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NextSeq® 500 Kit
Reference Guide
FOR RESEARCH USE ONLY
Revision History
Reagent Kit Overview
Kit Contents and Storage Requirements
Flow Cell Overview
Reagent Cartridge Overview
Buffer Cartridge Overview
Prepare the Reagent Cartridge
Prepare the Flow Cell
Next Steps
Technical Assistance
ILLUMINA PROPRIETARY
Part # 15048775 Rev. G
Catalog # FC-404-9001DOC
December 2014
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This document and its contents are proprietary to Illumina, Inc. and its affiliates ("Illumina"), and are intended
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for no other purpose. This document and its contents shall not be used or distributed for any other purpose
and/or otherwise communicated, disclosed, or reproduced in any way whatsoever without the prior written
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The instructions in this document must be strictly and explicitly followed by qualified and properly trained
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INCLUDING TO USERS OR OTHERS, AND DAMAGE TO OTHER PROPERTY.
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FOR RESEARCH USE ONLY
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Part #
Revision
Date
15048775
G
December
2014
Corrected total number of cycles possible for each kit
size. Each kit size includes enough reagents to
perform the number cycles listed on the label plus 2
index reads of 8 cycles each.
Reorganized description of reagent cartridge
contents.
15048775
F
September
2014
Added quantities to accessory box contents.
Updated URL for Safety Data Sheets (SDS) to
support.illumina.com/sds.html.
Updated NextSeq product markings from ™ to ®.
15048775
E
August
2014
Added statement that reagents are light sensitive and
to store reagents in a dark location.
Noted that NCS v1.2.2 is required for dual-index
sequencing.
15048775
D
May
2014
Corrected descriptions for reservoir positions #8
(custom Read 2 primer) and #9 (custom Index 1
primer).
Added NextSeq Accessory Box to kit contents.
Added instructions for preparing dual index primers
(BP13) and adding it to position #18 on the reagent
cartridge.
15048775
C
April
2014
Corrected NaOCl volume to 3 ml for position 28 of
the reagent cartridge.
Updated URL for Safety Data Sheets (SDS) to
support.illumina.com/sds.ilmn.
NextSeq 500 Kit Reference Guide
Description of Change
3
Revision History
Revision History
Part #
Revision
Date
15048775
B
February
2014
15048775
A
January
2014
4
Description of Change
Added reagent storage duration of up to 1 week at
2°C to 8°C.
Updated reagent cartridge label for reservoir #10 to
Load Library Here.
Added instructions for preparing a 0.03%–0.06%
dilution of NaOCl and loading onto the reagent
cartridge in reservoir #28.
Added illustrations to show good and bad clip
position on the flow cell.
Initial release.
Part # 15048775 Rev. G
Performing a run on the Illumina® NextSeq® 500 requires a single-use NextSeq 500 Kit.
Each kit includes 1 flow cell and the clustering and sequencing reagents required for
performing a sequencing run.
The flow cell, reagent cartridge, and buffer cartridge use radio-frequency identification
(RFID) for accurate consumable tracking and compatibility.
NextSeq 500 Kit Catalog Numbers
The NextSeq 500 Kit is available in a high output kit and a mid output kit.
} The high-output kit contains a high-output paired-end flow cell, a compatible
reagent cartridge, a buffer cartridge, and an accessory kit.
High Output Kits
NextSeq 500 High Output Kit (300 cycles)
NextSeq 500 High Output Kit (150 cycles)
NextSeq 500 High Output Kit (75 cycles)
Catalog #
Catalog # FC-404-1004
Catalog # FC-404-1002
Catalog # FC-404-1005
} The mid-output kit contains a mid-output paired-end flow cell, a compatible reagent
cartridge, a buffer cartridge, and an accessory kit.
Mid Output Kits
NextSeq 500 Mid Output Kit (300 cycles)
NextSeq 500 Mid Output Kit (150 cycles)
Catalog #
Catalog # FC-404-1003
Catalog # FC-404-1001
Kit Sizes
Kit size is based on the number of cycles that can be performed with the kit. Each kit
contains sufficient reagents for up to 2 index reads.
Kit Size
Number of Cycles
300 cycles
Provides reagents for up to 300 cycles (2 x 150 cycles) of sequencing and 2
index reads of 8 cycles each.
NextSeq 500 Kit Reference Guide
5
Reagent Kit Overview
Reagent Kit Overview
Kit Size
Number of Cycles
150 cycles
Provides reagents for up to 150 cycles (2 x 75 cycles) of sequencing and 2
index reads of 8 cycles each.
75 cycles
Provides reagents for up to 75 cycles (1 x 75 cycles) of sequencing and 2
index reads of 8 cycles each.
The NextSeq 500 flow cell is a paired-end flow cell. A single-read run can be performed
on a paired-end flow cell when the run is set up to perform only Read 1 and any index
reads. Make sure that the libraries to be sequenced are compatible with a paired-end
flow cell.
Kit Compatibility Labeling
Kit components are labeled with color-coded indicators to show compatibility between
flow cells and reagent cartridges. It is important to load the appropriate reagent cartridge
depending on the flow cell type that you are using for the sequencing run. The buffer
cartridge is universal.
To help identify compatible kit components in storage, each flow cell and reagent
cartridge label is marked with either High or Mid. The labeling appears on the outer
packaging of the kit component and on the individual kit component.
Always check the label markings when you prepare consumables for a run.
Kit Type
Marking on Label
High Output Kit Components
Mid Output Kit Components
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Part # 15048775 Rev. G
As soon as you receive your kit, promptly store the kit components at the indicated
temperature to ensure proper performance.
Reagents are sensitive to light. Store the reagent cartridge and buffer cartridge in a dark
location away from light.
Quantity
1
1
1
Kit Component
Reagent Cartridge
Buffer Cartridge
Flow Cell
Storage
-25°C to -15°C
15°C to 30°C
2°C to 8°C
Description
Clustering and sequencing reagents
Buffers and wash solution
Single-use paired-end flow cell
Accessory Box Contents
Quantity
1
1
Component
BP13
HT1
Storage
-25°C to -15°C
-25°C to -15°C
Description
NextSeq Dual Indexing Primers
Hybridization Buffer
BP13 is the Index 2 primer mix required for dual-indexed sequencing on the NextSeq
500. Load BP13 for all dual-indexed runs. If you are sequencing single-indexed libraries,
BP13 is not required.
NOTE
Dual indexing on the NextSeq 500 requires NextSeq Control Software (NCS) v1.2.2, or
later.
HT1 is used to dilute libraries before sequencing. For more information, see Denaturing
and Diluting Libraries for the NextSeq 500 (part # 15048776).
NextSeq 500 Kit Reference Guide
7
Kit Contents and Storage Requirements
Kit Contents and Storage Requirements
Flow Cell Overview
Figure 1 Flow Cell Cartridge
A
B
C
Lane pair A—Lanes 1 and 3
Lane pair B—Lanes 2 and 4
Flow 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. Lanes 1 and 3 (lane pair A) are
imaged at the same time. Lanes 2 and 4 (lane pair B) are imaged when imaging of lane
pair A is complete. Each lane is imaged in small imaging areas called tiles.
NOTE
Although the flow cell contains 4 lanes, 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.
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Part # 15048775 Rev. G
The reagent cartridge is a single-use consumable with RFID tracking and foil-sealed
reservoirs that are prefilled with clustering and sequencing reagents.
Figure 2 Reagent Cartridge
Before setting up the run, manually add prepared libraries into the designated reservoir
on the reagent cartridge. You can add a single library or a pool of indexed libraries. After
the run begins, libraries are transferred automatically from the reservoir to the flow cell.
The reagent cartridge includes several reservoirs reserved for the automatic post-run
wash. Wash solution is pumped from the buffer cartridge to the wash reservoirs in the
reagent cartridge, through the system, and then to the spent reagents cartridge.
Each reservoir on the reagent cartridge is numbered. Do not pierce the foil seals unless
instructed to do so to load libraries before a run, for example.
WARNING
This set of reagents contains formamide, an aliphatic amide that is a probable
reproductive toxin. Personal injury can occur through inhalation, ingestion, skin
contact, and eye contact. Dispose of containers and any unused contents in accordance
with the governmental safety standards for your region. For more information, see the
SDS for this kit at support.illumina.com/sds.html.
NextSeq 500 Kit Reference Guide
9
Reagent Cartridge Overview
Reagent Cartridge Overview
Reserved Reservoirs
Figure 3 Numbered Reservoirs
In addition to the reservoirs reserved for the automatic post-run wash, specific reservoirs
are reserved for loading libraries, optional custom primers, the dual-indexing primer,
and user-supplied sodium hypochlorite (NaOCl), which is required for the post-run
wash.
Position
7, 8, and 9
10
18
28
Description
Reserved for optional custom primers
Load libraries
For dual-indexed runs—Load BP13 Dual Index (Index 2) Primers
For all runs—Load 0.03%–0.06% sodium hypochlorite
For information about custom primers, see Using Custom Primers on the NextSeq 500 (part
# 15057456).
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Part # 15048775 Rev. G
Reagent Cartridge Overview
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.
Figure 4 Position 6
A
B
Protective rubber cover
Position 6
For more information, see the NextSeq 500 System User Guide (part # 15046563).
NextSeq 500 Kit Reference Guide
11
Buffer Cartridge Overview
Figure 5 Buffer Cartridge
The buffer cartridge is a single-use consumable containing 3 reservoirs prefilled with
buffers and wash solution. The contents of the buffer cartridge are sufficient for
sequencing 1 flow cell.
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Part # 15048775 Rev. G
To prepare the reagent cartridge for a sequencing run, first thaw the cartridge and then
inspect the larger reservoirs to make sure that reagents have thawed completely.
Thaw the Reagent Cartridge
1
Remove the reagent cartridge from -25°C to -15°C storage.
2
Place the reagent cartridge in a water bath containing enough room temperature
deionized water to submerge only the base of the reagent cartridge.
3
Allow the reagent cartridge to thaw in the room temperature water bath for
approximately 60 minutes or until it is thawed completely.
4
Remove the cartridge from the water bath and gently tap it on the bench to dislodge
water from the base of the cartridge. Gently dry the base of the cartridge.
Thaw BP13
1
Remove the tube of BP13 from -25°C to -15°C storage.
2
Thaw the tube of BP13 at room temperature for 30 minutes.
3
Invert the tube and visually inspect the contents to make sure that the primers have
thawed.
Inspect the Reagent Cartridge
1
Invert the reagent cartridge 5 times to mix the thawed reagents.
2
From the bottom of the reagent cartridge, visually inspect the reagents in the large
reservoirs in positions 29, 30, 31, and 32. Make sure that these positions are fully
thawed.
3
Gently tap the cartridge on the bench to reduce air bubbles in the reagents.
NextSeq 500 Kit Reference Guide
13
Prepare the Reagent Cartridge
Prepare the Reagent Cartridge
Load a Fresh Dilution of NaOCl
1
Prepare 3 ml of 0.03%–0.06% (w/v) NaOCl by combining the following volumes in a
microcentrifuge tube:
• 3%–6% NaOCl (30 µl)
• Laboratory-grade water (2970 µl)
2
Invert the tube several times to mix.
3
Use a low-lint lab tissue to wipe clean the foil seal covering reservoir #28.
4
Use a clean 1 ml pipette tip to pierce the foil seal covering reservoir #28.
5
Add 3 ml of 0.03%–0.06% NaOCl to the reservoir #28.
Figure 6 Load NaOCl
Load BP13
1
After thawing, invert the tube of BP13 several times to mix.
2
Use a low-lint lab tissue to wipe clean the foil seal covering reservoir #18.
3
Use a clean 1 ml pipette tip to pierce the foil seal covering reservoir #18.
4
Add the full contents of the tube of BP13 to reservoir #18.
Figure 7 Load BP13
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Part # 15048775 Rev. G
1
Remove a new flow cell package from 2°C to 8°C storage. Make sure that the flow
cell is compatible with the reagent cartridge as marked by High or Mid on the flow
cell packaging label.
2
Set the flow cell package aside at room temperature for 30 minutes. Do not remove
the flow cell from the packaging.
NOTE
Allow the flow cell to reach room temperature before proceeding. If the foil
packaging is intact, the flow cell can remain at room temperature up to 12 hours. You
can return the packaged flow cell to 2°C to 8°C storage for later use 1 time only.
Avoid repeated cooling and warming of the flow cell.
3
Put on a new pair of powder-free gloves.
4
Remove the flow cell from the foil packaging. After the foil packaging has been
opened, use the flow cell within the next 12 hours.
Figure 8 Remove from Foil Packaging
5
Open the clear plastic clamshell packaging and remove the flow cell.
NextSeq 500 Kit Reference Guide
15
Prepare the Flow Cell
Prepare the Flow Cell
Figure 9 Remove from Clamshell Package
6
Using a lint-free alcohol wipe or a lint-free tissue moistened with ethanol or
isopropanol, clean the glass surface of the flow cell. Dry the glass with a lint-free
tissue or lens paper.
Inspect the Flow Cell
Figure 10 Flow Cell Components
A
B
C
D
E
Port gaskets (4)
Retention clips (4)
Spring clips (4)
Flow cell cartridge frame
Carrier plate
1
Make sure that the flow cell ports are free of obstructions.
2
Make sure that the port gaskets are seated evenly and the rectangular white plastic
post is visible.
16
Part # 15048775 Rev. G
Make sure that the 4 small white retention clips are visibly snapped over the edge of
the black carrier plate.
If the plate is not secure under the clips, gently press the black carrier plate and
white cartridge frame together until the carrier plate snaps into place under the clips.
Figure 11 Retention Clip Inspection
A
B
4
Bad position—Retention clip is not snapped over the edge of the carrier plate.
Good position—Retention clip is snapped over the edge of the carrier plate.
Make sure that the 4 metal spring clips are flat against the black carrier plate.
Figure 12 Spring Clip Inspection
A
B
Bad position—Spring clip is not flat against the carrier plate.
Good position—Spring clip is flat against the carrier plate.
NextSeq 500 Kit Reference Guide
17
Prepare the Flow Cell
3
Next Steps
1
If applicable for your library type, denature and dilute your libraries as described in
the Denaturing and Diluting Libraries for the NextSeq 500 (part # 15048776).
2
Load your libraries onto the reagent cartridge as described in the NextSeq 500 System
User Guide (part # 15046563).
3
Using the NextSeq Control Software (NCS) interface, set up the run as described in
the NextSeq 500 System User Guide (part # 15046563).
Visit the NextSeq 500 support pages on the Illumina support website for access to
documentation, software downloads, frequently asked questions, and online training.
18
Part # 15048775 Rev. G
For technical assistance, contact Illumina Technical Support.
Table 1 Illumina General Contact Information
Website
Email
www.illumina.com
[email protected]
Table 2 Illumina Customer Support Telephone Numbers
Region
Contact Number
Region
North America
1.800.809.4566
Italy
Austria
0800.296575
Netherlands
Belgium
0800.81102
Norway
Denmark
80882346
Spain
Finland
0800.918363
Sweden
France
0800.911850
Switzerland
Germany
0800.180.8994
United Kingdom
Ireland
1.800.812949
Other countries
Contact Number
800.874909
0800.0223859
800.16836
900.812168
020790181
0800.563118
0800.917.0041
+44.1799.534000
Safety Data Sheets
Safety data sheets (SDSs) are available on the Illumina website at
support.illumina.com/sds.html.
Product Documentation
Product documentation in PDF is available for download from the Illumina website. Go
to support.illumina.com, select a product, then click Documentation & Literature.
NextSeq 500 Kit Reference Guide
Technical Assistance
Technical Assistance
*15048775*
Part # 15048775 Rev. G
Illumina
San Diego, California 92122 U.S.A.
+1.800.809.ILMN (4566)
+1.858.202.4566 (outside North America)
[email protected]
www.illumina.com