illumina NextSeq 550Dx User manual

NextSeq 550Dx Research Mode
Instrument Reference Guide
Document # 1000000041922 v01
March 2018
ILLUMINA PROPRIETARY
For Research Use Only. Not for use in diagnostic procedures.

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Document # 1000000041922 v01
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NextSeq 550Dx Research Mode Instrument Reference Guide

Revision History
Document Date Description of Change
Document #
1000000041922 v01
March
2018
Added information about the Illumina Proactive monitoring service in
the Customize System Settings section.
Document #
1000000041922 v00
November
2017
Initial release.
Document # 1000000041922 v01
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NextSeq 550Dx Research Mode Instrument Reference Guide

Table of Contents
Chapter 1 Overview 1
About this Guide 1
Introduction 1
Additional Resources 2
Instrument Components 2
Sequencing Consumables Overview 6
Chapter 2 Getting Started 11
Starting the Instrument 11
Customize System Settings 12
User-Supplied Consumables and Equipment 13
Chapter 3 Sequencing 15
Introduction 15
Sequencing Workflow 16
Prepare the Reagent Cartridge 16
Prepare the Flow Cell 17
Prepare Libraries for Sequencing 17
Set Up a Sequencing Run 18
Monitor Run Progress 24
Automatic Post-Run Wash 26
Chapter 4 Scanning 27
Introduction 27
Scanning Workflow 28
Download the DMAP Folder 28
Load the BeadChip Onto the Adapter 29
Set Up a Scan 30
Monitor Scan Progress 32
Chapter 5 Maintenance 35
Introduction 35
Perform a Manual Wash 35
Replace Air Filter 38
Software Updates 39
Reboot and Shut Down Options 40
Appendix A Troubleshooting 43
Introduction 43
Troubleshooting Files 43
Resolve Automatic Check Errors 44
Spent Reagents Container is Full 45
Rehybridization Workflow 46
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BeadChip and Scan Errors 48
Custom Recipes and Recipe Folders 49
RAID Error Message 49
Configure System Settings 50
Appendix B Real-Time Analysis 53
Real-Time Analysis Overview 53
Real-Time Analysis Workflow 54
Appendix C Output Files and Folders 59
Sequencing Output Files 59
Sequencing Output Folder Structure 61
Scanning Output Files 62
Scanning Output Folder Structure 63
Index 65
Technical Assistance 69
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NextSeq 550Dx Research Mode Instrument Reference Guide

Chapter 1 Overview
About this Guide 1
Introduction 1
Additional Resources 2
Instrument Components 2
Sequencing Consumables Overview 6
About this Guide
This instrument reference guide provides instructions on the use of the NextSeq 550Dx instrument in
research (RUO) mode.
Introduction
The Illumina®NextSeq™550Dx instrument is a single solution that provides a seamless transition
between high-throughput sequencing and array scanning.
Sequencing Features
uHigh-throughput sequencing—The NextSeq™550 instrument enables sequencing of exomes, whole
genomes, and transcriptomes and supports TruSeq™and Nextera™libraries.
uFlow cell types—Flow cells are available in configurations for high output and mid output. Each flow
cell type is kitted with a compatible prefilled reagent cartridge.
uReal-Time Analysis (RTA)—Integrated analysis software performs on-instrument data analysis,
which includes image analysis and base calling. The NextSeq 550Dx uses an implementation of
RTA called RTA v2, which includes important architecture and feature differences. For more
information, see Real-Time Analysis on page 53.
uBaseSpace®integration—The sequencing workflow is integrated with BaseSpace, the Illumina
genomics computing environment for data analysis, storage, and collaboration. For instruments
configured for BaseSpace, library information and run parameters are specified on the BaseSpace
Prep tab. Runs that were set up in BaseSpace appear on the instrument interface during run setup.
As the run progresses, output files are streamed in real time to BaseSpace or BaseSpace Onsite.
Array Scanning Features
uIntegrated array scanning in control software—The NextSeq 550Dx instrument allows you to
transition between array scanning and high-throughput sequencing on the same instrument using the
same control software.
uExtended imaging capability—The imaging system in the NextSeq 550Dx instrument includes
software and stage modifications that enable imaging of a larger surface area to accommodate
BeadChip scanning.
uBeadChip types—Compatible BeadChip types include CytoSNP-12, CytoSNP-850K, and Karyomap-
12.
uBeadChip adapter—A reusable BeadChip adapter enables easy loading of a BeadChip onto the
instrument.
uData Analysis—Use the BlueFuse®Multi software to analyze array data.
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Additional Resources
The following documentation is available for download from the Illumina website.
Resource Description
NextSeq 550Dx Instrument
Reference Guide (document #
1000000009513)
When in diagnostic mode, provides instructions for operating the instrument and
troubleshooting procedures.
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.
Denaturing and Diluting
Libraries for the NextSeq
System (document #
15048776)
Provides instructions for denaturing and diluting prepared libraries for a sequencing
run, and preparing an optional PhiX control. This step applies to most library types.
NextSeq Custom Primers
Guide (document #
15057456)
Provides information about using custom sequencing primers in place of Illumina
sequencing primers.
BaseSpace help
(help.basespace.illumina.com)
Provides information about using BaseSpace®and available analysis options.
Visit the NextSeq 550Dx instrument support page on the Illumina website for access to documentation,
software downloads, online training, and frequently asked questions.
Instrument Components
The NextSeq 550Dx instrument includes a touch screen monitor, a status bar, and 4 compartments.
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NextSeq 550Dx Research Mode Instrument Reference Guide

Figure 1 Instrument Components
AImaging compartment—Holds the flow cell for sequencing or the BeadChip adapter for scanning.
BTouch screen monitor—Enables on-instrument configuration and setup using the control 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 for sequencing or the BeadChip adapter for scanning. After loading the flow cell or BeadChip
adapter, 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 38.
NextSeq 550Dx Software
The instrument software includes integrated applications that perform sequencing runs or array
scanning.
uNextSeq Control Software (NCS)—The control software guides you through the steps to set up a
sequencing run or an array scan.
uReal-Time Analysis (RTA) software—For sequencing runs, RTA performs image analysis and base
calling during the run. The NextSeq 550Dx instrument uses RTA v2, which includes important
architecture and feature differences from earlier versions. For more information, see Real-Time
Analysis on page 53.
Status Icons
A status icon in the top-right corner of the control 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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NextSeq 550Dx Research Mode Instrument Reference Guide

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 NCS is restarted.
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 shutting down the instrument, see Shut Down the Instrument on page 41.
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.
Reusable BeadChip Adapter Overview
The reusable BeadChip adapter holds the BeadChip during scanning. The BeadChip is secured in the
recessed shelf of the adapter with the retention clip. Then, the BeadChip adapter is loaded onto the
stage in the imaging compartment.
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Figure 3 Reusable BeadChip Adapter
ABeadChip adapter
BRetention clip
Sequencing Consumables Overview
Performing a sequencing run on the NextSeq 550Dx instrument in research mode requires a single-use
NextSeq 500/550 Kit or NextSeq 550Dx High Output Reagent Kit. Each kit includes one flow cell and
the reagents required for a sequencing run.
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.
If you use a NextSeq 550Dx High Output Reagent Kit for a research mode run, all components must be
from the same kit lot. A NextSeq 500/550 Kit cannot be used for a diagnostic mode run.
Kit Compatibility Labeling
Kit components are labeled with color-coded indicators to show compatibility between flow cells and
reagent cartridges. Always use a compatible reagent cartridge and flow cell. The buffer cartridge is
universal.
Each flow cell and reagent cartridge is labeled High or Mid. Always check the label when you prepare
consumables for a run.
Kit Type Marking on Label
High Output Kit Components
Mid Output Kit Components
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Flow Cell Overview
Figure 4 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.
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 5 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.
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NextSeq 550Dx Research Mode Instrument Reference Guide

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 6 Numbered Reservoirs
Position Description
7, 8, and 9 Reserved for optional custom primers
10 Load libraries
For information about custom primers, see NextSeq Custom Primers Guide (document # 15057456) .
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 22.
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.
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NextSeq 550Dx Research Mode Instrument Reference Guide

Figure 7 Buffer Cartridge
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NextSeq 550Dx Research Mode Instrument Reference Guide

Document # 1000000041922 v01
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NextSeq 550Dx Research Mode Instrument Reference Guide

Chapter 2 Getting Started
Starting the Instrument 11
Customize System Settings 12
User-Supplied Consumables and Equipment 13
Starting the Instrument
Turn on the power toggle switch to the I(on) position.
Figure 8 Power Switch Located on Back of Instrument
1 Press the power button above the reagent compartment. The power button turns on the instrument
power and starts the integrated instrument computer and software.
By default, the instrument boots into diagnostic mode.
Figure 9 Power Button Located on Front of Instrument
2 Wait until the operating system has finished loading.
The NextSeq 550Dx Operating Software (NOS) launches and initializes the system automatically.
After the initialization step is complete, the Home screen opens.
3 Enter your Local Run Manager user name and password.
For information on Local Run Manager passwords, see the NextSeq 550Dx Instrument Reference
Guide (document # 1000000009513) .
4 Select Login.
The Home screen opens, with Sequence, Local Run Manager, Manage Instrument, and Perform
Wash icons.
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5 Use the Reboot to RUOcommand in NOSto safely shut down the instrument and reboot to research
mode.
uSelect Manage Instrument.
uSelect Reboot / Shut Down.
uSelect Reboot to RUO.
6 Wait until the operating system has finished loading.
The NCS launches and initializes the system automatically. After the initialization step is complete,
the Home screen opens.
7 If your system has been configured to require login credentials, log in to Windows using the user
name and password for your site.
NOTE
If you are not sure what mode the instrument is in, see Instrument Mode Indicators .
Instrument Mode Indicators
The following table lists instrument mode indicators on the NCSor NOS screen. For information on how
to switch from research mode to diagnostic mode, see Reboot and Shut Down Options on page 40.
Mode Home Screen Color Bar Status Icon Orientation
Diagnostic Mode Welcome to NextSeqDx Blue Horizontal
Research Mode Welcome to NextSeq Orange Vertical
Customize System Settings
The control software includes customizable system settings for the following.
uInput preferences
uAudio settings
uInstrument name
uRun setup preferences
uPurging of unused reagents
Customize Instrument Avatar and Nickname
1 From the Manage Instrument screen, select System Customization.
2 To assign a preferred avatar image for your instrument, select Browse and navigate to the image.
3 In the Nick Name field, enter a preferred name for the instrument.
4 Select Save to save settings and exit the screen.
The image and name appear at the upper-left corner of each screen.
Set Keyboard Option and Audio Indicator
1 From the Manage Instrument screen, select System Customization.
2 Select the Use on-screen keyboard checkbox to activate the on-screen keyboard for input to the
instrument.
3 Select the Play audio checkbox to turn on audio indicators for the following events.
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uUpon instrument initialization
uWhen a run is started
uWhen certain errors occur
uWhen user interaction is required
uWhen a run has finished
4 Select Save to save settings and exit the screen.
Set Run Setup Options
1 From the Manage Instrument screen, select System Customization.
2 Select the Use Advanced Load Consumables checkbox to enable the option to load all run
consumables from a single screen.
3 Select the Skip Pre-Run Check Confirmation checkbox to start sequencing or scanning
automatically after a successful automatic check.
4 Select Save to save settings and exit the screen.
Set Automatic Purge Option
1 From the Manage Instrument screen, select System Customization.
2 Select the Purge Consumables at End of Run checkbox to purge unused reagents from the
reagent cartridge to the spent reagents container automatically after each run.
NOTE
Purging consumables automatically adds additional time to the workflow.
3 Select Save to save settings and exit the screen.
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
1 N NaOH
(sodium hydroxide)
General lab supplier Library denaturation, diluted to 0.2N
200 mM Tris-HCl, pH7 General lab supplier Library denaturation
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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NextSeq 550Dx Research Mode Instrument Reference Guide

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
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
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
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NextSeq 550Dx Research Mode Instrument Reference Guide

Chapter 3 Sequencing
Introduction 15
Sequencing Workflow 16
Prepare the Reagent Cartridge 16
Prepare the Flow Cell 17
Prepare Libraries for Sequencing 17
Set Up a Sequencing Run 18
Monitor Run Progress 24
Automatic Post-Run Wash 26
Introduction
To perform a sequencing run on the NextSeq 550Dx instrument, prepare a reagent cartridge and flow
cell, and then follow the software prompts to set up and start the run. Cluster generation and sequencing
are performed on-instrument. After the run, an instrument wash begins automatically using components
already loaded on the instrument.
Cluster Generation
During cluster generation, single DNA molecules are bound to the surface of the flow cell, and then
amplified to form clusters.
Sequencing
Clusters are imaged using two-channel sequencing chemistry and filter combinations specific to each of
the fluorescently labeled nucleotides. After imaging of a tile on the flow cell is complete, the next tile is
imaged. The process is repeated for each cycle of sequencing. Following image analysis, the software
performs base calling, filtering, and quality scoring.
Monitor run progress and statistics from the control software interface, from the Run tab on BaseSpace,
or from a networked computer using the Sequencing Analysis Viewer (SAV) software. See Sequencing
Analysis Viewer on page 26.
Analysis
As the run progresses, the control software automatically transfers base call (BCL) files to BaseSpace
or the specified output location for secondary analysis.
Several analysis methods are available depending on your application. For more information, see the
BaseSpace help (help.basespace.illumina.com) .
Sequencing Run Duration
Sequencing run duration depends on the number of cycles performed. The maximum run length is a
paired-end run of 150 cycles each read (2 x 150), plus up to 8 cycles each for 2 index reads.
Number of Cycles in a Read
In a sequencing run, the number of cycles performed in a read is 1 more cycle than the number of
cycles analyzed. For example, a paired-end 150-cycle run performs reads of 151-cycles (2 × 151) for a
total of 302 cycles. At the end of the run, 2 x 150 cycles are analyzed. The extra cycle is required for
phasing and prephasing calculations.
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