical Note: The comments entered when saving changes in the Sample Trace View,
Resistance for this drug, studies. are displayed only in the Sample History comments field.
both notes above apply. • Review & Release of Results – a designated signatory section of the report.

+ Evidence of Resistance The protease inhibitor Patient Information:


for Protease Inhibitors (PI) evidence of Accession Number ----- Patient Information
estimates response to resistance interpretations Assay Operator Name ----- ID -----

ritonavir-boosted were developed to Institution Name


Physician
-----
-----
First Name
Middle Name
-----
-
regimens estimate the expected Field 1 ----- Last Name -----

virological response to Field 2


Date Drawn
----- Gender
Date of Birth
Not Available

standard doses of
protease inhibitors with Site Information:

pharmacokinetic boosting Institution Name Cooper Medical Suites


Department Name Lab Testing
by ritonavir. This has Lab Director Dr. S. Cooper
become the most Address 1 Suite 4A

common method of
Address 2 2311 N. Los Robles Avenue
Mail Stop -
administering each of the City Pasadena

protease inhibitors, State/Province


ZIP / Postal Code
California
-
except nelfinavir,1 to Country -

ensure adequate drug Phone


Fax
213-454-8100
-
levels in all patients. Contact Email sheldor@caltech.com

Boosted PIs are more Website URL -

active in the presence of


Comments:
resistance than non- -

boosted PIs.2,3

Review & Release of Results:

Signature / Date: Name(Print) / Title:

Notes:

ViroSeq HIV-1 Genotyping Software 27 of 54


• HIV-1 Resistance Mutation List – summarizes the mutations included within • Mutation Notations Key – a color key is used to define text conventions used in
the algorithm for the drugs identified under the Drug Resistance section of the the HIV-1 Resistance Mutation List to denote levels of resistance. Mutations
report. are represented using style, color, and punctuation conventions to indicate their
relevance to the development of drug resistance.
HIV-1 Resistance Mutation List:
N88S ,I50L
NRTI Class Mutations Included within the Algorithm
Mutation Notations Key:
EPIVIR® (lamivudine, 3TC) {Red Bold Curly Bracket} Presence of this mutation alone confers viral resistance
{M184V,M184I**} [Blue Bold-Italics Square Bracket] Presence of this mutation alone confers the possibility of viral resistance
[69ins**,K65R,Q151M] (Black Parenthesis) This mutation must appear with at least one other mutation to confer the possibility of viral resistance
(V75I*,69del***,K65N***,K219Q,K219R***,L210W,D67T***,D67S***,D67N*,Q151L***,D67E,D67H***,D67G***,K70T***,A62
V,T215Y,F116Y,M41L,F77L,K219E***,K70E***,67del***,K70G***,K70Q***,K70R,K70S***,K219N***,K70N***,T215F) <Green Angle Bracket> This mutation counters resistance

EMTRIVA® (emtricitabine, FTC)


* NOTE: This mutation has not been fully validated
{M184V,M184I**}
[69ins**,K65R,Q151M] ** NOTE: This mutation has not been clinically verified
(V75I*,69del***,K65N***,K219Q,K219R***,L210W,D67T***,D67S***,D67N*,Q151L***,D67E,D67H***,D67G***,K70T***,A62 *** NOTE: For this mutation, both notes above apply
V,T215Y,F116Y,M41L,F77L,K219E***,K70E***,67del***,K70G***,K70Q***,K70R,K70S***,K219N***,K70N***,T215F)

{Red Bold Curly Bracket} Presence of this mutation alone confers viral
VIDEX® (didanosine, ddI)
{K65R,L74I***,L74V,Q151M,69ins**}

resistance
[K65N***,69del***,Q151L***,67del***,V75M***,T69D,K70E***,K70G***,V75T***]
(K219Q,K219R***,A62V,T215S***,F116Y,T215Y,T215V***,M41L,T215L***,T215N***,T215E***,T215D***,T215C***,T215I**
*,T215F,V75I*,T69A***,T215A***,V75S***,L210W,D67T***,D67S***,D67N*,T69N***,V75A***,D67E,T69S***,D67H***,D67G
***,K70T***,M184V,F77L,K219E,M184I**,K70Q***,K70R,K70S***,K219N***,K70N***)
ZERIT® (stavudine, d4T) [Blue Bold-Italics Square Bracket] Presence of this mutation alone confers
{Q151M,T215Y,T215F,69ins**,V75M***,V75T***}
[K65R,69del***,Q151L***,T215S***,T215V***,T215L***,67del***,T215N***,T215E***,T215D***,T215C***,T215I***,T215A** the possibility of viral resistance
*,V75S***,V75A***,K70E***,K70R]

(Black Parenthesis) This mutation must appear with at least one other
(K65N***,K219Q,K219R***,A62V,F116Y,M41L,V75I*,T69A***,T69D,L210W,D67T***,D67S***,D67N*,T69N***,D67E,T69S*
**,D67H***,D67G***,K70T***,F77L,K219E,K70Q***,K70S***,K219N***,K70N***)
<M184V,M184I**>
ZIAGEN® (abacavir, ABC) mutation to confer the possibility of viral resistance
{69ins**,K65R,Q151M,Y115F*}
[L74I***,69del***,K65N***,L74V,Q151L***,K70E***,67del***,K70G***]
(V75I*,T215A***,K219Q,K219R***,L210W,D67T***,D67S***,D67N*,D67E,D67H***,D67G***,K70T***,M184V,A62V,T215S** <Green Angle Bracket> This mutation counters resistance
*,T215Y,F116Y,T215V***,M41L,T215L***,F77L,K219E,T215N***,V75T***,T215E***,M184I**,T215D***,K70Q***,T215C***,
K70R,K70S***,T215I***,K219N***,K70N***,T215F)
VIREAD® (tenofovir, TDF)
* NOTE: This mutation has not been fully validated
{69ins**,K65R}
[K65N***,Q151M,K70E***,K70G***]
(V75I*,L74I***,69del***,T215A***,K219Q,K219R***,L210W,D67T***,D67S***,D67N*,Q151L***,D67E,D67H***,D67G***,K70
** NOTE: This mutation has not been clinically validated
T***,A62V,T215S***,T215Y,F116Y,T215V***,M41L,T215L***,F77L,K219E,T215N***,67del***,T215E***,T215D***,K70Q***,
T215C***,K70R,K70S***,Y115F*,T215I***,K219N***,K70N***,T215F)
<M184V,M184I**>
*** NOTE: For this mutation, both notes above apply
• References – a list of the citations referred to in the sample report.
RETROVIR® (zidovudine, ZDV)
{69ins**,Q151M,T215Y,T215F}
[T215A***,Q151L***,T215S***,T215V***,T215L***,T215N***,67del***,T215E***,T215D***,T215C***,K70R,T215I***]
(V75I*,T69A***,K219Q,T69D,K219R***,L210W,D67T***,D67S***,D67N*,T69N***,D67E,D67H***,T69S***,D67G***,A62V,F
116Y,M41L,F77L,K219E,K219N***)
<K65R,K65N***,M184V,K70E***,M184I**> References:
NNRTI Class Mutations Included within the Algorithm
1. Panel on Antiretroviral Guidelines for Adults and Adolescents. Guidelines for the use of antiretroviral agents in HIV-1-infected adults and
INTELENCE® (etravirine, ETR) adolescents. Department of Health and Human Services. Available at http://aidsinfo.nih.gov/contentfiles/lvguidelines/AdultandAdolescentGL.pdf.
{Y181V***,Y181I*} 2. M Boffito, E Acosta, D Burger, CV Fletcher, C Flexner, R Garaffo, G Gatti, M Kurowski, CF Perno, G Paytavin, M Regazzi and D Back.
[M230L***,L100I*,K101P***,G190Q***,F227C***,Y181C,G190E***] Current status and future prospects of therapeutic drug monitoring and applied clinical pharmacology in antiretroviral therapy. Antiviral Therapy,
(E138G***,V106I***,H221Y***,E138A***,V179D***,V179E***,V179F***,Y188L**,M230I***,E138K***,V179L***,V179T***,K10 2005, 10:375-392
1H***,L100V***,A98G***,K101E*,G190T***,G190S*,G190V***,E138R***,G190A*,E138Q***,V90I***,G190C***) 3. N Shulman, A Zolopa, D Havir, A Hsu, C Renz, S Boller, P Jiang, R Rode, J Gallant, E Race, DJ Kempf and E Sun. Virtual inhibitory quotient
predicts response to fitonavir boosting of indinavir-based therapy in human immunodeficiency virus-infected patients with ongoing viremia.
Antimicrobial Agents Chemotherapy, 2002,46:3907-3916

NNRTI Class Mutations Included within the Algorithm

SUSTIVA® (efavirenz, EFV)


{V106M***,Y188L**,V106A**,K103N,K101P***,Y188C**,K103S***,G190Q***,K103H***,G190T***,G190S*,G190V***,G190
C***,G190E***}
Save a Report
[M230L***,Y181V***,Y188H***,L100I*,K103T***,P225H***,F227C***,K238T***,Y181I*,G190A*,Y181C]
(E138G***,V106I***,H221Y***,V179D***,V108I*,V179E***,V179F***,M230I***,E138K***,V179L***,K101Q***,V179T***,K101
H***,L100V***,A98G***,Y318F***,K238N***,F227L***,K101E*,E138R***,E138Q***,V90I***)
1. Click on the Save as PDF button to open the Save as dialog box.
VIRAMUNE® (nevirapine, NVP)
{M230L***,Y181V***,V106M***,Y188L**,V106A**,Y188H***,K103N,K101P***,Y188C**,K103T***,K103S***,G190Q***,K10 2. In the Save as dialog box, navigate to a location in which the file is to be
3H***,G190T***,G190S*,F227C***,G190V***,Y181I*,G190A*,G190C***,G190E***,Y181C}
[M230I***,L100I*,Y318F***,P225H***,F227L***,K101E*,K238T***]
(E138G***,V106I***,H221Y***,V179D***,V108I*,V179E***,V179F***,E138K***,V179L***,K101Q***,V179T***,K101H***,L10
saved.
0V***,A98G***,K238N***,E138R***,E138Q***,V90I***)
EDURANT® (rilpivirine, RPV) 3. Enter the file name and click on the Save button.
{Y181V***,Y188L**,K101P***,F227C***,Y181I*}
[M230L***,M230I***,E138K***,L100I*,G190Q***,Y181C,G190E***]
(E138G***,V106I***,H221Y***,E138A***,V179D***,V179E***,V179F***,V179L***,V179T***,K101H***,L100V***,A98G***,K1
01E*,G190T***,G190S*,G190V***,E138R***,G190A*,E138Q***,V90I***,G190C***) Print a Report
PI Class Mutations Included within the Algorithm

APTIVUS® (tipranavir, TPV)


[I84V,V82L***,I84A***,V82T]
1. Click on the Print button to open the Print dialog box.
(V82M**,V32I*,V82C***,L90M,V82F**,L33F*,I54A***,I47V*,L10I,K43T***,I54S***,T74P***,I54V,L10R**,I54T*,A71L***,I84C*
**,Q58E***,L10V*,I47A***,A71V,I54M***,L10Y***,A71T*,V82S**,M46L*,A71I***,N83D***,M46I)
<L76V***,L24I***,I50L***,I54L***,I50V**>
2. Select a printer from the list of available printers.
CRIXIVAN® (indinavir, IDV)
{I84A***} 3. Click on the OK button to print.
[I84V,L90M,I84C***,V82T,V82S**,L76V***,V82M**,V82A,V82C***,V82F**]
(M46V***,G48A***,V32I*,G73A***,F53L*,I54A***,L24I***,I47V*,L10I,L10F*,I54S***,I54V,T74P***,I54T*,I54M***,I54L***,G48

Export to Files
T***,G48S***,G48V,M46L*,G48Q***,G48L***,G48M***,M46I,N83D***,V82L***,N88S***,G73T***,G73S*,L10R**,A71L***,L1
0V*,I47A***,A71V,L10Y***,A71T*,G73C***,A71I***)
<I50L***>
FORTOVASE® / INVIRASE® (saquinavir, SQV)
{I84V,G48A***,I84C***,I84A***,G48T***,G48S***,G48V,G48Q***,G48L***,G48M***}
[L90M]
Analyzed sample data may be exported in three different formats for one or
(V82A,V82C***,G73A***,V82F**,F53L*,L24I***,I54A***,L10I,I54S***,G73T***,T74P***,G73S*,I54V,L10R**,I54T*,A71L***,L1
0V*,A71V,I54M***,A71T*,L10Y***,I54L***,V82T,V82S**,G73C***,M46L*,A71I***,N83D***,M46I)
more sample files using the Export to file function in the main window:
<L76V***,I50L***,I47A***>
KALETRA® (lopinavir + ritonavir, LPV) • XML, a text-based format
{I47A***}
[I84A***,I50V**,L76V***]
(M46V***,I84V,G48A***,V32I*,L90M,G73A***,F53L*,L33F*,I54A***,L24I***,I47V*,L10I,L10F*,I54S***,I54V,T74P***,I54T*,I8 • FASTA, Consensus nucleotide and/or amino-acid sequence
4C***,I54M***,I54L***,V82T,G48T***,V82S**,G48S***,G48V,M46L*,G48Q***,G48L***,G48M***,M46I,N83D***,V82M**,V82
L***,V82A,V82C***,V82F**,G73T***,G73S*,L10R**,A71L***,L10V*,A71V,L10Y***,A71T*,G73C***,A71I***)
<I50L***>
• Comma Separated Value (CSV), sample mutations
PREZISTA® (darunavir, DRV)
(V11I***,L76V***,G73T***,I84V,T74P***,G73S*,I84C***,V32I*,I84A***,L89V***,I47A***,G73A***,I54M***,V82F**,L33F*,I54L* Note: The Export to file function does not remove the sample data from the
**,I50V**,I47V*,G73C***,L10F*)
<I50L***,N88S***> project. To remove data, refer to Deleting Samples, Projects, and Repositories on
PI Class Mutations Included within the Algorithm
page 38.
REYATAZ® (atazanavir, ATV)
{I84V,I84C***,I50L***,I84A***,N88S***}
[G48A***,G48T***,G48S***,G48V,G48Q***,G48L***,G48M***]
(M46V***,V32I*,L90M,G73A***,F53L*,L33F*,I54A***,L24I***,L10I,I54S***,N88G***,I54V,T74P***,I54T*,I54M***,I54L***,V82
T,V82S**,M46L*,M46I,N83D***,V82M**,V82L***,V82A,V82C***,V82F**,N88T***,G73T***,G73S*,L10R**,A71L***,L10V*,A7
1V,L10Y***,A71T*,G73C***,A71I***)
<L76V***>
VIRACEPT® (nelfinavir, NFV)
{I84V,L90M,D30N,I84C***,I84A***,N88D*,N88S***}
[G48A***,I54A***,N88G***,I54S***,I54V,I54T*,I54M***,I54L***,V82T,G48T***,L23I***,V82S**,G48S***,G48V,M46L*,G48Q*
**,G48L***,G48M***,M46I,V82A,V82C***,V82F**,N88T***]
(M46V***,V32I*,K20T***,G73A***,L24I***,I47V*,L10I,L10F*,T74P***,N83D***,V82M**,V82L***,G73T***,G73S*,L10R**,A71
L***,L10V*,I47A***,A71V,L10Y***,A71T*,G73C***,A71I***)
<I50L***>
LEXIVA® (fosamprenavir, FPV)
{L76V***,I84V,I84C***,I84A***,I47A***,I54M***,I54L***,I50V**}
[V82F**,I47V*]
(M46V***,V11I***,V82M**,V82L***,V82A,V32I*,V82C***,L90M,G73A***,L33F*,I54A***,L10I,L10F*,I54S***,G73T***,T74P***,
G73S*,I54V,L10R**,I54T*,A71L***,L10V*,L89V***,A71V,L10Y***,A71T*,V82T,V82S**,G73C***,M46L*,A71I***,N83D***,M4
6I)
<N88S***,I50L***>

28 of 54 Software Manual
For the XML format, the default data file name is the sample name plus the suffix of To import a .VAS file
“_report”. When multiple samples are selected for export, each sample file is output
Note: The .VAS file may not be imported into the same project in which the original
into a separate XML file.
data file resides.
1. Select a project for import of the .VAS file.
2. From the main window, select Tools > Import Analyzed Sample.
3. Select the .VAS file to be imported and click Open. The .VAS file is
imported into the selected project.

For FASTA and CSV formats, the default file name is the sample name when a
single sample is exported. For the Consensus Amino Acid Sequence (FASTA)
format, the file name is automatically appended with the suffix of “_AA” to
distinguish the file from the Consensus Nucleotide Sequence (FASTA) format.
When multiple samples are selected for export to either FASTA or CSV, the sample
data are combined into a single output file.
1. Select one or more samples from the main window.
2. Click on the Export to file button in the toolbar, or right-click on the sample
and select Export to file from the drop-down menu to open the Export
dialog box.
3. Files are saved to the directory shown in the Directory text box. To change
the directory, click on the Browse button and select a new destination.
4. Choose one or more of the options:
a. Sample Report (XML)
b. Consensus Nucleotide Sequence (FASTA)
c. Consensus Amino Acid Sequence (FASTA)
d. Mutations (CSV)
Note: When exporting multiple samples, enter a file name for the FASTA
and/or CSV file(s).
5. Verify that the exported file(s) was/were generated.

Export Analyzed Samples


Analyzed samples may be exported in a .VAS file format that may be imported into
the ViroSeq software for further review.
Note: The Export Analyzed Sample function does not remove the sample data
from the project. To remove data, refer to Deleting Samples, Projects, and
Repositories on page 38.
Note: The exported file (.VAS) may not be imported into the same project in which
the original data file resides.
1. Select one or more samples from the main window.
2. In the main window, go to the Tools drop-down menu and select Export
Analyzed Sample.
3. Files are saved to the directory shown in the Directory text box. To change
the directory, click on the Browse button and select a new destination.
4. Click on OK. Each sample file is exported as a separate .VAS file.

ViroSeq HIV-1 Genotyping Software 29 of 54


Section 5: Bibliography

1. Panel on Antiretroviral Guidelines for Adults and Adolescents. Guidelines for


the use of antiretroviral agents in HIV-1-infected adults and adolescents.
Department of Health and Human Services. Available at
http://aidsinfo.nih.gov/contentfiles/lvguidelines/AdultandAdolescentGL.pdf.
2. Boffito, M., et al. 2005. Current status and future prospects of the therapeutic
drug monitoring and applied clinical pharmacology in antiretroviral therapy.
Antiretroviral Therapy 10:375-392.
3. Shulman, M, et al. 2002. Virtual inhibitory quotient predicts response to
ritonovir boosting of indinavir-based therapy in human immunodeficiency
virus-infected patients with ongoing viremia. Antimicrobial Agents
Chemotherapy 46:3907-3916.

30 of 54 Software Manual
Appendix A: User Manage Users

Account Management
Users can be added or existing accounts may be edited using the Manage
Users button located in the main toolbar. Existing user accounts may be edited or
disabled, but cannot be deleted.

Administrators can create multiple user accounts with different roles. The roles,
Administrator and Lab Technician, provide users with different capabilities and
responsibilities. Only users with an Administrator credential can access the
Manage Users function.

Software License Renewal


Role User Privileges Account Status

Renewing before the license expires Administrator Able to change all account • Active
When the software license is within 30 days from expiring, a message box displays settings and cannot be made • Change Password
when the application is launched. A user can renew the license key prior to the inactive or disabled.
expiration of the current license.
Lab Technician No user management • Active
1. From the main application window, select Help > License & Registration.
privileges. • Inactive
2. Follow the steps in Registration and Activation on page 6 to complete
registration information and to save the registration file. • Change Password
3. E-mail the saved registration file to softwareregistration@celera.com. • Disabled

4. Once the license key is received, save the License.key file to a location for
later use.
Adding a New User
5. From the main application window, select Help > License & Registration.
1. Click on the Add button to create a new user.
6. Click on the Browse... button to access the Open dialog box.
2. Enter a user ID in the User ID text box (minimum of six alphanumeric
7. Navigate to the location that contains the License.key file, double-click on characters).
the License.key file, then click on the OK button.
Note: The User ID field cannot be edited once the user account is saved.
3. Choose a role for the user using the drop-down menu. The options are
Renewing after the license has expired Administrator or Lab Technician.
After the license has expired, a message box displays when the application is 4. Enter the user’s name in the Full Name text box (a minimum of three
launched and the License & Registration window opens to allow the user to renew alphanumeric characters is required).
the license key. The ViroSeq software is inoperable if the license is expired.
5. Choose the user’s account status. The options are Active, Inactive, or
1. After the license has expired and the software is launched, the License & Change Password.
Registration dialog box will display.
• Active: new users are not prompted to change passwords when logging
2. Follow the steps in Registration and Activation on page 6 to complete in for the first time.
registration information and save the registration file.
• Inactive: new users will not be able to access their account until the
3. E-mail the saved registration file to softwareregistration@celera.com. account status is changed to Active or Change Password.
4. Once the license key is received, save the License.key file to a location for • Change Password: new users are prompted to change passwords when
later use. logging in for the first time.
5. Launch the software to display the License & Registration dialog box. 6. Enter a password for the account. The case-sensitive password must consist
6. Click on the Browse... button to access the Open dialog box. of at least 6 alphanumeric characters. The underscore (_) symbol may be
7. Navigate to the location that contains the License.key file, double-click on included in the password; no other symbols are allowed.
the License.key file, then click on the OK button. Note: If Change Password is selected as the Account Status, ensure that
a new user is supplied with their temporary password.
7. Click the Save button.

Editing an Existing User Account


1. Select an existing user from the left side of the screen.
2. Click on the Edit button to change the selected user’s information.
a. User ID – Not editable.
b. Role – Select from the following options:
• Admin
• Lab Technician
c. Full Name – The user’s full name may be edited, but must contain at
least three alphanumeric characters.

ViroSeq HIV-1 Genotyping Software 31 of 54


d. Account Status – Select from the following options:
• Active – Active accounts are able to log into and utilize the
software.
• Inactive (applicable to Lab Technician accounts only) – Inactive
accounts are unable to log into the software.
• Change Password – Forces the user to perform a password change
at the next log in.
e. Password / Re-enter – Enter and verify a new user password.
Note: The case-sensitive password must consist of at least 6
alphanumeric characters. The underscore (_) symbol may be included
in the password; no other symbols are allowed.
3. Click on the Save button.

Disabling a Lab Technician Account


1. Select an existing Lab Technician account from the left side of the screen.
2. Click on the Disable button to change the selected user’s status.
3. Click on the Save button.
Note: Disabled Lab Technician accounts may be re-activated by an
Administrator.

32 of 54 Software Manual
Appendix B: Insertions Out-of-frame Insertions and Deletions
An out-of-frame insertion or deletion (indel) is composed of a number of bases that

and Deletions
is not divisible by three.
When the ViroSeq software detects an out-of-frame indel, a red Review flag is
displayed in the sample section along with the status message, “Out of frame
indel(s) are detected.” Indels can be reviewed in the Sample Trace View window
by selecting the Insertion and/or Deletion POI filter.
An example of a two base, out-of-frame insertion is shown below.

The ViroSeq HIV-1 Genotyping Software identifies base insertions and deletions
(indel), both in-frame and out-of-frame. Indels are reported at the codon positions in
which they are detected. Users may edit at the segment level and replace an indel
with a base. The edited base(s) can be further reviewed by selecting the Edited POI
filter to display the user edits in the Sample Trace View and in the Variants table.
IMPORTANT! Any insertion of one or more codons at or between codons 67 to
71 are interpreted by the ViroSeq software as an indication of the RT codon 69
insertion mutation.

Indel Symbol
A dash marker (-) represents the presence of an indel in the Sample Trace View
window as indicated below:

Location of the Dash Symbol (-) Explanation


Reference Translation An amino acid insertion within the
sample at the indicated position Three-base Deletion Example
Consensus Translation An amino acid deletion within the sample In the example below, a three-base deletion begins in the third position of frame 2
at the indicated position and extends into positions 1 and 2 of frame 3. The ViroSeq software would not flag
this sample as an out-of-frame indel. No translation is provided at the Consensus
Reference Sequence A base insertion at the corresponding
Translation for codons 91 and 92; ViroSeq software would report deletions for both
position within the Consensus Sequence
codons in the Sample Trace View window and Sample Report.
Consensus Sequence A base deletion at the corresponding
position within the Reference Sequence

In-frame Insertions and Deletions


An in-frame insertion or deletion (indel) is composed of a number of bases that is
divisible by three. Indels can be reviewed in the Sample Trace View window by
selecting the Insertion and/or Deletion POI filter.
An example of a nine-base in-frame insertion is shown below.

ViroSeq HIV-1 Genotyping Software 33 of 54


Insertion Adjustment for Multiples of Example 1: Adjusted Reference Sequence and Translation (in ViroSeq)

Three
When a base insertion occurs as a multiple of three and begins at the second or third
position within a codon, the ViroSeq software performs an adjustment to correctly
identify the insertions and mutation, and to also avoid downstream misalignments
that would otherwise be identified as mismatches. An adjustment is made to the
Reference Sequence by virtually shifting the base insertion to align within the
frame(s); the direction of the base insertion shift is dependent upon the starting
position of the insertion. The Reference Translation is subsequently adjusted per the
adjusted Reference Sequence.
Note: The adjusted Reference Sequence is applied only to the Reference
Translation; an adjustment to the base insertion in the Sample Trace View is not
displayed.
Note: The Reference Sequence without an Adjustment examples within this section
are provided for illustrative purposes only and are not displayed in the ViroSeq
software.

Example 1: Leading Frame Adjustment


In the first diagram below, Example 1: Reference Sequence without an Adjustment, a
six-base insertion begins at the third position of frame 1 and extends into frames 2
and 3. The insertion spans across three codons and would be reported as a three-
codon insertion of 69insS, 69insS, and 69insE. In addition, the insertion would cause
a frameshift and downstream translations would be identified as mismatches.
In the second diagram, Example 1: Adjusted Reference Sequence and Translation,
the ViroSeq software has virtually shifted the insertion to align it within the codon
boundaries. For this example, the insertion is shifted to the right by one base
position, replacing the base T in the third position of frame 3. The base T is shifted to
the third position of frame 1 and codon 68 includes the three bases, AGT, and is
identified as the amino acid S. The ViroSeq software subsequently reports a two-
codon insertion of 69insS and 69insE, and the mutation T69S. No Reference
Translation is provided for frames 2 and 3.

Example 1: Reference Sequence without an Adjustment

Example 1: Adjusted Reference Sequence and Translation

34 of 54 Software Manual
Example 2: Trailing Frame Translation
Example 2: Adjusted Reference Sequence and Translation (in ViroSeq)
In the first diagram below, Example 2: Reference Sequence without an Adjustment, a
six-base insertion begins at the second position of frame 2 and extends into frames 3
and 4. The insertion spans across three codons and would be reported as a three-
codon insertion of 69insS, 69insE, and 69insS; a mutation would be reported as
T69K. In addition, the insertion would cause a frameshift and downstream base calls
would be identified as mismatches.
In the second diagram, Example 2: Adjusted Reference Sequence and Translation,
the ViroSeq software adjusts the Reference Sequence by virtually shifting the
insertion to the left by one base position, replacing the base A in the first position of
frame 2. The base A is shifted to the first position of frame 4 and codon 69 includes
the three bases, ACT, and is identified as the amino acid T. The ViroSeq software
subsequently reports a two-codon insertion of 69insS and 69insE, and the mutation
T69S. No Reference Translation is provided for frames 2 and 3.
Example 2: Reference Sequence without an Adjustment

Example 2: Adjusted Reference Sequence and Translation

ViroSeq HIV-1 Genotyping Software 35 of 54


Auto-correction  false deletion at codon 69
mixture call
When the ViroSeq software identifies a mobility issue within a segment, an auto-
correction is performed to avoid a misalignment. The auto-correction notation is a
dash marker (-) inserted at the segment level that acts as a placeholder and is not a
true base deletion. In some cases, the user may choose to edit the segment (refer to
Example 2, below).

Example 1
The ViroSeq software has detected a mobility shift in segment A and has performed mobility shift
an auto-correction at position 136’3. No user-edits are required.

mobility shift

 auto-correction
The user may edit the auto-correction notation as to avoid a false deletion at codon
69. For this example, the user may consider the following edits:
• substituting an “A” in place of the dash marker at position 69’1 in segments
F and G
• substituting a “G” in place of the “R” in segments F and G at position 70’3
The user edits are tracked by ViroSeq software as shown in the sample trace below.
The Consensus Translation at codon 69 is now a “K” and is no longer reported as the
mutation, 69delH.
Example 2
A mobility shift of an “A” peak in both segments F and G cause the following to
occur:
an auto-correction is performed at position 69'1 in both segments F and G,
a mixture call “R” is made at position 70'3, and
a false deletion at codon 69 of the Consensus Translation (69delH).

36 of 54 Software Manual
Appendix C: Data System Memory Requirement
Workspaces, repositories, projects, and samples may be created or added as long as
the computer has 5 GB of free disk space available. When the available disk space is
Management less than 5 GB, an error message is displayed to alert the end-user (refer to Appendix
E: Troubleshooting for additional information).

Workspace

Sample Data File Management


In ViroSeq HIV-1 Genotyping Software v3.0, sample data files are organized into
workspaces, repositories, and projects.

Data Organization
:RUNVSDFH

A workspace is at the top level of data organization. A workspace is a user-defined


location in which data can be imported, analyzed, and reviewed. Refer to Create a
Workspace on page 7 for instructions on how to set up a workspace.
• Workspaces can only be defined by a user with Administrator privileges.
• One workspace can be used by a single user (e.g., private) or multiple users,
based upon the Windows access rights to a given folder location.
5HSRVLWRU\ • Users (e.g., Administrator or Lab Technician) only have access to workspaces in
which their user account was defined.
• To enable workspace selection when the ViroSeq software is launched, refer to
Settings on page 8.
• Each workspace has defined Analysis Settings and Naming Configuration
settings.
• The workspace path is displayed at the bottom of the Explorer panel in the main
3URMHFW
window.
• Workspaces cannot be renamed, archived, or deleted.

Repository
Example of Workspace with Multiple A repository is the second level of data
Repositories and Projects organization in the ViroSeq HIV-1
Genotyping Software v3.0 that is created
:RUNVSDFH and named by the end-user. Repositories
are contained within a workspace and are
designed to allow the user to create logical
groupings of data.
• One or more repositories may be
created within a workspace.
• Repositories may be defined by
5HSRVLWRU\B$ 5HSRVLWRU\B% 5HSRVLWRU\B& Administrators and Lab Technician
users.
• Repositories may be deleted by
Administrators and Lab Technician
3URMHFWB$ 3URMHFWB$ 3URMHFWB$ 3URMHFWB$ 3URMHFWB% 3URMHFWB% 3URMHFWB% 3URMHFWB& users.
• Repositories cannot be renamed once created.

ViroSeq HIV-1 Genotyping Software 37 of 54


Project Accessing an Archived Workspace
A project folder is the third level of To access analyzed data from an archived workspace, unzip the workspace in the
data management used in the ViroSeq same location from which it was deleted. The ViroSeq software will find the re-
HIV-1 Genotyping Software v3.0 that instated folder the next time the software is launched.
is created and named by the end-user.
One or more project folders are
contained within a repository to assist Deleting Samples, Projects, and
the end-user with data management.
• A repository may contain one or
Repositories
more projects. The Delete function may be used to delete sample files, projects, or repositories.
Both Administrator and Lab Technician users may use the Delete function as
• Projects may be defined by
described in this section.
Administrators and Lab
Technician users. IMPORTANT! Each deletion must be confirmed by the user as deleted items
cannot be retrieved.
• Projects may be deleted by Administrators and Lab Technician users.
• Projects cannot be renamed once created.
Deleting Samples
A single sample or multiple samples files can be deleted.
Sample Data Files (.ab1)
Sample data files (.ab1) are contained within a project folder.
To delete a single sample file
• A sample is composed of seven primer files, where the primer name is • Click on the sample to be deleted and click on the Delete button in the toolbar, or
designated by the letter A, B, C, D, F, G, or H. • Right-click on the sample to be deleted and click on the Delete option in the
• A project may contain one or more samples. drop-down menu.

• Sample files may be imported or deleted by Administrators and Lab Technician To delete multiple sample files
users.
• Select a range of sample files by clicking on a sample file and then, holding the
The ViroSeq HIV-1 Genotyping Software v3.0 supports data files (.ab1) generated Shift button, click on another sample file; all of the sample files between the two
by: will be selected. Click on the Delete button in the toolbar or right-click to
• Applied Biosystems 3130 series Genetic Analyzers with Data Collection v3.0 display the drop-down menu and select the Delete option to delete the selected
and Sequencing Analysis Software v5.2 or above, with KB Basecaller v1.2 or files.
above, and • Select multiple, non-contiguous sample files by holding the Ctrl key and then
• ABI PRISM 3100 Genetic Analyzer with Data Collection v1.1 or v1.0.1 or the clicking on select sample files. Click on the Delete button in the toolbar or right-
ABI PRISM 3100-Avant Genetic Analyzer with Data Collection v1.0 that have click to display the drop-down menu and select the Delete option to delete the
been re-analyzed using the Sequencing Analysis Software v5.2 or above, with selected files.
KB Basecaller v1.2 or above.
Refer to External Software Requirements on page 3 for additional information. Deleting Projects
A single project or multiple projects can be deleted. Deleting a project will delete all
of the sample files contained within the project.
Archiving Analyzed Data
Analyzed data may be archived when the ViroSeq software requires more available To delete a single project folder
disk space for operation. A minimum of 5 GB of available disk space is required for • Click on the project to be deleted and click on the Delete button in the toolbar, or
operation of the ViroSeq software. Archiving is performed by zipping the workspace
• Right-click on the project to be deleted and click on the Delete option in the
folder and deleting the original, non-compressed workspace folder using Windows
drop-down menu.
file management functions.
Note: Archiving and deleting a workspace is not recorded as an action in the Audit To delete multiple project folders
Trail.
• Select a range of project folders by clicking on a project and then, while holding
Workspace folders can be accessed using Windows file management functions. The down the Shift button, click on another project; all of the project folders between
Windows file path for the workspace is displayed at the bottom of the ViroSeq the two will be selected. Click on the Delete button in the toolbar or right-click
Explorer panel in the main window. to display the drop-down menu and select the Delete option to delete the selected
1. Exit from the ViroSeq software. folders.
2. Navigate to the workspace location on the computer. • Select multiple, non-contiguous project folders by holding down the Ctrl key
3. Using the Windows zip function, select the workspace folder and create a and then clicking on select project folders. Click on the Delete button in the
zipped file. toolbar or right-click to display the drop-down menu and select the Delete option
to delete the selected folders.
4. After the zipped file has been created, delete the original Workspace folder.
Note: When a workspace is deleted, the ViroSeq software displays a
message, noting that the workspace as not found or inaccessible. Do not
select the deleted workspace. Select a different workspace or create a new
workspace in the ViroSeq software.

38 of 54 Software Manual
Deleting Repositories • Testing laboratory
A single repository or multiple repositories can be deleted. Deleting a repository will – name of the laboratory that processed the sample
delete all of the projects contained within the repository. – the name of the laboratory director
– the name of the laboratory department that processed the sample
To delete a single repository – the laboratory mailstop of the department that processed the sample
• Click on the project to be deleted and click on the Delete button in the toolbar, or – the site address of the laboratory
• Right-click on the project to be deleted and click on the Delete option in the – the city where the laboratory is located.
drop-down menu. – the state or province where the laboratory is located
– the zip or postal code where the laboratory is located
To delete multiple repositories
– the country where the laboratory is located
• Select a range of repositories by clicking on a repository and then, while holding
– the contact phone number for the laboratory
down the Shift button, click on another repository; all of the repositories
between the two will be selected. Click on the Delete button in the toolbar or – the fax number for the laboratory
right-click to display the drop-down menu and select the Delete option to delete – the contact email for the laboratory
the selected repositories. – the website of the laboratory
• Select multiple, non-contiguous repositories by holding down the Ctrl key and • Drug resistance
then clicking on select repositories. Click on the Delete button in the toolbar or – NRTI drug class
right-click to display the drop-down menu and select the Delete option to delete
• mutations found
the selected repositories.
• drug names
– NNRTI drug class
Export to File Formats • mutations found
• drug names
XML – PI drug class
A report exported in XML format is populated with the following XML tags with the • mutations found
corresponding information: • drug names
• software name and version – Additional mutations
• project id – the sample file hierarchy (repository/project/sample name) • protease
• Patient information • reverse transcriptase
– patient identification number • reference sequence
– first name of the patient • consensus sequence
– middle name of the patient
– last name of the patient Consensus Nucleotide Sequence or Consensus Amino Acid
– sample accession number Sequence (FASTA)
– gender of the patient The FASTA file is an ASCII-formatted text file that contains file name information
– date of birth of the patient and a sequence.
– age of the patient
Comma Separated Values (CSV)
• Ordering physician
A report exported in a CSV format contains the following information:
– name of the physician that ordered the test performed
• Column 1 – sample name
– name of the institution where the physician is located
• Column 2 – the type of mutation (i.e., Resistance Mutations or Additional
• Assay information
Mutations)
– the date that the patient sample was drawn
• Column 3 – resistance mutations per drug class and additional mutations by
– the name of the operator who performed the assay
region (protease or reverse transcriptase)
– any information entered into Fields 1 and 2 of Patient Information
• Column 4 or more – detected mutations
– any comments made about the sample by the operator

ViroSeq HIV-1 Genotyping Software 39 of 54


Appendix D: Example
Data Set

This appendix provides a description of the example data set that is included with the
software. The example data set is stored in the Sample Data folder within the
application installation folder (C:\Programs\Celera\ViroSeq HIV 3.0\Sample
Data).
Users may import, analyze and compare their results with the expected results
described for each example.

Drug-Resistance
The Drug-Resistance sample is flagged red in the sample section, that indicates a
region of no coverage. The last base of the RT gene (335’3) has no data, indicated by
a dark line in the segment map region of the Sample Trace View. The Sample
Report provides the following Drug Resistance mutations: Edit
• NRTI Class - M41L, E44A, D67N, L74V, V118I, M184V, L210W, T215Y, Segment H of the Edit sample has flat peaks for about 200 base positions in the 3’
K219R end. Potential misalignments that could be caused by these peaks are auto-corrected
• NNRTI Class - K101E, V108I, H221Y by the ViroSeq software. The autocorrections are indicated by deletion markers (-)
• PI Class - L10I, M46I, I47V, A71V, L76V, I84A highlighted in yellow in the segment.

40 of 54 Software Manual
Segment H also has noisy data in the middle of the segment that results in several
mismatches between the segment base call and the consensus base call in some base
INDEL
positions. The mismatches are highlighted in yellow at the segment level. The INDEL sample is marked with a red review flag for out-of-frame indels and
segment D reports a status of “no signal”. Segment D is not used to generate the
mismatch deletion consensus sequence.

The ViroSeq software detects the drug resistant T69ins mutation in this sample.
There is a 2-codon insertion before codon 69 of the RT gene.

ViroSeq HIV-1 Genotyping Software 41 of 54


The ViroSeq software also identifies two single codon deletions in the Protease
gene. These are not true deletions or drug resistant positions: these deletions are a
HIV-3
result of auto correction for misalignment due to a mobility issue. These can be The HIV-3 sample contains one drug resistant mutation.
corrected, if needed, by editing the bases: • NRTI Class - None
• 69delH – Segments F, A, and G provide three-fold coverage in this region. • NNRTI Class - K103N
There is a mobility shift of an ‘A’ peak in segment F and segment G in the • PI Class - None
base position 69’1 that causes a mixture call ‘R’ at position 70’3 and a
deletion in position 69’1 in the two segments. This then results in a deletion
at codon 69.

This can be manually corrected by replacing the deletion in 69’1 by an ‘A’


and replacing the ‘R’ in 70’3 by a ‘G’ in segments F and G.
• 86delG – Segments F, A, and G provide three-fold coverage in this region.
There is a mobility shift of an ‘A’ peak in segment F and segment G in the
base position 86’3 that causes a mixture call ‘M’ at position 87’1 and a
deletion in position 86’3 in the two segments. This then results in a deletion
at codon 86.

This can be manually corrected by replacing the deletion in 86’3 by an ‘A’ and
replacing the ‘M’ in 87’1 by a ‘C’ in segments F and G.

42 of 54 Software Manual
Example of a Sample Report
A sample report example is provided within this section. For a description of the sample report contents, refer to Reports on page 26.

ViroSeq® HIV-1 Antiretroviral Drug Resistance Report

Patient ID: ----- Institution Name: Cooper Medical Suites

Accession Number: ----- Report Generated by: Administrator

Sample Name: Drug-Resistance Report Date & Time: Apr 25, 2013 9:55:32 AM

Drug Resistance:
NRTI Class Evidence of Resistance
EMTRIVA® (emtricitabine, FTC) Resistance***
EPIVIR® (lamivudine, 3TC) Resistance***

RETROVIR® (zidovudine, ZDV) Resistance***


VIDEX® (didanosine, ddI) Resistance***

VIREAD® (tenofovir, TDF) Resistance***

ZERIT® (stavudine, d4T) Resistance***

ZIAGEN® (abacavir, ABC) Resistance***

NNRTI Class Evidence of Resistance

EDURANT® (rilpivirine, RPV) Possible Resistance***


INTELENCE® (etravirine, ETR) Possible Resistance***
SUSTIVA® (efavirenz, EFV) Resistance***

VIRAMUNE® (nevirapine, NVP) Resistance***

PI+ Class Evidence of Resistance

APTIVUS® (tipranavir, TPV) Possible Resistance***


CRIXIVAN® (indinavir, IDV) Resistance***

FORTOVASE® / INVIRASE® (saquinavir, SQV) Resistance***

KALETRA® (lopinavir + ritonavir, LPV) Resistance***

LEXIVA® (fosamprenavir, FPV) Resistance***

PREZISTA® (darunavir, DRV) Possible Resistance***


REYATAZ® (atazanavir, ATV) Resistance***

VIRACEPT® (nelfinavir, NFV) Resistance***

* NOTE: At least one mutation used to determine Evidence of Resistance for this drug has not been fully validated.
** NOTE: At least one mutation used to determine Evidence of Resistance for this drug has not been clinically verified.
*** NOTE: For at least one mutation used to evaluate Evidence of Resistance for this drug, both notes above apply.
+ Evidence of Resistance for Protease Inhibitors estimates response to ritonavir-boosted regimens. Refer to section titled "Notes on Evidence of Resistance"

Notes on Evidence of Resistance:


Resistance Mutations present constitute a high level of genetic evidence for viral resistance
Possible Resistance Mutations present suggest the possibility of viral resistance
None There is insufficient evidence for viral resistance

The protease inhibitor (PI) evidence of resistance interpretations were developed to estimate the expected virological response to standard doses of protease inhibitors with
pharmacokinetic boosting by ritonavir. This has become the most common method of administering each of the protease inhibitors, except nelfinavir (ref. 1), to ensure adequate drug
levels in all patients. Boosted PIs are more active in the presence of resistance than non-boosted PIs. (ref. 2,3)

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ViroSeq HIV-1 Genotyping Software Page 43 of 54


ViroSeq® HIV-1 Antiretroviral Drug Resistance Report

Patient ID: ----- Institution Name: Cooper Medical Suites

Accession Number: ----- Report Generated by: Administrator

Sample Name: Drug-Resistance Report Date & Time: Apr 25, 2013 9:55:32 AM

Drug Resistance Mutations Identified:


NRTI Class:

M41L, D67N, L74V, M184V, L210W, T215Y, K219R

NNRTI Class:

K101E, K103R, V108I, V179D, H221Y

PI Class:

L10I, M46I, I47V, A71V, L76V, I84A

Additional Mutations:
Additional Mutations: The following amino acids differing from the reference sequence (HXB-2, accession number K03455) at the indicated
codon positions were identified and may be useful as a baseline determination of virus genotype.

Protease

V3I, G16A, K20R, M36I, S37D, R41K, Q61N, I62V, L63P, C67Y, I93L

RT

K20R, V35M, T39A, K43E, E44A, V118I, I202M, H208Y, R211K, L214F, R277K, T286A, I326V

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Page 44 of 54 Software Manual


ViroSeq® HIV-1 Antiretroviral Drug Resistance Report

Patient Information:
Accession Number ----- Patient Information
Assay Operator Name ----- ID -----
Institution Name ----- First Name -----
Physician ----- Middle Name -
Field 1 ----- Last Name -----
Field 2 ----- Gender Not Available
Date Drawn Date of Birth

Site Information:
Institution Name Cooper Medical Suites
Department Name Lab Testing
Lab Director Dr. S. Cooper
Address 1 Suite 4A
Address 2 2311 N. Los Robles Avenue
Mail Stop -
City Pasadena
State/Province California
ZIP / Postal Code -
Country -
Phone 213-454-8100
Fax -
Contact Email sheldor@caltech.com
Website URL -

Comments:
-

Review & Release of Results:

Signature / Date: Name(Print) / Title:

Notes:

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ViroSeq HIV-1 Genotyping Software Page 45 of 54


ViroSeq® HIV-1 Antiretroviral Drug Resistance Report

HIV-1 Resistance Mutation List:


NRTI Class Mutations Included within the Algorithm

EPIVIR® (lamivudine, 3TC)


{M184V,M184I**}
[69ins**,K65R,Q151M]
(V75I*,69del***,K65N***,K219Q,K219R***,L210W,D67T***,D67S***,D67N*,Q151L***,D67E,D67H***,D67G***,K70T***,A62
V,T215Y,F116Y,M41L,F77L,K219E***,K70E***,67del***,K70G***,K70Q***,K70R,K70S***,K219N***,K70N***,T215F)
EMTRIVA® (emtricitabine, FTC)
{M184V,M184I**}
[69ins**,K65R,Q151M]
(V75I*,69del***,K65N***,K219Q,K219R***,L210W,D67T***,D67S***,D67N*,Q151L***,D67E,D67H***,D67G***,K70T***,A62
V,T215Y,F116Y,M41L,F77L,K219E***,K70E***,67del***,K70G***,K70Q***,K70R,K70S***,K219N***,K70N***,T215F)
VIDEX® (didanosine, ddI)
{K65R,L74I***,L74V,Q151M,69ins**}
[K65N***,69del***,Q151L***,67del***,V75M***,T69D,K70E***,K70G***,V75T***]
(K219Q,K219R***,A62V,T215S***,F116Y,T215Y,T215V***,M41L,T215L***,T215N***,T215E***,T215D***,T215C***,T215I**
*,T215F,V75I*,T69A***,T215A***,V75S***,L210W,D67T***,D67S***,D67N*,T69N***,V75A***,D67E,T69S***,D67H***,D67G
***,K70T***,M184V,F77L,K219E,M184I**,K70Q***,K70R,K70S***,K219N***,K70N***)
ZERIT® (stavudine, d4T)
{Q151M,T215Y,T215F,69ins**,V75M***,V75T***}
[K65R,69del***,Q151L***,T215S***,T215V***,T215L***,67del***,T215N***,T215E***,T215D***,T215C***,T215I***,T215A**
*,V75S***,V75A***,K70E***,K70R]
(K65N***,K219Q,K219R***,A62V,F116Y,M41L,V75I*,T69A***,T69D,L210W,D67T***,D67S***,D67N*,T69N***,D67E,T69S*
**,D67H***,D67G***,K70T***,F77L,K219E,K70Q***,K70S***,K219N***,K70N***)
<M184V,M184I**>
ZIAGEN® (abacavir, ABC)
{69ins**,K65R,Q151M,Y115F*}
[L74I***,69del***,K65N***,L74V,Q151L***,K70E***,67del***,K70G***]
(V75I*,T215A***,K219Q,K219R***,L210W,D67T***,D67S***,D67N*,D67E,D67H***,D67G***,K70T***,M184V,A62V,T215S**
*,T215Y,F116Y,T215V***,M41L,T215L***,F77L,K219E,T215N***,V75T***,T215E***,M184I**,T215D***,K70Q***,T215C***,
K70R,K70S***,T215I***,K219N***,K70N***,T215F)
VIREAD® (tenofovir, TDF)
{69ins**,K65R}
[K65N***,Q151M,K70E***,K70G***]
(V75I*,L74I***,69del***,T215A***,K219Q,K219R***,L210W,D67T***,D67S***,D67N*,Q151L***,D67E,D67H***,D67G***,K70
T***,A62V,T215S***,T215Y,F116Y,T215V***,M41L,T215L***,F77L,K219E,T215N***,67del***,T215E***,T215D***,K70Q***,
T215C***,K70R,K70S***,Y115F*,T215I***,K219N***,K70N***,T215F)
<M184V,M184I**>
RETROVIR® (zidovudine, ZDV)
{69ins**,Q151M,T215Y,T215F}
[T215A***,Q151L***,T215S***,T215V***,T215L***,T215N***,67del***,T215E***,T215D***,T215C***,K70R,T215I***]
(V75I*,T69A***,K219Q,T69D,K219R***,L210W,D67T***,D67S***,D67N*,T69N***,D67E,D67H***,T69S***,D67G***,A62V,F
116Y,M41L,F77L,K219E,K219N***)
<K65R,K65N***,M184V,K70E***,M184I**>
NNRTI Class Mutations Included within the Algorithm

INTELENCE® (etravirine, ETR)


{Y181V***,Y181I*}
[M230L***,L100I*,K101P***,G190Q***,F227C***,Y181C,G190E***]
(E138G***,V106I***,H221Y***,E138A***,V179D***,V179E***,V179F***,Y188L**,M230I***,E138K***,V179L***,V179T***,K10
1H***,L100V***,A98G***,K101E*,G190T***,G190S*,G190V***,E138R***,G190A*,E138Q***,V90I***,G190C***)

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Page 46 of 54 Software Manual


ViroSeq® HIV-1 Antiretroviral Drug Resistance Report

NNRTI Class Mutations Included within the Algorithm

SUSTIVA® (efavirenz, EFV)


{V106M***,Y188L**,V106A**,K103N,K101P***,Y188C**,K103S***,G190Q***,K103H***,G190T***,G190S*,G190V***,G190
C***,G190E***}
[M230L***,Y181V***,Y188H***,L100I*,K103T***,P225H***,F227C***,K238T***,Y181I*,G190A*,Y181C]
(E138G***,V106I***,H221Y***,V179D***,V108I*,V179E***,V179F***,M230I***,E138K***,V179L***,K101Q***,V179T***,K101
H***,L100V***,A98G***,Y318F***,K238N***,F227L***,K101E*,E138R***,E138Q***,V90I***)
VIRAMUNE® (nevirapine, NVP)
{M230L***,Y181V***,V106M***,Y188L**,V106A**,Y188H***,K103N,K101P***,Y188C**,K103T***,K103S***,G190Q***,K10
3H***,G190T***,G190S*,F227C***,G190V***,Y181I*,G190A*,G190C***,G190E***,Y181C}
[M230I***,L100I*,Y318F***,P225H***,F227L***,K101E*,K238T***]
(E138G***,V106I***,H221Y***,V179D***,V108I*,V179E***,V179F***,E138K***,V179L***,K101Q***,V179T***,K101H***,L10
0V***,A98G***,K238N***,E138R***,E138Q***,V90I***)
EDURANT® (rilpivirine, RPV)
{Y181V***,Y188L**,K101P***,F227C***,Y181I*}
[M230L***,M230I***,E138K***,L100I*,G190Q***,Y181C,G190E***]
(E138G***,V106I***,H221Y***,E138A***,V179D***,V179E***,V179F***,V179L***,V179T***,K101H***,L100V***,A98G***,K1
01E*,G190T***,G190S*,G190V***,E138R***,G190A*,E138Q***,V90I***,G190C***)
PI Class Mutations Included within the Algorithm

APTIVUS® (tipranavir, TPV)


[I84V,V82L***,I84A***,V82T]
(V82M**,V32I*,V82C***,L90M,V82F**,L33F*,I54A***,I47V*,L10I,K43T***,I54S***,T74P***,I54V,L10R**,I54T*,A71L***,I84C*
**,Q58E***,L10V*,I47A***,A71V,I54M***,L10Y***,A71T*,V82S**,M46L*,A71I***,N83D***,M46I)
<L76V***,L24I***,I50L***,I54L***,I50V**>
CRIXIVAN® (indinavir, IDV)
{I84A***}
[I84V,L90M,I84C***,V82T,V82S**,L76V***,V82M**,V82A,V82C***,V82F**]
(M46V***,G48A***,V32I*,G73A***,F53L*,I54A***,L24I***,I47V*,L10I,L10F*,I54S***,I54V,T74P***,I54T*,I54M***,I54L***,G48
T***,G48S***,G48V,M46L*,G48Q***,G48L***,G48M***,M46I,N83D***,V82L***,N88S***,G73T***,G73S*,L10R**,A71L***,L1
0V*,I47A***,A71V,L10Y***,A71T*,G73C***,A71I***)
<I50L***>
FORTOVASE® / INVIRASE® (saquinavir, SQV)
{I84V,G48A***,I84C***,I84A***,G48T***,G48S***,G48V,G48Q***,G48L***,G48M***}
[L90M]
(V82A,V82C***,G73A***,V82F**,F53L*,L24I***,I54A***,L10I,I54S***,G73T***,T74P***,G73S*,I54V,L10R**,I54T*,A71L***,L1
0V*,A71V,I54M***,A71T*,L10Y***,I54L***,V82T,V82S**,G73C***,M46L*,A71I***,N83D***,M46I)
<L76V***,I50L***,I47A***>
KALETRA® (lopinavir + ritonavir, LPV)
{I47A***}
[I84A***,I50V**,L76V***]
(M46V***,I84V,G48A***,V32I*,L90M,G73A***,F53L*,L33F*,I54A***,L24I***,I47V*,L10I,L10F*,I54S***,I54V,T74P***,I54T*,I8
4C***,I54M***,I54L***,V82T,G48T***,V82S**,G48S***,G48V,M46L*,G48Q***,G48L***,G48M***,M46I,N83D***,V82M**,V82
L***,V82A,V82C***,V82F**,G73T***,G73S*,L10R**,A71L***,L10V*,A71V,L10Y***,A71T*,G73C***,A71I***)
<I50L***>
PREZISTA® (darunavir, DRV)
(V11I***,L76V***,G73T***,I84V,T74P***,G73S*,I84C***,V32I*,I84A***,L89V***,I47A***,G73A***,I54M***,V82F**,L33F*,I54L*
**,I50V**,I47V*,G73C***,L10F*)
<I50L***,N88S***>

FOR IN VITRO DIAGNOSTIC USE ViroSeq® HIV-1 Genotyping Software v3.0.0.28 Page 5 of 6

ViroSeq HIV-1 Genotyping Software Page 47 of 54


ViroSeq® HIV-1 Antiretroviral Drug Resistance Report

PI Class Mutations Included within the Algorithm

REYATAZ® (atazanavir, ATV)


{I84V,I84C***,I50L***,I84A***,N88S***}
[G48A***,G48T***,G48S***,G48V,G48Q***,G48L***,G48M***]
(M46V***,V32I*,L90M,G73A***,F53L*,L33F*,I54A***,L24I***,L10I,I54S***,N88G***,I54V,T74P***,I54T*,I54M***,I54L***,V82
T,V82S**,M46L*,M46I,N83D***,V82M**,V82L***,V82A,V82C***,V82F**,N88T***,G73T***,G73S*,L10R**,A71L***,L10V*,A7
1V,L10Y***,A71T*,G73C***,A71I***)
<L76V***>
VIRACEPT® (nelfinavir, NFV)
{I84V,L90M,D30N,I84C***,I84A***,N88D*,N88S***}
[G48A***,I54A***,N88G***,I54S***,I54V,I54T*,I54M***,I54L***,V82T,G48T***,L23I***,V82S**,G48S***,G48V,M46L*,G48Q*
**,G48L***,G48M***,M46I,V82A,V82C***,V82F**,N88T***]
(M46V***,V32I*,K20T***,G73A***,L24I***,I47V*,L10I,L10F*,T74P***,N83D***,V82M**,V82L***,G73T***,G73S*,L10R**,A71
L***,L10V*,I47A***,A71V,L10Y***,A71T*,G73C***,A71I***)
<I50L***>
LEXIVA® (fosamprenavir, FPV)
{L76V***,I84V,I84C***,I84A***,I47A***,I54M***,I54L***,I50V**}
[V82F**,I47V*]
(M46V***,V11I***,V82M**,V82L***,V82A,V32I*,V82C***,L90M,G73A***,L33F*,I54A***,L10I,L10F*,I54S***,G73T***,T74P***,
G73S*,I54V,L10R**,I54T*,A71L***,L10V*,L89V***,A71V,L10Y***,A71T*,V82T,V82S**,G73C***,M46L*,A71I***,N83D***,M4
6I)
<N88S***,I50L***>
Mutation Notations Key:
{Red Bold Curly Bracket} Presence of this mutation alone confers viral resistance
[Blue Bold-Italics Square Bracket] Presence of this mutation alone confers the possibility of viral resistance
(Black Parenthesis) This mutation must appear with at least one other mutation to confer the possibility of viral resistance
<Green Angle Bracket> This mutation counters resistance

* NOTE: This mutation has not been fully validated


** NOTE: This mutation has not been clinically verified
*** NOTE: For this mutation, both notes above apply

References:
1. Panel on Antiretroviral Guidelines for Adults and Adolescents. Guidelines for the use of antiretroviral agents in HIV-1-infected adults and
adolescents. Department of Health and Human Services. Available at http://aidsinfo.nih.gov/contentfiles/lvguidelines/AdultandAdolescentGL.pdf.
2. M Boffito, E Acosta, D Burger, CV Fletcher, C Flexner, R Garaffo, G Gatti, M Kurowski, CF Perno, G Paytavin, M Regazzi and D Back.
Current status and future prospects of therapeutic drug monitoring and applied clinical pharmacology in antiretroviral therapy. Antiviral Therapy,
2005, 10:375-392
3. N Shulman, A Zolopa, D Havir, A Hsu, C Renz, S Boller, P Jiang, R Rode, J Gallant, E Race, DJ Kempf and E Sun. Virtual inhibitory quotient
predicts response to fitonavir boosting of indinavir-based therapy in human immunodeficiency virus-infected patients with ongoing viremia.
Antimicrobial Agents Chemotherapy, 2002,46:3907-3916

FOR IN VITRO DIAGNOSTIC USE ViroSeq® HIV-1 Genotyping Software v3.0.0.28 Page 6 of 6

Page 48 of 54 Software Manual


Appendix E: Example of Diminished Peaks

Troubleshooting

The following table provides recommended corrective action to address problems


and error messages that may be encountered. If additional assistance is required,
refer to How to Obtain Services and Support on page 2.

Event Recommended Action

File import failure Refer to the Software Messages and


Warnings section for Import Data.

Invalid segment Refer to the Software Messages and


Warnings section for Invalid Segment

Sample Trace View does not Click in the Sample Trace View to
automatically refresh after being refresh the window.
resized.

Diminished peaks may appear across These peaks are primarily caused by
the entire sequence segment or may the presence of dye blobs at the Example of Diminished Peaks with Peak Height Increased
appear in limited regions within a beginning, middle or end of a
sequence segment. sequence segment. Less frequently,
the diminished peaks are caused by
low signal intensity.
Adjust the peak height of the
segment(s) in which the diminished
peaks appear, using the ViroSeq
software. Referr to Adjust the
Electropherogram Display on
page 19.

ViroSeq HIV-1 Genotyping Software 49 of 54


Software Messages and Warnings Affected Message Recommended Action
Software messages, including status and sample status messages, and warnings are Function
displayed with the occurrence of specific events. The following table provides a
Workspace The selected directory is Make sure that the
summary of messages with a recommended corrective action.
(continued) unreachable or does not Workspace directory is
Affected exist. Please select a correctly identified and the
Message Recommended Action different directory to be folder is present.
Function
used as the Workspace
Installation Administrator level Login to the computer as a
privileges are required to Windows OS Administrator The workspace used in the Make sure that the
install this application. user. last session of this Workspace directory is
Please logout and login as application is not found or correctly identified and the
an Administrator to install is inaccessible. folder is present. If the
this application. Workspace has been archived
and deleted, create a new
This system does not meet Make sure that the computer Workspace (refer to Create a
the minimum memory meets the minimum memory Workspace on page 7).
requirements. A minimum requirements before
of 1 GB of RAM for 32-bit installing the ViroSeq This workspace is in a If other users will need to use
OS or 2 GB of RAM for 64- software. private directory and will this workspace, create a
bit OS is required to install not be accessible by any workspace in a location
this application. other user on this available to other users.
computer. Make sure that the
There is not enough disk Check the computer hard Workspace folder is in a non-
space left for the proper disk and make sure that a user specific Windows
operation of the minimum of 5 GB of space location.
application. are available.
There is not enough disk Make sure that the available
Out of Disk Space Disk Check the computer hard space left for the proper disk space is greater than 5
space required for the disk and make sure that a operation of the GB. Refer to Archiving
installation exceeds minimum of 5 GB of space application. Analyzed Data on page 38.
available disk space. are available.
A workspace requires a Make sure that the available
A minimum screen Check to make sure that the minimum of 5 GB of disk space is greater than 5
resolution of 1024x768 is computer’s screen resolution available disk space GB. Refer to Archiving
required to install this is set to 1024 x 768 or higher. Analyzed Data on page 38.
application.
Password Password must have a Enter a password with a
System The memory available to Make sure that the available minimum length.The minimum of 6 characters.
Memory the application has reached memory in the system is minimum required is 6.
a very low level. The greater than 5 MB RAM.
application is unable to Change The password you re- Check and make sure that the
perform the requested Password entered does not match the password entered is the same
action unless more memory specified password; Please in both fields.
is made available to the try again.
application. Invalid user Id or Check and enter the correct
Application Another instance of the Close the current active password. Re-enter your User ID and/or Password.
application seems to be application and relaunch the user Id and password
running. Please exit it application to continue. Login SEC_E0002: Invalid user Check and enter the correct
before launching again. Id or password. Re-enter User ID and/or Password.
License Invalid license key. The license key used might your user Id and password.
be a non-ViroSeq v3.0 SEC_E0003: This user Contact your Administrator
license or a license generated account has been disabled. to make the account active or
for a different computer. Use Contact your use a different active
a ViroSeq v3.0 license key Administrator for account.
generated for the computer assistance.
used.
Settings The currently displayed Check the valid input range
Workspace The selected workspace is Make sure that the selected Window page contains invalid for the field displayed at the
not compatible with the Workspace folder was values. top of the window and enter
current application. Please created using ViroSeq v3.0 a valid value.
select another workspace. software.
Report An error has occurred Use an image for the report
Settings trying to process the logo that is not more than 48
selected file. x 48 pixels in dimensions.

50 of 54 Software Manual
Affected Message Recommended Action Affected Message Recommended Action
Function Function

Import You have selected a To avoid an extended and Patient Date of birth is later than Enter a date prior to today’s
Samples directory that may include time-consuming search, click Information today's date date.
a large number of No to exit the operation. (continued)
subdirectories. Including Click Browse and select a
The input field is over limit Enter no more than 32
subdirectories may take a specific subdirectory from
very long time to search for which files may be imported. of length of 32 characters in the field.
supported files. Are you Unable to acquire The Patient Information for
sure you want to include appropriate locks for this a sample cannot be edited if
subdirectories? operation it may be already the Sample Trace View
locked by another user or window for the sample is
Project already has Click OK to close the dialog
process. open. Close the Sample
<sample name>. Please and select a different project
import sample into another for import of the analyzed <sample name> No lock Trace View window and
acquired then edit the Patient
project. sample file.
Information.
Status: Unsupported Check the sample file (.ab1)
naming convention names and set the correct Sample This sample could not be Click OK to close the error
Analysis analyzed successfully and message. Check the Status
Naming Configuration to
so can not be opened in the column. If the status is “no
identify the primer name in
the file name. Trace View. signal” for all segments, re-
run the sample.
If the Naming
Configuration is correct, Re-analyze This operation will discard Click Yes to proceed with the
then the sample file(s) (.ab1) sample all previous edits made to operation or click Cancel to
may be corrupt or the version this sample (<sample stop further action.
of KB Basecaller does not name>). Would you like to
meet software requirements. continue?
Re-analyze sample files
Unable to acquire A sample cannot be re-
using KB Basecaller v1.2 or
appropriate locks for this analyzed if the Sample
higher.
operation it may be already Trace View window for the
Invalid Status: Short segment The segment is shorter than locked by another user or sample is open. Choose
Segment 100 bases after process. Cancel to close the Analysis
Sample Status: After auto-
autotrimming. Remove this <sample name> No lock Settings dialog box. Close
trimming this segment is
below the minimum segment and add a valid acquired the Sample Trace View
segment. window and then proceed
requirement of 100 bases
with the sample re-analysis.
Status: Poor quality The average QV is less than
20. Remove this segment and User attempts The Drug Logic has Select Yes to proceed with
Sample Status: Average to open the changed since this sample the re-analysis of the sample
quality value for this add a valid segment.
Sample was last analyzed. If you with the updated Drug Logic.
segment is below 20 Trace View choose to re-analyze, Select No to proceed to the
Status: No signal No signal was detected for changes will be saved Sample Trace View without
this segment. Remove this automatically and previous re-analysis of the sample.
segment and add a valid results will no longer be
available. Do you want to Select Cancel to stop further
segment. action.
re-analyze the sample using
All Segments Status: Analysis failed No valid segments were the current drug logic?
Invalid Sample Status: This sample detected. Remove all
segments and add valid Remove Can not remove the last At least one segment is
does not have any valid
segments. Segment segment. required to assemble a
segments
sample. If the sample is no
Add No source files found in the Use the Browse button to longer needed, delete it from
Segment selected directory or any of choose a directory or the sample section in the
its subdirectories subdirectory that contains main window.
valid source files (.ab1). Trimming Segment will be too short if A segment cannot be
Patient Comments are limited to a Reduce the text content to Segment trimmed from here trimmed if it would make the
Information maximum of 500 500 characters or less. segment shorter than 100
characters. bases long.

Date Drawn is later than Enter a date prior to today’s Export to file A file already exists with Click Yes to overwrite the
today's date date. the name: <filename> Do file. Click No to exit the
you want to overwrite it? dialog and re-export the file
with a different file name.

ViroSeq HIV-1 Genotyping Software 51 of 54


Affected Message Recommended Action
Function

Export to file The selected directory is in If other users will need to use
(continued) a private directory and will this workspace, create a
not be accessible by any workspace in a location
other user on this available to other users.
computer. Make sure that the selected
export folder is in a non-user
specific Windows location.

Export <Filename> exists. Click Overwrite to replace


Analyzed Overwrite old file or the existing file, click
Sample rename new file? Rename to provide a
different name for the file, or
click Cancel to exit the
operation.

Repository Deleting this repository will Click Yes to delete the


also delete all its content; repository and its contents.
this action cannot be Click Cancel to exit the
undone. Proceed with the operation.
deletion?

Project or Deleting a project or a Click Yes to delete the


Sample sample will remove them project or sample, or click
from the application. Cancel to exit the dialog.
Samples will have to be
imported again to be used.
Proceed with the deletion?

52 of 54 Software Manual
Appendix F: Glossary .FASTA
An ASCII-formatted text file that contains file name information and a
sequence.The text-based format represents nucleotide sequences. The base pairs and
amino acids are represented by single-letter codes.

Frame
An organization of a nucleotide sequence into consecutive, non-overlapping triplet
sets of bases.

.ab1 Indel
A file type produced by Applied BioSystems software for sequencing data Abbreviation for insertion and deletion mutations.
analysis.The file type used for import by the ViroSeq HIV-1 Genotyping Software
v3.0. In-frame Indel
An in-frame insertion or deletion composed of a number of bases that is divisible by
Additional Mutation three.
The number of identified mutations that are not used in the calculation of drug
resistance. Insertion
Unexpected nucleotide base(s) that has been added to a known sequence.
Administrator
One of two user roles defined within the ViroSeq HIV-1 Genotyping Software v3.0. IUB
An administrator is able to change all settings. An administrator user account cannot International Union of Biochemists
be made inactive or disabled.
Lab Technician
ASCII One of two user roles defined within the ViroSeq HIV-1 Genotyping Software. Lab
American Standard Code for Information Interchange. A simple text format that is Technicians do not have the ability to create, edit, or disable user accounts, edit the
recognizable across many platforms and computers. site information, or edit the report settings.

Base Count License Key


The total number of bases detected in the segment. A unique product key required for software installation. The ViroSeq HIV-1
Genotyping Software license key is only for use on the computer system on which
Base Call the application is installed and registered (individual license key).
The nucleotide identity assigned to a signal peak in an electropherogram.
Misalignment or Misaligned Segment
Codon A segment that is not aligned with the Reference Sequence.
A triplet of adjacent nucleotide bases that specify a given amino acid.
Mismatched Base Calls
Codon Notation Base calls that differ between the consensus and reference sequence.
Codon position and base location within the codon (e.g., codon 101'1).
Mixture or Mixed Bases
Consensus Sequence More than one nucleotide base detected at a single base position.
A combined sequence of nucleotide bases that is created from an alignment of
individual sequences. The consensus sequence is created by polling each codon Navigation Bar
position in the sequences and placing the base with the highest count in the A graphical element showing POIs and providing a means to navigate to the
consensus sequence. corresponding data.

Consensus Translation Out-of-frame Indel


The amino acid sequence translated from the consensus nucleotide sequence. An out-of-frame insertion or deletion composed of a number of bases that is not
divisible by three.
.CSV
Comma Separated Value. A comma is used as a delimiter between values, to PDF
separate information that is to be saved and later retrieved from within a database. Portable Document Format.

Deletion POI or Position of Interest


Expected nucleotide base that is removed or missing from a known sequence. Position of Interest. An area within a segment identified by the ViroSeq HIV-1
Genotyping Software that requires the user’s attention.
Dye Blob(s)
A large peak or peaks that correlate to the presence of unincorporated dye Primer Identification
terminators, which may or may not obscure the sample sequence. The matching of a sequence to a known primer.

Electropherogram Project
The processed fluorescent signals of sequencing dyes computed by the data The third level of data management in the ViroSeq HIV-1 Genotyping Software
collection and analysis software provided with sequencing instrumentation. v3.0, contained within a repository. A project contains sample data files (.ab1) and
allows users to separate files into logical groupings.
Explorer Panel
Contains a list of all of the available repositories and projects.

ViroSeq HIV-1 Genotyping Software 53 of 54


QV
Quality Value. An established metric for determining the quality of sequencing data.
• For the segment QV, a QV > 20 indicates that the probability of a base miscall is
< 1.0%.

Reading Frames
A division of a nucleotide sequence into consecutive, non-overlapping triplets.

Reference Sequence
The reference nucleotide base sequence to which the consensus nucleotide sequence
is compared. The ViroSeq reference sequence is based on HXB-2 (GenBank
K03455).

Reference Translation
The amino acid sequence translated from the reference sequence.

Repository
The second level of data management in the ViroSeq HIV-1 Genotyping Software
v3.0, contained within a workspace. A repository contains one or more projects. A
repository allows for the logical grouping of separate projects.

Sample File
A file containing the sample sequence data, generated in the Sequencing Analysis
Software.

Sample Trace View


A window in the ViroSeq HIV-1 Genotyping Software that allows users to view and
edit the nucleotide sequences of a file.

Start Point Parameter


In the Sequencing Analysis Software, the Start Point parameter is the raw data
point where the basecalling starts in the sample file, which is usually the same point
as the beginning of the first base peak.

Stop Point Parameter


In the Sequencing Analysis Software, the Stop Point parameter specifies where the
last raw data point is to be included in the basecalling; this is the last data point in the
file.

Workspace
The highest level of data management in the ViroSeq HIV-1 Genotyping Software
v3.0. A workspace is a user-defined location in which data can be imported,
analyzed, and reviewed. Each workspace has unique software settings. One
workspace can be used by a single user (e.g., private) or multiple users, based upon
the Windows access rights to a given folder location. Users (e.g., Administrator or
Lab Technician) only have access to workspaces in which their user account was
defined. Workspaces contain repositories.

.VAS
ViroSeq Analyzed Sample. The format used by the ViroSeq HIV-1 Genotyping
Software v3.0 for export and import of a single analyzed sample.

.XML
Extensible Markup Language. A simple text-based format used to represent
structured information. This flexible text format is used in exchange of data on the
Web and elsewhere.

54 of 54 Software Manual
© 2013 Celera Corporation. All rights reserved.
TRADEMARKS
Algorithm Advisor, Celera and its Spirit Design, and ViroSeq are trademarks and registered trademarks of Celera
Corporation or its subsidiaries in the U.S. and/or certain other countries.
ABI PRISM, Applied Biosystems, and KB are trademarks and registered trademarks of Life Technologies
Corporation or its subsidiaries in the U.S. and/or certain other countries.
Microsoft and Windows are registered trademarks of Microsoft Corporation.
All other trademarks are the sole property of their respective owners.

DRAFT
May 8, 2013 3:52 pm, SW Manual Back.fm
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