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HiSeq® 3000/4000 Cluster Kit
Reference Guide
FOR RESEARCH USE ONLY
Reagent Kit Overview
Kit Contents and Storage Requirements
cBot Reagent Plate
User-Supplied Consumables
Patterned Flow Cell
Prepare the Flow Cell
Prepare Clustering Reagents
Next Steps for Clustering
Prepare Indexing and Paired-End Reagents
Paired-End and Indexing Reagent Positions
Next Steps for Sequencing
Technical Assistance
ILLUMINA PROPRIETARY
Part # 15066495 Rev. A
Catalog # PE-410-9001DOC
February 2015
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This document and its contents are proprietary to Illumina, Inc. and its affiliates ("Illumina"), and are intended
solely for the contractual use of its customer in connection with the use of the product(s) described herein and
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
consent of Illumina. Illumina does not convey any license under its patent, trademark, copyright, or commonlaw rights nor similar rights of any third parties by this document.
The instructions in this document must be strictly and explicitly followed by qualified and properly trained
personnel in order to ensure the proper and safe use of the product(s) described herein. All of the contents of
this document must be fully read and understood prior to using such product(s).
FAILURE TO COMPLETELY READ AND EXPLICITLY FOLLOW ALL OF THE INSTRUCTIONS
CONTAINED HEREIN MAY RESULT IN DAMAGE TO THE PRODUCT(S), INJURY TO PERSONS,
INCLUDING TO USERS OR OTHERS, AND DAMAGE TO OTHER PROPERTY.
ILLUMINA DOES NOT ASSUME ANY LIABILITY ARISING OUT OF THE IMPROPER USE OF THE
PRODUCT(S) DESCRIBED HEREIN (INCLUDING PARTS THEREOF OR SOFTWARE) OR ANY USE OF
SUCH PRODUCT(S) OUTSIDE THE SCOPE OF THE EXPRESS WRITTEN LICENSES OR PERMISSIONS
GRANTED BY ILLUMINA IN CONNECTION WITH CUSTOMER'S ACQUISITION OF SUCH PRODUCT
(S).
FOR RESEARCH USE ONLY
© 2015 Illumina, Inc. All rights reserved.
Illumina, 24sure, BaseSpace, BeadArray, BlueFish, BlueFuse, BlueGnome, cBot, CSPro, CytoChip,
DesignStudio, Epicentre, GAIIx, Genetic Energy, Genome Analyzer, GenomeStudio, GoldenGate,
HiScan, HiSeq, HiSeq X, Infinium, iScan, iSelect, MiSeq, NeoPrep, Nextera, NextBio, NextSeq, Powered
by Illumina, SeqMonitor, SureMDA, TruGenome, TruSeq, TruSight, Understand Your Genome, UYG,
VeraCode, verifi, VeriSeq, the pumpkin orange color, and the streaming bases design are trademarks of
Illumina, Inc. and/or its affiliate(s) in the U.S. and/or other countries. All other names, logos, and other
trademarks are the property of their respective owners.
To cluster a flow cell on the cBot for subsequent sequencing on the HiSeq 4000 or HiSeq
3000, you need 1 HiSeq 3000/4000 PE Cluster Kit.
Kit Name
Catalog #
HiSeq 3000/4000 PE Cluster Kit
PE-410-1001
NOTE
The kit includes a denaturation step before clustering on the cBot. Libraries are denatured
in the 8-tube strip before adding the ExAmp reaction mix.
HiSeq 3000/4000 Cluster Kit Reference Guide
3
Reagent Kit Overview
Reagent Kit Overview
Kit Contents and Storage Requirements
When you receive your kit, promptly store the kit components at the indicated
temperature to ensure proper performance.
Reagents are sensitive to light. Store reagents in a dark location away from light.
Cluster Kit Box 1
Box 1 contains components used on the cBot. RSB is used to dilute libraries.
Reagent
Quantity
Storage
Description
cBot Reagent
Plate
1
-25°C to -15°C
cBot reagent plate, PE
RSB
1
-25°C to -15°C
Resuspension Buffer
EPX1
1
-25°C to -15°C
Enhanced Pattern Cluster Mix 1
EPX2
1
-25°C to -15°C
Enhanced Pattern Cluster Mix 2
EPX3
1
-25°C to -15°C
Enhanced Pattern Cluster Mix 3
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Part # 15066495 Rev. A
Box 2 contains reagents and sequencing primers used for indexing and paired-end
resynthesis on the HiSeq.
Reagent
Quantity
Storage
Description
HRM
1
-25°C to -15°C
HT Resynthesis Mix
HLM2
1
-25°C to -15°C
HT Linearization Mix 2
HAM
1
-25°C to -15°C
HT Amplification Mix
HPM
1
-25°C to -15°C
HT Amp Premix
HDR
1
-25°C to -15°C
HT Denaturation Mix
(contains formamide)
HP11
1
-25°C to -15°C
Primer Mix – Read 2
HP14
1
-25°C to -15°C
Indexing Primer Mix
Accessory Kit
The accessory kit contains components used for setting up a sequencing run on the
HiSeq.
Accessory
Quantity
Purpose
Flow cell gaskets
(package of 4)
1
Use to replace the flow cell gaskets between runs;
replace before a maintenance wash.
Flow cell storage tube with
storage buffer, 50 ml
1
Use to store the flow cell after clustering and
before sequencing.
Funnel bottle caps
(package of 8)
1
Use to replace SBS reagent bottle caps before
loading reagents onto the instrument.
HiSeq 3000/4000 Cluster Kit Reference Guide
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Kit Contents and Storage Requirements
Cluster Kit Box 2
cBot Reagent Plate
Figure 1 cBot Reagent Plate
The cBot reagent plate contains 12 rows with 8 deep wells each. Each reagent occupies a
full row of 8 wells. Not all rows of the reagent plate contain reagent.
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.
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Part # 15066495 Rev. A
The following user-supplied consumables are used for preparation and loading of
reagents.
Component
Supplier
Purpose
1 N NaOH
General lab supplier
Use for library denaturation step.
200 mM Tris-HCl, pH 8.0
General lab supplier
Use for library denaturation step
after diluting to 0.1 N NaOH.
8-tube strip with caps,
0.2 ml
Fisher Scientific,
catalog # AB-0264*
Use for the ExAmp Reaction and
library mix on the cBot.
Microcentrifuge tube,
1.5 ml
VWR,
catalog #20170-038*
Use for preparation of the ExAmp
Reaction master mix.
Laboratory-grade water
Millipore or
General lab supplier
Use for library denaturation step.
*or equivalent
HiSeq 3000/4000 Cluster Kit Reference Guide
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User-Supplied Consumables
User-Supplied Consumables
Patterned Flow Cell
The flow cell used on the HiSeq 4000 and HiSeq 3000 is a patterned flow cell with
billions of ordered nanowells. The wells are manufactured into the glass of the flow cell
allowing for the generation of sequencing clusters in an ordered arrangement. Clusters
are aligned closely together, increasing the number of output reads and amount of
sequencing data generated. The patterned flow cell contains 8 lanes with 2 swaths per
lane, and has the same general dimensions as other HiSeq high-output flow cells.
The patterned flow cell is provided in the HiSeq 3000/4000 PE Cluster Kit, and is
shipped dry in a foil-wrapped package. Inside the foil package, the flow cell is packaged
in a protective plastic clamshell case.
Figure 2 Example of Clusters on a Patterned Flow Cell
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Part # 15066495 Rev. A
1
Remove a new flow cell package from 2°C to 8°C storage. Do not remove the flow
cell from the packaging.
2
Set the flow cell package aside at room temperature for at least 30 minutes.
NOTE
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
Peel open the foil package from the end with the angled seal.
Figure 3 Open Foil Packaging
HiSeq 3000/4000 Cluster Kit Reference Guide
9
Prepare the Flow Cell
Prepare the Flow Cell
5
Remove the clear clamshell package from the foil packaging.
Figure 4 Remove From Foil Packaging
6
Open the clear plastic clamshell packaging and remove the flow cell.
Figure 5 Remove Flow Cell From Clamshell Package
7
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.
8
Set aside until you are ready to load the flow cell onto the cBot when prompted by
the cBot software.
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Part # 15066495 Rev. A
Clustering reagents provided in the HiSeq 3000/4000 PE Cluster Kit are used on the cBot.
To prepare reagents, thaw the cBot reagent plate and prepare the ExAmp master mix.
Best Practices
} Wear a fresh pair of gloves when preparing clustering reagents.
} The clear plastic lid on the reagent plate protects the foil seal from being damaged or
punctured during thawing. Remove the lid only when you are ready to load the cBot
reagent plate onto the cBot.
} Always prepare freshly diluted NaOH for denaturing libraries for cluster generation.
This step is essential to the denaturation process.
} To prevent small pipetting errors from affecting the final NaOH concentration,
prepare at least 1 ml of freshly diluted 0.1 N NaOH.
} After completing the reagent preparation steps, proceed to setting up the cBot for
clustering and load reagents when prompted.
Thaw the cBot Reagent Plate
The cBot reagent plate takes approximately 60 minutes to thaw using a room
temperature water bath. Alternatively, you can thaw reagents at 2°C to 8°C overnight,
not to exceed 16 hours.
1
Remove the cBot reagent plate from -25°C to -15°C storage.
2
Place the reagent plate in a water bath containing enough room temperature
deionized water to submerge only the reagent plate base. Allow reagents to thaw for
60 minutes. Make sure that reagents have thawed before proceeding.
Thaw EPX1, EPX2, EPX3, and RSB
The following reagents are used to create the ExAmp master mix and dilute libraries.
1
Remove EPX1, EPX2, EPX3, and RSB from -25°C to -15°C storage.
2
Thaw each tube at room temperature for approximately 10 minutes. Do not vortex.
3
When thawed, place on ice until you are ready to prepare the ExAmp master mix.
HiSeq 3000/4000 Cluster Kit Reference Guide
11
Prepare Clustering Reagents
Prepare Clustering Reagents
Prepare a Fresh Dilution of NaOH
CAUTION
Using freshly diluted NaOH is essential in denaturing libraries for cluster generation on
the HiSeq.
1
Prepare 1 ml of 0.1 N NaOH by combining the following volumes in a
microcentrifuge tube:
• Laboratory-grade water (900 µl)
• Stock 1 N NaOH (100 µl)
2
Invert the tube several times to mix.
NOTE
A fresh dilution of 0.1 N NaOH is required for denaturing libraries and a PhiX control.
After denaturing libraries, you can set aside the remaining NaOH to prepare a PhiX
control within the next 12 hours. Otherwise, discard the remaining dilution of NaOH.
Denature Libraries and Add Optional PhiX Control
The library loading concentration depends on the libraries to be sequenced. The
following instructions apply to supported Illumina libraries and assume an insert size
typical for the associated library type. Make sure that you dilute to a concentration
appropriate for the library type.
} Too high DNA loading concentration leads to reduced %PF.
} Too low DNA loading concentration leads to reduced or unacceptable %PF, and
high % duplicates that negatively affect depth of coverage.
1
Dilute the library or pooled libraries to the appropriate concentration for the library
type.
Library Type
Dilution
TruSeq Nano DNA
Dilute to 2–3 nM in RSB.
TruSeq DNA PCR-Free
Dilute to 1–2 nM in RSB.
Nextera Rapid Capture Exome
Dilute to 2–3 nM in RSB.
TruSeq Stranded Total RNA
Dilute to 2–3 nM in RSB.
TruSeq Stranded mRNA
Dilute to 2–3 nM in RSB.
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Part # 15066495 Rev. A
[Optional] Spike-in 1% nondenaturedIllumina PhiX control to nondenatured libraries.
Library Type
Spike-In
TruSeq Nano DNA
Add 200–300 pM PhiX to 2–3 nM library.
TruSeq DNA PCR-Free
Add 100–200 pM PhiX to 1–2 nM library.
Nextera Rapid Capture Exome
Add 200–300 pM PhiX to 2–3 nM library.
TruSeq Stranded Total RNA
Add 200–300 pM PhiX to 2–3 nM library.
TruSeq Stranded mRNA
Add 200–300 pM PhiX to 2–3 nM library.
3
Label a new 8-tube strip #1 through #8.
4
To denature the library, add the following volumes to the labeled 8-tube strip in the
order listed:
a Add 5 µl of nondenatured library into the bottom of each well.
b Add 5 µl of freshly diluted 0.1 N NaOH. Using a P10 or P20 pipette set to 5 µl,
slowly pipette up and down 10 times to mix the reaction.
c Incubate for 8 minutes at room temperature.
d Add 5 µl of 200 mM Tris-HCl pH 8.0. Using a P10 or P20 pipette set to 5 µl,
slowly pipette up and down 10 times to mix the reaction.
5
Cap and set aside the 8-tube strip containing the denatured library on ice until you
are ready to add the ExAmp master mix.
CAUTION
To ensure proper performance, do not exceed 30 minutes before adding the ExAmp
master mix.
Prepare the cBot Reagent Plate
1
Invert the reagent plate several times to mix the thawed reagents.
2
Vortex the plate for approximately 10 seconds to dislodge trapped air bubbles.
3
Tap the reagent plate on a hard surface 5–10 times to collect reagent droplets at the
bottom of the tubes. Alternatively, pulse centrifuge the reagent plate.
HiSeq 3000/4000 Cluster Kit Reference Guide
13
Prepare Clustering Reagents
2
4
Set the reagent plate aside on ice until you are ready to load it onto the cBot. Make
sure that you remove the clear plastic cover from the cBot reagent plate before
loading, and do not puncture the foil seals.
Prepare the ExAmp Reaction
Prepare the ExAmp reaction master mix immediately before use.
CAUTION
Do not refreeze ExAmp reagents after thawing.
1
Invert each tube of EPX1 and EPX2 several times each to make sure that the reagents
are thoroughly mixed.
NOTE
EPX3 does not move when inverted. This condition is normal due to the viscosity of
EPX3.
2
Briefly centrifuge EPX1, EPX2, and EPX3. Do not vortex.
3
Add the following volumes to a 1.5 ml tube in the order listed:
a Add 210 µl EPX1.
b Add 30 µl EPX2. Using a P1000 pipette set to 200 µl, slowly pipette up and
down 10 times to mix the reaction. Do not vortex.
NOTE
Due to the viscosity of ExAmp reagents, especially EPX2 and EPX3, aspirate and
dispense reagents slowly to ensure accurate pipetting.
c
d
Add 110 µl EPX3. Using a P1000 pipette set to 200 µl, slowly pipette up and
down 10 times to mix the reaction. Make sure that you do not introduce air
bubbles.
Briefly centrifuge the reaction. Do not vortex.
NOTE
The solution can be cloudy, which is normal. If the mixture separates into a cloudy
portion and a transparent portion, repeat the pipette mixing step to make sure that
the solution is uniform.
4
Add 35 µl of the master mix into the bottom of each well of the 8-tube strip
containing denatured and neutralized libraries. Change tips between samples.
5
Using a multichannel P100 or P200 pipette set to 40 µl, slowly pipette up and down
10 times to mix the reaction. Do not vortex.
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Part # 15066495 Rev. A
Make sure that the tubes are capped, and then briefly centrifuge the 8-tube strip.
7
Set aside the 8-tube strip containing the ExAmp master mix and library solution on
ice until you are ready to load it onto the cBot.
CAUTION
Do not exceed 15 minutes before loading the 8-tube strip containing the ExAmp
master mix and library solution onto the cBot.
8
Promptly proceed to the cBot setup steps. When prompted by the cBot software,
carefully remove the caps and place the 8-tube strip in the template row on the cBot.
HiSeq 3000/4000 Cluster Kit Reference Guide
15
Prepare Clustering Reagents
6
Next Steps for Clustering
After you have prepared the flow cell and clustering reagents, you are ready to load
them onto the cBot when prompted by the cBot software. For cBot workflow instructions,
see the cBot System Guide (part # 15006165).
Clustering takes approximately 3 hours. After clustering, the patterned flow cell can be
stored in the flow cell storage buffer provided in the kit for up to 48 hours at 2°C to 8°C.
During clustering on the cBot, prepare SBS and paired-end reagents for the HiSeq 4000
or HiSeq 3000. For SBS reagent preparation instructions, see the HiSeq 3000/4000 SBS Kit
Reference Guide (part # 15066494).
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Part # 15066495 Rev. A
Indexing and paired-end reagents are loaded onto the instrument at the beginning of the
run and used during indexing reads and the Read 2 resynthesis step of a sequencing
run.
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.
Thaw Indexing and Paired-End Reagents
1
Remove the following reagents from -25°C to -15°C storage: HAM, HDR, HLM2,
HP11, HP14, HPM, and HRM.
NOTE
For non-indexed libraries, HP14 is not required.
2
Thaw reagents in a beaker filled with room temperature deionized water for about
20 minutes, or until reagents are fully thawed.
3
After reagents have thawed, place HAM, HLM2, and HRM on ice. Do not place
HDR on ice.
Prepare HAM, HDR, HLM2, HP11, HP14, HPM, and HRM
1
Invert each tube several times to mix the reagent.
2
Centrifuge the reagent at 1000 rpm for 1 minute.
3
For HAM, HLM2, and HRM—Set aside on ice.
For HDR, HP11, HP14, and HPM—Set aside at room temperature.
HiSeq 3000/4000 Cluster Kit Reference Guide
17
Prepare Indexing and Paired-End Reagents
Prepare Indexing and Paired-End Reagents
Paired-End and Indexing Reagent Positions
Use the following information for reference. For reagent loading instructions, see the
system guide for the HiSeq 4000 or HiSeq 3000.
Figure 6 Paired-End Reagent Rack
Table 1 Paired-End Reagent Positions
Position
Reagent
Description
10
ml PW1 or laboratory-grade water
19
PW1
10 ml PW1 or laboratory-grade water
18
PW1
Indexing Primer Mix
17
HP14
Primer Mix – Read 2
16
HP11
HT Denaturation Reagent
15
HDR
HT Amplification Premix
14
HPM
HT Amplification Mix
13
HAM
10 ml PW1 or laboratory-grade water
12
PW1
HT Linearization Mix 2
11
HLM2
HT Resynthesis Mix
10
HRM
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Part # 15066495 Rev. A
After you have prepared paired-end reagents, you are ready to load them onto the HiSeq.
For reagent loading and sequencing workflow instructions, see the HiSeq 4000 System
Guide (part # 15066496) or the HiSeq 3000 System Guide (part # 15066493).
Make sure that you use the compatible version of SBS kit and HiSeq Control Software
(HCS).
Cluster Kit Version
SBS Kit Version
HCS/RTA Version
HiSeq 3000/4000 PE Cluster Kit
HiSeq 3000/4000 SBS Kit
HCS v3.3/RTA v2.3, or later
HiSeq 3000/4000 Cluster Kit Reference Guide
19
Next Steps for Sequencing
Next Steps for Sequencing
Notes
For technical assistance, contact Illumina Technical Support.
Table 2 Illumina General Contact Information
Website
Email
www.illumina.com
[email protected]
Table 3 Illumina Customer Support Telephone Numbers
Region
Contact Number
Region
North America
1.800.809.4566
Italy
Australia
1.800.775.688
Netherlands
Austria
0800.296575
New Zealand
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
0800.451.650
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.
HiSeq 3000/4000 Cluster Kit Reference Guide
Technical Assistance
Technical Assistance
*15066495*
Part # 15066495 Rev. A
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