illumina NextSeq 550Dx User manual

NextSeq 550Dx
Instrument Reference Guide
Document # 1000000009513 v00
November 2017
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FAILURE TO COMPLETELY READ AND EXPLICITLY FOLLOW ALL OF THE INSTRUCTIONS CONTAINED HEREIN
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Table of Contents
Chapter 1 Overview 1
Sequencing Features 1
Additional Resources 1
Instrument Components 1
Sequencing Consumables Overview 3
User-Supplied Consumables and Equipment 6
Chapter 2 NextSeq 550Dx Software 9
NextSeq 550Dx Software Overview 9
Local Run Manager Overview 11
User Passwords 16
Chapter 3 Getting Started 19
Starting the Instrument 19
Customize System Settings 20
Reboot and Shut Down Options 21
Chapter 4 Sequencing 23
Introduction 23
Sequencing Workflow 24
Create Run with Local Run Manager Software 24
Prepare the Reagent Cartridge 25
Prepare the Flow Cell 25
Prepare Libraries for Sequencing 25
Load Libraries onto the Reagent Cartridge 26
Set Up a Sequencing Run 26
Monitor Run Progress 31
View Run and Sample Data 33
Possible Run States 34
Requeue or Stop Analysis 34
Automatic Post-Run Wash 35
Chapter 5 Maintenance 37
Introduction 37
Perform a Manual Wash 37
Replace Air Filter 40
Appendix A Administrative Settings and Tasks 43
Introduction 43
User Management 43
System Settings 45
Module Settings 46
Audit Trails 47
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Appendix B Troubleshooting 49
Introduction 49
System Check 49
Troubleshooting Files 51
Resolve Automatic Check Errors 51
Spent Reagents Container is Full 52
RAID Error Message 53
Configure System Settings 53
Appendix C Real-Time Analysis 55
Real-Time Analysis Overview 55
Real-Time Analysis Workflow 56
Appendix D Output Files and Folders 59
Sequencing Output Files 59
Output Folder Structure 61
Index 63
Technical Assistance 67
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Chapter 1 Overview
Sequencing Features 1
Additional Resources 1
Instrument Components 1
Sequencing Consumables Overview 3
User-Supplied Consumables and Equipment 6
Sequencing Features
uHigh-throughput sequencing—The NextSeq 550Dx instrument enables sequencing of DNA
libraries.
uReal-Time Analysis (RTA)—Performs image processing and base calling. For more information, see
Real-Time Analysis on page 55.
uOn-instrument data analysis capability— Local Run Manager software analysis modules specified
for the run can analyze run data.
uDual Boot—The NextSeq 550Dx instrument contains separate hard drives that support diagnostic
(Dx) and research (RUO) modes.
Additional Resources
The following documentation is available for download from the Illumina website.
Resource Description
NextSeq 550Dx Instrument Site
Prep Guide (document #
1000000009869)
Provides specifications for laboratory space, electrical requirements, and environmental
considerations.
NextSeq 550Dx Instrument Safety
and Compliance Guide (document
# 1000000009868)
Provides information about operational safety considerations, compliance statements,
and instrument labeling.
RFID Reader Compliance Guide
(document # 1000000030332)
Provides information about the RFID reader in the instrument, compliance certifications,
and safety considerations.
NextSeq 550Dx Research Mode
Instrument Reference Guide
(document # 1000000041922)
Provides information about operating the NextSeq 550Dx instrument in research mode.
Instrument Components
The NextSeq 550Dx instrument includes a touch screen monitor, a status bar, and 4 compartments.
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Figure 1 Instrument Components
AImaging compartment—Holds the flow cell during a sequencing run.
BTouch screen monitor—Enables on-instrument configuration and setup using the operating software
interface.
CStatus bar—Indicates instrument status as processing (blue), requires attention (orange), or ready to
sequence (green).
DBuffer compartment—Holds the buffer cartridge and the spent reagents container.
EReagent compartment—Holds the reagent cartridge.
FAir filter compartment—Holds the air filter. Access the filter from the back of the instrument.
GPower button—Powers the instrument and the instrument computer on or off.
Imaging Compartment
The imaging compartment houses the stage, which includes three alignment pins for positioning the flow
cell. After loading the flow cell, the imaging compartment door closes automatically and moves
components into position.
Reagent and Buffer Compartments
Setting up a sequencing run on the NextSeq 550Dx instrument requires access to the reagent
compartment and buffer compartment to load run consumables and empty the spent reagents container.
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Figure 2 Reagent and Buffer Compartments
AReagent compartment door—Encloses the reagent compartment with a latch under the lower-right
area of the door. The reagent compartment holds the reagent cartridge.
BReagent cartridge—The reagent cartridge is a prefilled single-use consumable.
CBuffer cartridge—The buffer cartridge is a prefilled single-use consumable.
DSpent reagents container—Spent reagents are collected for disposal after each run.
EBuffer compartment door—Encloses the buffer compartment with a latch under the lower-left corner of
the door.
Air Filter Compartment
The air filter compartment holds the air filter and is located in the back of the instrument. Replace the air
filter every 90 days. For information on replacing the filter, see Replace Air Filter on page 40.
Power Button
The power button on the front of the NextSeq 550Dx turns on power to the instrument and instrument
computer. The power button performs the following actions depending on the state of instrument power.
By default, the NextSeq 550Dx boots into diagnostic mode.
For information on initial power up of the instrument, see Starting the Instrument on page 19.
For information on shutting down the instrument, see Shut Down the Instrument on page 21.
Power State Action
Instrument power is off Briefly press the button to turn on the power.
Instrument power is on Briefly press the button to turn off the power. A dialog box appears on the screen to confirm
a normal instrument shutdown.
Instrument power is on Press and hold the power button for 10 seconds to cause a hard shutdown of the instrument
and instrument computer.
Use this method to turn off the instrument only if the instrument is unresponsive.
NOTE
Turning off the instrument during a sequencing run ends the run immediately. Ending a run is final. Run
consumables cannot be reused and sequencing data from the run is not saved.
Sequencing Consumables Overview
The sequencing consumables required to run the NextSeq 550Dx are provided separately in a single-use
kit. Each kit includes one flow cell, a reagent cartridge, a buffer cartridge, and library dilution buffer. For
more information, see the package insert NextSeq 550Dx High Output Reagent Kit v2 .
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The flow cell, reagent cartridge, and buffer cartridge use radio-frequency identification (RFID) for
accurate consumable tracking and compatibility.
NOTE
Keep sequencing consumables stored in their boxes until ready for use.
Required Storage for Sequencing Consumables
Item (1 per run) Storage Requirement
Library dilution buffer -25°C to -15°C
Reagent cartridge -25°C to -15°C
Buffer cartridge 15°C to 30°C
Flow cell cartridge 2°C to 8°C
Flow Cell Overview
Figure 3 Flow Cell Cartridge
ALane pair A—Lanes 1 and 3
BLane pair B—Lanes 2 and 4
CFlow cell cartridge frame
The flow cell is a glass-based substrate on which clusters are generated and the sequencing reaction is
performed. The flow cell is encased in a flow cell cartridge.
The flow cell contains 4 lanes that are imaged in pairs.
uLanes 1 and 3 (lane pair A) are imaged at the same time.
uLanes 2 and 4 (lane pair B) are imaged when imaging of lane pair A is complete.
Although the flow cell has 4 lanes, only a single library or set of pooled libraries is sequenced on the
flow cell. Libraries are loaded onto the reagent cartridge from a single reservoir and transferred
automatically to the flow cell to all 4 lanes.
Each lane is imaged in small imaging areas called tiles. For more information, see Flow Cell Tiles on
page 59.
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Reagent Cartridge Overview
The reagent cartridge is a single-use consumable with RFIDtracking and foil-sealed reservoirs that are
prefilled with clustering and sequencing reagents.
Figure 4 Reagent Cartridge
The reagent cartridge includes a designated reservoir for loading prepared libraries. After the run begins,
libraries are transferred automatically from the reservoir to the flow cell.
Several reservoirs are reserved for the automatic post-run wash. Wash solution is pumped from the
buffer cartridge to the reserved reservoirs, through the system, and then to the spent reagents container.
WARNING
This set of reagents contains potentially hazardous chemicals. Personal injury can occur
through inhalation, ingestion, skin contact, and eye contact. Wear protective equipment,
including eye protection, gloves, and laboratory coat appropriate for risk of exposure. Handle
used reagents as chemical waste and discard in accordance with applicable regional, national,
and local laws and regulations. For additional environmental, health, and safety information, see the
SDS at support.illumina.com/sds.html.
Reserved Reservoirs
Figure 5 Numbered Reservoirs
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Position Description
7, 8, and 9 Reserved for optional custom primers
10 Load libraries
Removable Reservoir in Position #6
The prefilled reagent cartridge includes a denaturation reagent in position 6 that contains formamide. To
facilitate safe disposal of any unused reagent after the sequencing run, the reservoir in position 6 is
removable. For more information, see Remove Used Reservoir from Position #6 on page 30.
Buffer Cartridge Overview
The buffer cartridge is a single-use consumable containing three reservoirs that are prefilled with buffers
and wash solution. The contents of the buffer cartridge are sufficient for sequencing one flow cell.
Figure 6 Buffer Cartridge
Library Dilution Buffer Overview
The library dilution buffer is in the NextSeq 550Dx Accessory Box. Use the buffer as needed to dilute
libraries according to the library preparation instructions to achieve recommended cluster densities.
User-Supplied Consumables and Equipment
The following consumables and equipment are used for consumables preparation, sequencing, and
instrument maintenance.
User-Supplied Consumables for Sequencing Runs
Consumable Supplier Purpose
Alcohol wipes, 70% Isopropyl
or
Ethanol, 70%
VWR, catalog#95041-714
(or equivalent)
General lab supplier
Flow cell cleaning and general purpose
Lab tissue, low-lint VWR, catalog#21905-026
(or equivalent)
Flow cell cleaning and general purpose
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User-Supplied Consumables for Instrument Maintenance
Consumable Supplier Purpose
NaOCl, 5%
(sodium hypochlorite)
Sigma-Aldrich, catalog # 239305
(or laboratory-grade equivalent)
Washing the instrument using the
manual post-run wash; diluted to 0.12%
Tween 20 Sigma-Aldrich, catalog # P7949 Washing the instrument using manual
wash options; diluted to 0.05%
Water, laboratory-grade General lab supplier Washing the instrument (manual wash)
Air filter Illumina, catalog # 20022240 Cleaning the air the instrument takes in
for cooling
User-Supplied Equipment
Item Source
Freezer, -25°C to -15°C, frost-free General lab supplier
Refrigerator, 2°C to 8°C General lab supplier
Guidelines for Laboratory-Grade Water
Always use laboratory-grade water or deionized water to perform instrument procedures. Never use tap
water. Use only the following grades of water or equivalents:
uDeionized water
uIllumina PW1
u18Megohms (MΩ) water
uMilli-Q water
uSuper-Q water
uMolecular biology grade water
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Chapter 2 NextSeq 550Dx Software
NextSeq 550Dx Software Overview 9
Local Run Manager Overview 11
User Passwords 16
NextSeq 550Dx Software Overview
The software described in this chapter is used to configure, run, and analyze data from the NextSeq
550Dx instrument.The instrument software includes integrated applications that perform sequencing
runs. Software updates are performed by Illumina personnel.
uLocal Run Manager software— Integrated software solution for creating a run and analyzing results
(secondary analysis). The software also provides sample tracking and controls user permissions.
For more information, see Local Run Manager Overview on page 11
uNextSeq 550Dx Operating Software (NOS)—Controls instrument operation.
uThe software is preinstalled on the NextSeq 550Dx, and runs on-instrument. The NOS performs
the run according to the parameters specified in the Local Run Manager software module.
uBefore you start the sequencing run, you select a run that you created with Local Run Manager.
The NOS software interface guides you through the steps to load the flow cell and reagents.
uDuring the run, the software operates the flow cell stage, dispenses reagents, controls fluidics,
sets temperatures, captures images of clusters on the flow cell, and provides a visual summary
of quality statistics. You can monitor the run in NOS, or in Local Run Manager.
uReal-Time Analysis (RTA) software— RTA performs image analysis and base calling, also referred
to as primary analysis, during the run. For more information, see Real-Time Analysis on page 55.
Required Disk Space
The integrated instrument computer has approximately 1.5TB of storage capacity.
Before starting a run, the software checks available disk space. The amount of disk space required
depends on the Local Run Manager analysis module. If there is not enough disk space for the run, a
software prompt appears. The message indicates how much disk space is required for the run and how
much disk space must be cleared before the run can proceed. If there is insufficient space, delete run
folders that do not need to be reanalyzed in Local Run Manager. For more information, see Delete a Run
Folder on page 15.
Status Icons
A status icon in the top-right corner of the NOS signals any change in conditions during run setup or
during the run.
Status Icon Status Name Description
Status OK System is normal.
Processing System is processing.
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Status Icon Status Name Description
Warning A warning has occurred.
Warnings do not stop a run or require action before proceeding.
Error An error has occurred.
Errors require action before proceeding with the run.
Service Needed A notification requiring attention has occurred. Refer to the message for additional
information.
When a change in condition occurs, the icon blinks to alert you. Select the icon to view a description of
the condition. Select Acknowledge to accept the message and Close to close the dialog box.
NOTE
Acknowledging a message resets the icon, and the message is grayed out. The message is still
visible to the user if they select the icon, but disappears once NOS is restarted.
Navigation Bar Icons
The minimize NOS icon is in the top-right corner of the operating software interface and is only visible to
admin users.
Access
Icon
Icon Name Description
Log Off Select to log off of the software.
Minimize NOS Select to minimize NOS to access Windows applications and folders.
This icon appears for admin users only.
NOSHome Screen
The NOSHome Screen features the following four icons.
uSequence— Select to start a sequencing run from a pre-defined run list.
uLocal Run Manager—Select to launch Local Run Managerfor creating a run, monitoring run status,
analyzing sequencing data, and viewing results. See Local Run Manager Overview on page 11.
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uManage Instrument— Select to control system settings, reboot to research mode, or shut down the
instrument software.
uPerform Wash— Select to perform a Quick Wash or the Manual Post-Run Wash.
Local Run Manager Overview
The Local Run Managersoftware is an on-instrument integrated solution for creating a run, monitoring
status, analyzing sequencing data, and viewing results.
The Local Run Manager has the following features:
uRuns as a service on the instrument computer and integrates with the instrument operating
software.
uRecords samples to be sequenced.
uProvides a run setup interface specific to the analysis module.
uPerforms a series of analysis steps specific to the selected analysis module.
uShows analysis metrics in the form of tables and graphs upon completion of analysis.
Viewing Local Run Manager
The Local Run Manager interface is viewed within NOS or through a web browser. The supported web
browser is Chromium.
NOTE
If you use an unsupported browser, download the supported browser when prompted on the "Confirm
Unsupported Browser" message. Select "here" to download the supported version of Chromium.
View from a Networked Computer
1 Open a Chromium web browser on a computer with access to the same network as the instrument
and connect using the instrument IP address or instrument name. For example,
http://myinstrument.
View on the Instrument Monitor
1 To view the Local Run Manager interface on the instrument monitor, select one of the following
options:
uFrom the NOSHome Screen, select Local Run Manager.
Click the X in the upper right corner to return to NOS when done.
uSelect the Minimize NOSicon, open the Chromium web browser that is on the instrument, and
enter http://localhost in the address bar.
Only admin users can minimize NOS.
Sequencing During Analysis
The NextSeq 550Dx system computing resources are dedicated to either sequencing or analysis. If a
new sequencing run is needed on the NextSeq 550Dx before secondary analysis of an earlier run is
complete, the user can cancel secondary analysis from within Local Run Manager and then start a new
sequencing run.
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To restart the analysis performed by Local Run Manager, use the Requeue feature on the Local Run
Manager interface after the new sequencing run is complete. At that point, secondary analysis starts
from the beginning. See Requeue or Stop Analysis on page 34.
Local Run Manager Workflow
Using the Local Run Manager interface, create a run. Because the specific steps
vary for each analysis module, see the workflow guide for the analysis module you
are using for step-by-step instructions.
Using NOS, select the planned run, load consumables, and start sequencing.
Local Run Manager loads the specified analysis module and performs data
analysis.
Using the Local Run Manager interface, select a completed run to view the results
page.
Dashboard Overview
After you log in to the Local Run Manager software, the dashboard opens. From the dashboard, you can
perform the following tasks:
uTrack sequencing and analysis status
uSort and filter runs
uCreate and edit runs
uStop or requeue analysis
uView analysis results
uHide runs
uPin runs
The dashboard lists all runs performed on the instrument, 10 runs per page. Use the navigation arrows
at the bottom of the list to scroll through pages.
Each run listed includes the run name, analysis module, run status, and the date that the run was last
modified. To sort runs, click the column names.
uRun Name / ID—Lists the assigned run name and links to the run results pages. After analysis is
complete, the analysis folder name is added to the run name column.
uModule—Lists the analysis module assigned to the run.
uStatus—Lists the status of the run and includes a progress status bar. For more information, see
Possible Run States on page 34.
uLast Modified—Lists the date and time that a run or analysis session was last modified. By default,
the Active Runs page is sorted by this column.
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Local Run Manager Icons
The following icons are used in Local Run Manager to perform various tasks. Note that depending on the
size of the screen, some icons are condensed under the More Options icon.
Icon Name Description
Copy to
clipboard
Copies a field to the computer clipboard.
Delete run
folder
Deletes the data from a run to clear hard drive space.
Edit Enables editing of run parameters before sequencing.
Edit output
folder
location
Enables editing of run folder path.
Edit users Enables editing of passwords and users.
Export Command to export items.
Hide run Moves a run from the active runs pages to the hidden runs page. View hidden runs using
the menu at the lower right corner of the active runs page.
Locked Indicates that the run is being sequenced, or another user is editing a run in another
browser session.
More
options
Opens available options for an entry on the dashboard. Depending on the size of the
screen, the dots can look more round.
Pin Pins a run to the active runs page to ensure that the run folder will not be deleted.
Restore run Moves a run from the hidden runs page to the active runs page.
Relink run
folder
Enables a run to be relinked after a deleted run folder is restored. The run can then be
requeued for analysis.
Requeue Restarts analysis of the selected run.
Run is
pinned
Indicates that a run has been pinned to the active runs page.
Run deleted Indicates a deleted run.
Save output
folder
location
Save edits to the path of the output run folder.
Search Opens the search field to search run names and sample IDs. Depending on the size of
the screen, the icon can be condensed underneath the more options icon.
Show filters Shows filters.
Stop
analysis
Stops analysis by the Local Run Manager module.
Unpin Unpin a run.
User Opens the user account menu.
Warnings
detected
See the notification text for information about warnings.
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Active Runs Page
The dashboard opens to show the Active Runs page. Across the top of the Active Runs page, a
summary of active runs provides a count of runs in each of the following run status categories. To filter
active runs by status, click the number.
uReady—Indicates that the run is ready for sequencing.
uIn Progress—Indicates that the run is in the process of sequencing or data analysis.
uStopped or Unsuccessful—Indicates that analysis was stopped manually or that analysis was
unsuccessful.
uComplete—Indicates that analysis was successfully completed.
uTotal—The total number of runs on the Active Runs page.
Filter Runs
1 Click a run status category in the summary of active runs to filter the Active Runs page as follows.
Ready—Filters the list to show only runs that are ready for sequencing.
In Progress—Filters the list to show only runs that are in progress.
Stopped or Unsuccessful—Filters the list to show only runs that were stopped or unsuccessful.
Complete—Filters the list to show only runs that are complete.
Total—Removes filters and shows all active runs.
Hide and Restore Runs
1 From the Active Runs page, click the More Options icon, and then click Hide.
2 When prompted to confirm the move, click Hide.
The run is moved to the Hidden Runs page.
3 To show the Hidden Runs page, click the Active Runs drop-down arrow and select Hidden Runs.
4 From the Hidden Runs page, click the Restore icon.
5 When prompted to confirm the move, click Restore.
The run is restored to the Active Runs page.
6 To show the Active Runs page, click the Hidden Runs drop-down arrow and select Active Runs.
Search for Runs or Samples
1 From the dashboard navigation bar, click the Search icon.
2 In the search field, enter a run name or sample ID.
As you type, a list of possible matches appears to assist your search.
3 Select a match from the list or press Enter.
uIf you searched on a run name, the Run Overview tab opens.
uIf you searched on a sample ID, the Samples and Results tab opens.
For more information, see View Run and Sample Data on page 33.
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Edit a Run
1 From the Active Runs page, click the More Options icon next to the run name that you want to
edit.
2 Click Edit.
3 When prompted to confirm the action, click Continue.
4 Edit run parameters as needed.
5 When finished, click Save Run.
The Last Modified dates for the run is updated on the Active Runs page.
Pin a Run
Pinned runs are prevented from being deleted or hidden. Arun cannot be pinned if the run is hidden or its
run folder has been deleted.
1 From the Active Runs page, click the more options icon next to a run.
2 Select Pin.
Delete Run Folder and Hide are disabled. Pin is replaced with Unpin.
Delete a Run Folder
You can manually delete run folders to manage storage space. The run cannot be pinned and must be in
one of the following states:
uSequencing Complete
uPrimary Analysis Complete
uPrimary Analysis Unsuccessful
uSequencing Errored
uAnalysis Errored
uAnalysis Complete
uSequencing Stopped
uAnalysis Stopped
1 From the Active Runs page, click the more options icon next to the run name that you want to
delete.
2 Select Delete Run Folder.
The Delete Run Folder option is replaced with Relink Run Folder. The Run Deleted icon appears
on the run.
Relink a Run Folder
Only an admin user can relink deleted run folders.
1 Copy the run folder back to its original location.
If the run is not saved in its original location, the software displays an error message following an
attempt to restore.
2 Hover over the more options icon next to the run.
3 Select Relink Run Folder.
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4 Confirm to proceed and restore the run.
The run is restored to the state it was in before the run folder was deleted.
User Passwords
To access the Local Run Managerinterface, you need a valid user name and password to log in to the
system. Only an admin user can assign user credentials.
NOTE
User accounts are specific to an instrument. User passwords are not universal across multiple
instruments.
When your password is approaching an expiration date, a message appears at the top of the interface to
remind you to reset your password.
My Account
From My Account, you can view your user name, your assigned role, permissions, and change your
password.
After you have logged in for the first time, you can change your existing password at any time from the
My Account window.
Your current password appears encrypted. Therefore, you have to know your current password to
change it to a new password. Forgotten passwords require assistance from a system administrator or
admin user.
Password States
The following are possible password states:
uTemporary password—When an admin user creates a user account, the admin user assigns a
temporary password for the new user.
uUser password—Upon first access, a new user is prompted to change the temporary password from
the login screen to a user-assigned password.
uForgotten password—If a user forgets a password, an admin user can reassign a temporary
password that can be changed upon next access.
uUsed password—Any user cannot reuse a password for up to five password cycles.
uUser lockout—The number of attempts to log in using an invalid password is configured by an admin
user. If a user exceeds the number of allowed attempts, the user account is locked. Only an admin
user can unlock the account, or assign a temporary password.
Change Your Password
1 From the navigation bar at the top of the interface, click the User icon next to your name.
NOTE
Depending on the size of the screen you are viewing the software on, the User icon may be
condensed underneath More Options .
2 From the drop-down list, click My Account.
3 Click the Edit icon in the Password section.
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