illumina NovaSeq 6000 User manual

NovaSeq 6000
Sequencing System Guide
Document # 1000000019358 v07 Material # 20023471
April 2018
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For Research Use Only. Not for use in diagnostic procedures.

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Document # 1000000019358 v07 Material # 20023471
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NovaSeq 6000 Sequencing System Guide

Revision History
Document Date Description of Change
Material # 20023471
Document #
1000000019358 v07
April 2018 Clarified usage of the library tube to mix reagents in the boost step
before sequencing.
Added a symbol descriptions table for symbols on the consumables or
consumable packaging.
Added information about the Illumina Proactive monitoring service in
the Run Setup Modes section.
Added information about the NovaSeq LIMSAPI.
Updated software descriptions to NovaSeq Control Software v1.4.0
Updated typical number of reads passing filter for S2 flow cells.
Updated recommended loading concentrations for the NovaSeq Xp
workflow.
Updated instructions for opening the flow cell packaging.
Clarified procedure for loading libraries onto the flow cell.
Added note about availability of the instrument for starting a
maintenance wash.
Added information about the staggered start countdown timer.
Updated instructions on how to add or remove SRPrules.
Document #
1000000019358 v06
February
2018
Added note in Flow Cell section to indicate software version 1.3.1 is
required when using an S1 flow cell.
Updated descriptions and Standard volume in table in Library Loading
Methods .
Added caution in Reagent Kit Components .
Added 0.5 and 1.5 ml tubes, pipettes tips for 20, 200, 1000 ul pipettes
to Consumables table. Added graduated cylinder to Equipment table.
Added Prepare the Flow Cell section in Chapters 4 and 5, moved steps
from Chapter 6 to these sections.
Updated total volume for the S1 flow cell in Chapter 4.
Added Recommended Library Pool Plexity table to Create a
Normalized Library Pool in Chapter 4.
Updated Thaw SBS and Cluster Cartridges steps in Chapters 4 and 5.
Clarified thawing instructions in Prepare Flow Cell .
Updated thawing information in NovaSeq Xp Recommended Loading
Concentrations .
Updated Recommended Library Pool Plexity table in Create a
Normalized Library Pool in Chapter 5.
Added sentence specifying that flow cell must be used within 12 hours
after removing it from packaging in NovaSeq Xp Workflow Summary
and Prepare Flow Cell .
Document #
1000000019358 v05
December
2017
Added clarification about empty library tube for Xp in the Sequencing
Workflow diagram.
In Denature Library and Option PhiX Control for the Standard workflow,
updated Tris-HCI volumes in table for step 5.
In Prepare the ExAmp Master Mix for the NovaSeq Xp workflow, added
note after step 4 to indicate vortexing is required for best results.
In Load Libraries onto the Flow Cell for the NovaSeq Xp workflow,
added reminder after step 3 to load samples slowly.
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NovaSeq 6000 Sequencing System Guide

Document Date Description of Change
Material # 20023471
Document #
1000000019358 v04
October
2017
Added individual lane loading to the list of instrument features.
Consumables - added the NovaSeq Xp 2-Lane Kit and NovaSeq Xp 4-
Lane kit. Added NovaSeq Xp 2-Lane Manifold Pack and NovaSeq 4-
Lane Manifold Pack.
Equipment - added the NovaSeq Xp Flow Cell Dock and P200 Pipette
for NovaSeq Xp Workflow
Added a Preparing Consumables chapter for the NovaSeq Xp workflow
Moved Empty Used Reagent Bottles from the Sequencing chapter to
the beginning of the NovaSeq Standard Workflow and NovaSeq Xp
Workflow chapters.
Updated Pooled Library Concentration table and Recommended
Loading Concentration table for the Standard workflow.
Material # 20020483
Document #
1000000019358 v03
September
2017
Updated software descriptions to NovaSeq Control Software v1.2,
which includes support for the S1 and S4 flow cells.
Added disk space requirements for a dual flow cell run for S1 and S4
flow cells.
Specified the naming requirement for certain *.json files.
Reorganized kit overview information in a Kits and Accessories
chapter. This chapter covers configurations, components, and
compatibility labeling for the reagent and library loading kits.
Added the NovaSeq 6000 Reagent Kit to the user-supplied
consumables.
Updated pool and denature library instructions to include information
for S1 and S4 flow cells.
Updated the instructions for thawing reagent cartridges to require a 2-
hour water bath for S1 and S2, and a four-hour water bath for S4.
Updated descriptions of the library tube, reagent cartridges, and flow
cells to include S4 components.
Added section on automatic software updates in the Maintenance
chapter.
Replaced the reference to Reducing Whole-Genome Data Storage
Footprint (Pub. No. 970-2012-013) with NovaSeq Series and HiSeq X
Ten Data Quality Comparison (Pub.No.770-2017-010) .
Added note to step 3 in Enter Run Parameters in Chapter 6.
Updated Flow Cell Tiles section to include S1 and S4 tile information.
Document # 1000000019358 v07 Material # 20023471
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Document Date Description of Change
Material # 20018871
Document #
1000000019358 v02
April
2017
Added the following information:
• Illumina-supplied consumables required for a run.
• Storage conditions of reagent kit components.
• Recommendations for library loading concentration.
• NaOHdilution for two flow cells.
• Step to bring the flow cell to room temperature before loading.
• Glove-changing step after emptying the used reagent bottles.
• Configuration of LIMS output for third-party LIMS systems.
• Naming convention for sample sheets.
• Process Management icons and troubleshooting.
• Appendix containing Windows security features and configuration
instructions.
• Contact information for technical assistance.
Increased reagent cartridge thaw time to 4 hours.
Updated PhiX spike-in instructions to change 1% PhiX spike-in volume
to 0.9 µl and use 10 mM Tris-HCl, pH 8.5 to dilute 10 nM PhiX.
Updated instructions to clean the flow cell and flow cell stage only
when particulate is visible.
Updated maintenance wash frequency to every 14 days.
Reorganized and consolidated consumable preparation instructions to
improve continuity.
Renamed the French doors to the liquid compartment doors.
Material # 20018406
Document #
1000000019358 v01
March
2017
Corrected the name of a column on the Process Management screen
to Sequencing.
Material # 20015871
Document #
1000000019358 v00
February
2017
Initial release.
Document # 1000000019358 v07 Material # 20023471
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NovaSeq 6000 Sequencing System Guide

Table of Contents
Chapter 1 Overview 1
Introduction 1
Additional Resources 2
Sequencing Overview 2
Sequencing Workflow 3
Instrument Components 5
Chapter 2 Kits and Accessories 10
Kits Overview 10
Reagent Kit Components 11
NovaSeq Xp Kit Components 14
NovaSeq Xp Flow Cell Dock 15
Symbol Descriptions 16
Chapter 3 Getting Started 18
Start the Instrument 18
Configure Settings 18
User-Supplied Consumables and Equipment 23
Chapter 4 Standard Workflow: Preparing Consumables 27
Methods 27
Library Guidelines 27
Thaw SBSand Cluster Cartridges 27
Empty Used Reagent Bottles 28
Prepare Flow Cell 30
Pool and Denature Libraries for Sequencing 30
Chapter 5 NovaSeq Xp Workflow: Preparing Consumables 34
NovaSeq Xp Workflow Summary 34
Methods 35
Library Guidelines 35
Thaw SBSand Cluster Cartridges 35
Empty Used Reagent Bottles 36
Prepare Flow Cell 38
Pool, Denature, and Load Libraries for Sequencing 38
Chapter 6 Sequencing 47
Set Up a Sequencing Run 47
Monitor Run Progress 52
Delete the Run 53
Detach Position #30 53
Automatic Post-Run Wash 54
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Chapter 7 Maintenance 55
Preventive Maintenance 55
Perform a Maintenance Wash 55
Staggered Start of Runs on Flow Cell A and Flow Cell B 58
Software Updates 59
Appendix A Troubleshooting 60
Troubleshooting Resources 60
Troubleshooting Files 60
Pre-Run Check Errors 60
Process Management Troubleshooting 61
Run Failure Before Clustering 61
End a Run 62
Shut Down the Instrument 63
Appendix B Real-Time Analysis 64
Real-Time Analysis Overview 64
Real-Time Analysis Workflow 66
Appendix C Output Folders and Files 69
Sequencing Output Folder Structure 69
Sequencing Output Files 70
Appendix D Windows Security 71
Security Configurations 71
Password Requirements 71
Windows Firewall 71
Enhanced Mitigation Experience Toolkit 71
Software Restriction Policies 72
Index 75
Technical Assistance 79
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NovaSeq 6000 Sequencing System Guide

Chapter 1 Overview
Introduction 1
Additional Resources 2
Sequencing Overview 2
Sequencing Workflow 3
Instrument Components 5
Introduction
The Illumina®NovaSeq™ 6000 Sequencing System packages scalable throughput and flexible sequencing
technology into a production-scale platform with the efficiency and cost-effectiveness of a benchtop system.
Features
uScalable sequencing—The NovaSeq 6000 scales up to production-level sequencing with high-quality
data for a broad range of applications.
uAdjustable output—The NovaSeq 6000 is a dual flow cell system with a broad output range. Sequence
one flow cell, or sequence two flow cells with different read lengths simultaneously. Mix and match three
types of flow cells and different read lengths.
uPatterned flow cell—A patterned flow cell generates tightly spaced clusters.The reduced spacing
between nanowells increases cluster density and data output.
uOnboard ExAmp mixing—The NovaSeq 6000 mixes the ExAmp reagents with library, amplifies the library,
and performs cluster generation for a streamlined sequencing workflow.
uIndividual lane loading—The NovaSeq Xp flow cell dock allows preloading of libraries into individual lanes
of the flow cell and reduces the library loading volume.
uHigh-throughput line scanning—The NovaSeq 6000 uses one camera with bidirectional scanning
technology to quickly image the flow cell in two color channels simultaneously.
uReal-Time Analysis (RTA)—The NovaSeq 6000 uses an implementation of RTA called RTA3. This
integrated software analyzes images and calls bases.
uBaseSpace™ Sequence Hub integration—The sequencing workflow is integrated with BaseSpace
Sequence Hub, the Illumina genomics computing environment for data analysis, storage, and
collaboration. As the run progresses, output files are streamed to the environment in real time.
uBaseSpace Clarity LIMS ready—Improve operational efficiency with end-to-end tracking of samples and
reagents, automated workflows, and integrated instrument operation.
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Additional Resources
Visit the NovaSeq 6000 Sequencing System support pages on the Illumina website for software downloads,
training resources, information about compatible Illumina products, information on recycling of consumables
and packaging, and the most recent versions of this system guide and the following documentation. Always
check support pages for the latest versions.
Resource Description
Custom Protocol
Selector
A wizard for generating customized end-to-end documentation that is tailored to the library prep
method, run parameters, and analysis method used for the sequencing run.
NovaSeq Series Site
Prep Guide
(document #
1000000019360)
Provides specifications for laboratory space, electrical requirements, and environmental and
network considerations.
NovaSeq Series
Safety and
Compliance Guide
(document #
1000000019357)
Provides information about operational safety considerations, compliance statements, and
instrument labeling.
RFID Reader
Compliance Guide
(document #
1000000002699)
Provides information about the RFID reader in the instrument, including compliance
certifications and safety considerations.
NovaSeq Series
Custom Primers
Guide (document #
1000000022266)
Provides information about replacing Illumina sequencing primers with custom sequencing
primers.
Sequencing Overview
Cluster Generation
During cluster generation, single DNAmolecules are bound to the surface of the flow cell and simultaneously
amplified to form clusters. For the Standard workflow, the ExAmp master mix is mixed with the libraries
onboard the instrument before cluster generation. For the NovaSeq Xp workflow, the ExAmp reagents and
libraries are mixed and delivered to the flow cell outside of the instrument. Volumes vary by flow cell type and
workflow.
Sequencing
Clusters are imaged using bidirectional scanning and two-channel sequencing chemistry. The camera uses
sensors that detect red and green light to image each swath and simultaneously generate red and green
images of the whole swath. After imaging, base calling is performed for clusters within each tile based on the
ratio of red to green signal for each cluster, which is based on location determined by the patterned flow cell.
This process is repeated for each cycle of sequencing.
Analysis
As the run progresses, the control software automatically transfers base call (*.cbcl) files to the specified
output folder location for data analysis.
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Several analysis methods are available and depend on your application. For more information, visit the
BaseSpace Sequence Hub support page on the Illumina website.
Sequencing Workflow
Thaw SBSand cluster reagent cartridges.
Pool and denature libraries. For the Standard workflow, add libraries to the library tube. For the NovaSeq
Xp workflow, load ExAmp/library mix onto the flow cell.
For both workflows, load the library tube into the thawed cluster cartridge.
From the software interface, select Sequence and specify a dual or single flow cell run.
Unload consumables from the previous run and load new consumables for the current run.
Specify run parameters from the Run Setup screen. If BaseSpace Sequence Hub is configured, sign in
from the Log In screen.
After the pre-run checks are completed, the run starts automatically.
Monitor the run from the Sequence screen, BaseSpace Sequence Hub if run monitoring is enabled, or a
network computer using Sequencing Analysis Viewer.
Data are transferred to the specified output folder.
An instrument wash begins automatically when sequencing is complete.
Library Loading Methods
Libraries are loaded onto a NovaSeq 6000 Flow Cell using one of the following two methods, depending on
the workflow selected. Setting up a sequencing run differs based on the workflow. Make sure that you
always follow the instructions for your method. See Standard Workflow: Preparing Consumables on page 27
and NovaSeq Xp Workflow: Preparing Consumables on page 34.
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Workflow Library Pool Loading and ExAmp
Mixing Method
Individual Lane Addressability and
Data Analysis
Loading Volume*
S1–S2–S4
Modes (µl)
Standard A single library pool is loaded into the
library tube, mixed onboard in the library
tube with the ExAmp reagents, and
automatically delivered to the flow cell
for clustering and sequencing. A boost
step before sequencing uses reagents
in the cluster cartridge and the library
tube to create a conditioning mix that
helps increase clustering efficiency.
A single library pool is distributed, and
sequenced, across all lanes of the flow
cell. Reads from all lanes are analyzed
in aggregate.
150–225–465 µl
(entire flow cell)
NovaSeq
Xp
One or more libraries (the number
corresponds to the number of flow cell
lanes) are manually mixed with ExAmp
reagents outside of the instrument and
directly loaded into individual lanes of
the flow cell using the NovaSeq Xp flow
cell dock. The filled flow cell is then
loaded onto the instrument for
clustering and sequencing. A boost
step before sequencing uses the empty
library tube to mix reagents from the
cluster cartridge to create a
conditioning mix that helps increase
clustering efficiency.
Each library is loaded into a separate
lane of the flow cell, which is then
sequenced. Different pools, aliquots of
the same pool, or arbitrary
combinations can be used. Reads from
the different lanes are analyzed
individually or in aggregate,
accordingly.
27–33–45 µl
(individual lane)
Table 1 Library Loading Methods
*The NovaSeq Xp workflow requires a 25–50% lower concentration of denatured libraries compared to the Standard workflow.
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NovaSeq 6000 Sequencing System Guide

Instrument Components
The NovaSeq 6000 Sequencing System comprises a touch screen monitor, a status bar, a power button with
adjacent USBports, and three compartments.
Figure 1 External Components
ATouch screen monitor—Displays the control software interface for system configuration and run setup and
monitoring.
BOptics compartment—Contains the optical components that enable dual surface imaging of flow cells.
CLiquids compartment—Contains reagent and buffer cartridges, and bottles for used reagents.
DFlow cell compartment—Holds the flow cells.
EStatus bar—Indicates flow cell status as ready to sequence (green), processing (blue), or needs attention
(orange).
FPower and USB ports—Provides access to the power button and USBconnections for peripheral
components.
Flow Cell Compartment
The flow cell compartment contains the flow cell stage, which holds flow cell A on the left and flow cell Bon
the right. Each side has four clamps that automatically position and secure the flow cell.
An optical alignment target mounted on the flow cell stage diagnoses and corrects optical problems. When
prompted by the control software, the optical alignment target realigns the system and adjusts camera focus
to improve sequencing results.
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NovaSeq 6000 Sequencing System Guide

Figure 2 Flow Cell Stage
ASide A flow cell holder
BSide B flow cell holder
CFlow cell clamp (one of four per side)
DOptical alignment target
The software controls the opening and closing of the flow cell compartment door. The door opens
automatically to load a flow cell for a run or maintenance wash. After loading, the software closes the
compartment door, moves the flow cell into position, and engages the clamps and vacuum seal. Sensors
verify the presence and compatibility of the flow cell.
Liquids Compartment
Setting up a run requires accessing the liquids compartment to load reagents and buffer and empty used
reagent bottles. Two doors enclose the liquids compartment, which is divided into two matching sides for
flow cell A and flow cell B.
Figure 3 Liquids Compartment Components
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ASmall used reagent bottle—Holds used reagents from the cluster cartridge, with a cap holder for easy cap
storage.
BLarge used reagent bottle—Holds used reagents from the SBS and buffer cartridges, with a cap holder for
easy cap storage.
CReagent chiller—Refrigerates the SBS and cluster cartridges.
DReagent chiller drawer—Color-coded positions hold the SBS cartridge on the left (gray label) and the cluster
cartridge on the right (orange label).
EBuffer drawer—Holds the large used reagent bottle on the left and the buffer cartridge on the right.
Used Reagents
The fluidics system is designed to route cluster cartridge reagents, which are potentially hazardous, to the
small used reagent bottle. Reagents from the SBS and buffer cartridges are routed to the large used reagent
bottle. However, cross-contamination between used reagent streams can occur. For safety, assume that
both used reagent bottles contain potentially hazardous chemicals. The safety data sheet (SDS) provides
detailed chemistry information.
NOTE
If the system is configured to collect used reagents externally, the stream to the large used reagent bottle is
routed externally. Cluster cartridge reagents always go to the small used reagent bottle.
System Software
The instrument software suite includes integrated applications that perform sequencing runs, on-instrument
analysis, and related functions.
uNovaSeq Control Software (NVCS)—Guides you through the steps to set up a sequencing run, controls
instrument operations, and displays statistics as the run progresses. To demonstrate proper unloading
and loading of consumables, NVCS plays instructional videos during run setup.
uReal-Time Analysis (RTA)—Performs image analysis and base calling during a run. NovaSeq 6000 uses
RTA3, which incorporates architecture, security, and other feature enhancements to optimize
performance. For more information, see Real-Time Analysis on page 64.
uUniversal Copy Service—Copies output files from RTA3 and NVCS to the output folder throughout a run. If
applicable, the service also transfers data to BaseSpace Sequence Hub. If the Universal Copy Service is
interrupted during a run, the service makes multiple attempts to reconnect and automatically resume
data transfer.
Status Icons
A status icon on the control software interface indicates run status. A number on the icon indicates the
number of conditions for a status.
When a run status changes, the icon blinks to alert you. Select the icon to view a description of the condition.
Select Acknowledge to clear the message, and then Close to close the dialog box.
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Status
Icon
Status Name Description
Status okay System is normal.
Processing System is processing.
Warning A warning has occurred and attention is required.
Warnings do not stop a run or require action before proceeding.
Error An error has occurred.
Errors require action before proceeding with the run.
Table 2 NVCS Status Icons
Process Management
The Process Management screen provides access to the Compute Engine (CE) and hard drive (C:\). Use the
screen to monitor run progress, delete runs, and otherwise manage disk space. Never delete files and folders
directly from C:\.
Process Management displays available disk space, space used on CE and C:\, and the status of runs using
disk space. Run Date and Name columns identify each run. Run Status, BaseSpace, and Network columns
show the status of each process for a run.
Process Icon Description
Run Status The run is in progress.
The run has completed sequencing.
Network Files are being copied to the output folder on the network.
All files are copied to the output folder on the network.
Not applicable because the run is not configured to upload to a network output folder or
the upload status is unknown.
To troubleshoot, see Process Management Troubleshooting on page 61.
Table 3 Process Management Status Icons
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Process Icon Description
BaseSpace Files are being uploaded to BaseSpace Sequence Hub.
All files are uploaded to BaseSpace Sequence Hub.
Not applicable because the run is not configured to upload to BaseSpace Sequence
Hub or the upload status is unknown.
To troubleshoot, see Process Management Troubleshooting on page 61.
Before a flow cell run can begin, the minimum space requirements for CE and C:\ must be met.
NOTE
For single flow cell runs, the minimum space requirements are half of those shown in the following table.
Flow cell CE Space per cycle C:\ Space per flow cell pair
S1 1.35 Gb 20 Gb
S2 2.7 Gb 20 Gb
S4 4.3 Gb 40 Gb
Table 4 Minimum Space Requirements for CE and C:\ For Dual Flow Cell Runs
To calculate the total space required in the CE for the run, multiply the value under 'CE Space per cycle' by
the sum of the Read 1, Read 2, Index 1, and Index 2 length values (see Enter Run Parameters on page 50).
For example, for a paired-end 150 cycle, dual flow cell S4 run with both indexes 8 bases long, the space
required on the CE is ( 151 * 2 + 8 * 2 ) * 4.3 = 1.37 Tb.
For information on clearing disk space, see Delete the Run on page 53.
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Chapter 2 Kits and Accessories
Kits Overview 10
Reagent Kit Components 11
NovaSeq Xp Kit Components 14
NovaSeq Xp Flow Cell Dock 15
Symbol Descriptions 16
Kits Overview
Performing a run on the NovaSeq 6000 requires a NovaSeq 6000 Reagent Kit. The NovaSeq Xp workflow
also requires a NovaSeq Xp Kit. These kits are available in the following configurations.
For a complete list of items needed for a run, see User-Supplied Consumables and Equipment on page 23.
Kit Name Illumina Catalog #
NovaSeq 6000 S4 Reagent Kit (300 cycles) 20012866
NovaSeq 6000 S2 Reagent Kit (300 cycles) 20012860
NovaSeq 6000 S2 Reagent Kit (200 cycles) 20012861
NovaSeq 6000 S2 Reagent Kit (100 cycles) 20012862
NovaSeq 6000 S1 Reagent Kit (300 cycles) 20012863
NovaSeq 6000 S1 Reagent Kit (200 cycles) 20012864
NovaSeq 6000 S1 Reagent Kit (100 cycles) 20012865
NovaSeq Xp 2-Lane Kit 20021664
NovaSeq Xp 4-Lane Kit 20021665
Table 5 Kit Configurations
Compatibility Labeling
To identify compatible kit components, flow cells and cartridges are labeled with symbols that show the kit
mode:S1,S2, or S4. NovaSeq Xp manifolds support multiple modes and are labeled either 2-lane (for S1 and
S2 flow cells) or 4-lane (for S4 flow cells).
Components with different modes cannot be used in the same run. For example, do not pair S1 cartridges
with an S2 flow cell.
Kit Mode
Marking
on
Label
Description
S1 kit
components
S1 flow cell generates up to 1.6 billion single reads passing filter with output up to 500 Gb at
2 x 150 bp. The S1 kit provides fast sequencing of fewer samples for most high throughput
applications.
S2 kit
components
S2 flow cell generates up to 4.1 billion single reads passing filter with output up to 1250 Gb
at 2 x 150 bp. The S2 flow cell provides fast sequencing for most high-throughput
applications, with a greater number of reads than an S1 flow cell for more sequencing
output.
S4 kit
components
S4 flow cell generates up to 10 billion single reads passing filter with output up to 3000 Gb at
2 x 150 bp. It is a 4 lane version of the flow cell designed for maximum output. It enables
cost-effective whole-genome sequencing across a range of species and depths of
coverage.
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The NovaSeq Reagent Kits product page on the Illumina website provides detailed specifications for each
mode.
Reagent Kit Components
Each NovaSeq 6000 Reagent Kit contains the following components. Each component uses radio-frequency
identification (RFID) for accurate consumable tracking and compatibility.
When you receive your kit, promptly store components at the indicated temperature to ensure proper
performance.
Quantity Kit Component Storage Temperature
1 Library tube 15°C to 30°C
1 Flow cell 2°C to 8°C
1 Buffer cartridge 15°C to 30°C
1 Cluster cartridge -25°C to -15°C
1 SBScartridge -25°C to -15°C
Table 6 Kit Components
CAUTION
Avoid dropping cartridges. Injury may occur if dropped. Skin irritation may occur if reagents leak from
cartridges. Inspect cartridges for cracks before use.
Library Tube
The NovaSeq 6000 library tube is a 16 mm tube that fits into position #8 of the cluster cartridge. Position #8 is
labeled Library Tube and circled in orange for easy identification. The tube has a threaded cap that allows
storage of libraries when necessary. Ensure that the cap has been removed before loading into the cluster
cartridge.
Figure 4 Library Tube
The library tube is used one of two ways, depending on workflow:
uStandard—Pooled and denatured libraries are added to the library tube, which is then loaded uncapped
into the cluster cartridge. After the run begins, the instrument mixes the libraries with ExAmp reagents in
the library tube, which are then transferred automatically to the flow cell.
uNovaSeq Xp—The empty, uncapped library tube is loaded into the cluster cartridge. During the run,
reagents are mixed in the library tube before distribution to the flow cell.
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NovaSeq 6000 Sequencing System Guide

Flow Cell
The NovaSeq 6000 flow cell is a patterned flow cell encased in a cartridge. The flow cell is a glass-based
substrate containing billions of nanowells in an ordered arrangement, which increases the number of output
reads and sequencing data. Clusters are generated in the nanowells from which sequencing is then
performed.
Each flow cell has multiple lanes for sequencing pooled libraries. The S1 and S2 flow cells have two lanes
each, and the S4 flow cell has four. Each lane is imaged in multiple swaths, and the software then divides the
image of each swath into smaller portions called tiles. For more information, see Flow Cell Tiles on page 65.
NOTE
If you are using an S1 flow cell, make sure to use NovaSeq Control Software v1.3.1, or later.
Figure 5 Flow Cells
AFlow cell cartridge
BFour-lane flow cell (S4)
CTwo-lane flow cell (S1 and S2)
The underside of each flow cell has four gaskets. Libraries and reagents enter the flow cell lanes through the
gaskets on the inlet end of the flow cell. Used reagents are expelled from the lanes through the gaskets at the
outlet end.
NOTE
Avoid touching the gaskets when handling the flow cell.
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Figure 6 Inverted Flow Cell
AOutlet end
BInlet end
CGasket (one of four)
Buffer, Cluster, and SBSCartridges
The NovaSeq 6000 buffer, cluster, and SBScartridges have foil-seal reservoirs prefilled with reagents,
buffers, and wash solution. One of each cartridge type is included with the reagent kit.
The cartridges load directly onto the instrument, and are color-coded and labeled to reduce loading errors.
Guides in the reagent chiller and buffer drawers ensure proper orientation.
Cartridge Description
NovaSeq 6000 buffer cartridge Prefilled with sequencing buffers and weighs up to 6.8 kg (15 lbs). A plastic handle
facilitates carrying, loading, and unloading. Indentations in the top plate allow
cartridges to be stacked.
NovaSeq 6000 cluster cartridge Prefilled with clustering, indexing, and paired-end reagents as well as wash
solution. Includes a designated position for the library tube. Orange labeling
distinguishes the cluster cartridge from the SBS cartridge.
NovaSeq 6000 SBScartridge Prefilled with sequencing reagents at volumes specific to the number of cycles the
kit supports (300, 200, or 100). Each of the three reagent positions has an adjacent
position reserved for the automatic post-run wash. Gray labeling distinguishes the
SBScartridge from the cluster cartridge.
Table 7 Reagent Cartridges
Document # 1000000019358 v07 Material # 20023471
For Research Use Only. Not for use in diagnostic procedures.
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NovaSeq 6000 Sequencing System Guide