Professional Documents
Culture Documents
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
• 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.
Key to Symbols
For In Vitro
= Diagnostic Use = Catalog Number
= Manufacturer = Distributor
2 of 54 Software Manual
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.
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
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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.
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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.
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.
Settings
The next step in set up is to customize the settings:
• Analysis Settings
• General Settings
• Naming Configuration
• Report Settings
• Site Information
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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.
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• 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.
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.
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.
sample section
Explorer panel
Audit Trail /
Sample History
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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.
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.
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Sample Trace View Window
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.
• 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.
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.
• 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.
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.
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
$&KHFNWKH'1$ %&UHDWHD1HZ &&UHDWHD1HZ repositories may be created within a workspace. Each repository can
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$QDO\VLV'DWD $FFHVVDQ([LVWLQJ DQ([LVWLQJ
contain one or more projects.
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Create a New Repository
1. In the ViroSeq software, click on the Add New Repository button in the
main toolbar, or right-click in the Explorer panel and select Add New
Repository from the drop-down menu.
2. In the Data Repository dialog box, input a name for the repository in the
'&RQILUPILOH
1DPLQJ (,PSRUW'DWD Display Name text field.
)$QDO\]H'DWD
&RQILJXUDWLRQ )LOHV
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3. Click on the OK button.
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.
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.
7. After all of the POIs have been confirmed, click on the Save button to
preserve changes.
-----
Institution Name:
Report Generated by:
Cooper Medical Suites
Administrator
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***
* 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"
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)
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.
standard doses of
protease inhibitors with Site Information:
common method of
Address 2 2311 N. Los Robles Avenue
Mail Stop -
administering each of the City Pasadena
boosted PIs.2,3
Notes:
{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
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.
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.
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.
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:
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.
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 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
Data Organization
:RUNVSDFH
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.
• 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.
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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
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.
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.
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***
* 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"
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)
FOR IN VITRO DIAGNOSTIC USE ViroSeq® HIV-1 Genotyping Software v3.0.0.28 Page 1 of 6
Sample Name: Drug-Resistance Report Date & Time: Apr 25, 2013 9:55:32 AM
NNRTI Class:
PI Class:
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
FOR IN VITRO DIAGNOSTIC USE ViroSeq® HIV-1 Genotyping Software v3.0.0.28 Page 2 of 6
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:
-
Notes:
FOR IN VITRO DIAGNOSTIC USE ViroSeq® HIV-1 Genotyping Software v3.0.0.28 Page 3 of 6
FOR IN VITRO DIAGNOSTIC USE ViroSeq® HIV-1 Genotyping Software v3.0.0.28 Page 4 of 6
FOR IN VITRO DIAGNOSTIC USE ViroSeq® HIV-1 Genotyping Software v3.0.0.28 Page 5 of 6
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
Troubleshooting
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.
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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.
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.
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.
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.
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.
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.
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