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HiSeq X™ HD Reagent Kit v2
Reference Guide
FOR RESEARCH USE ONLY
Reagent Kit Overview
Kit Contents and Storage (Single-Pack Kit)
Kit Contents and Storage (20-Pack Kit)
Kit Compatibility Labeling
cBot Reagent Plate Contents
User-Supplied Consumables
HiSeq X Patterned Flow Cell
Prepare the Flow Cell
Prepare Clustering Reagents
Next Steps
Prepare SBS Reagents
SBS Reagent Positions
Prepare Indexing and Paired-End Reagents
Paired-End and Indexing Reagent Positions
Next Steps
Technical Assistance
ILLUMINA PROPRIETARY
Part # 15058861 Rev. A
Catalog # FC-501-9002DOC
October 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
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,
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GRANTED BY ILLUMINA IN CONNECTION WITH CUSTOMER'S ACQUISITION OF SUCH PRODUCT
(S).
FOR RESEARCH USE ONLY
© 2014 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, ForenSeq, MiSeq, MiSeqDx, MiSeq FGx, NeoPrep,
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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.
The HiSeq X™ HD Reagent Kit v2 is available in a single-pack kit and a 20-pack kit:
} Single-pack kit—Each kit includes consumables for sequencing 2 flow cells.
Consumables are packaged in 2 complete sets, each set supports 1 flow cell.
} 20-pack kit—Each kit includes consumables for sequencing 20 flow cells.
Consumables are packaged to support 4 flow cells at a time.
Kit Name
Catalog #
HiSeq X HD Reagent Kit v2 (300 cycles)
FC-501-2001
HiSeq X HD Reagent Kit v2 (300 cycles), 20 pack
FC-501-2021
Consumables include the flow cell, cBot manifold, clustering reagents used on the cBot,
and flow cell gaskets, funnel caps, and sequencing reagents used on the HiSeq X.
Storage Requirements
As soon as you receive your kit, promptly store the kit components at the indicated
temperature to ensure proper performance.
The following kit components containing SBS reagents are shipped at 15°C to 30°C.
However, when you receive your kit, store the contents at 2°C to 8°C.
} Single-pack kit—SBS Kit Box 1
} 20-pack kit—SBS Kit Box A, B, and C
Reagents are sensitive to light. Store reagents in a dark location away from light.
Workflow Changes Using v2 Kits
The HiSeq X HD Reagent Kit v2 introduces a denaturation step before clustering on the
cBot. Using the previous kit version, non-denatured libraries are added to the ExAmp
reaction in an 8-tube strip. Using the v2 kit, libraries are denatured in the 8-tube strip
before adding the ExAmp reaction mix.
Using the 20-pack kit requires that you cluster 4 flow cells at a time. Each tube of
ExAmp reagent is sufficient for preparing 4 flow cells. Make sure that 4 cBot instruments
are available to begin clustering of the flow cells before preparing the ExAmp reaction.
HiSeq X HD Reagent Kit v2 Reference Guide
3
Reagent Kit Overview
Reagent Kit Overview
Kit Contents and Storage (Single-Pack Kit)
The single-pack kit is packaged in the following configuration.
Kit Component
Quantity
PE Cluster Kit v2 (Box 1 of 2)
2
PE Cluster Kit v2 (Box 2 of 2)
2
Patterned Flow Cell
2
cBot Manifold
2
SBS Kit v2 (Box 1 of 2)
2
SBS Kit v2 (Box 2 of 2)
2
Accessory Kit v2
2
PE 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 Patterned Cluster Mix 1
EPX2
1
-25°C to -15°C
Enhanced Patterned Cluster Mix 2
EPX3
1
-25°C to -15°C
Enhanced Patterned Cluster Mix 3
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Part # 15058861 Rev. A
Kit Contents and Storage (Single-Pack Kit)
PE Cluster Kit Box 2
Box 2 contains reagents and sequencing primers used for indexing and paired-end
resynthesis on the HiSeq X.
Reagent
Quantity
Storage
Description
PRM
1
-25°C to -15°C
Patterned Resynthesis Mix
PLM2
1
-25°C to -15°C
Patterned Linearization Mix 2
PAM
1
-25°C to -15°C
Patterned Amplification Mix
PPM
1
-25°C to -15°C
Patterned Amp Premix
PDR
1
-25°C to -15°C
Patterned Denaturation Mix
(contains formamide)
HP11
1
-25°C to -15°C
Read 2 Primer Mix
HP12
1
-25°C to -15°C
Index i7 Primer Mix
SBS Kit Box 1
Box 1 contains sequencing buffers used on the HiSeq X.
Reagent
Quantity
Storage
Description
PW1
1
2°C to 8°C
Wash Buffer
PB1
1
2°C to 8°C
Patterned SBS Buffer 1
PB2
3
2°C to 8°C
Patterned SBS Buffer 2
* PB1 is sensitive to light. Store PB1 in a dark location away from light.
HiSeq X HD Reagent Kit v2 Reference Guide
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SBS Kit Box 2
Box 2 contains sequencing reagents used on the HiSeq X.
Reagent
Quantity
Storage
Description
PCM
1
-25°C to -15°C
Patterned Cleavage Mix
PIM
1
-25°C to -15°C
Patterned Incorporation Mix
PSM
1
-25°C to -15°C
Patterned Scan Mix
* PIM is sensitive to light. Store PIM in a dark location away from light.
Accessory Kit
The accessory kit contains components used for setting up a sequencing run on the
HiSeq X.
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.
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Part # 15058861 Rev. A
Kit Contents and Storage (20-Pack Kit)
Kit Contents and Storage (20-Pack Kit)
The packaging configuration of the 20-pack kit significantly reduces storage space
requirements. The 20-pack kit is packaged in the following configuration.
Kit Component
Quantity
PE Cluster Kit v2 (Box A)
1
PE Cluster Kit v2 (Box B)
1
PE Cluster Kit v2 (Box C)
1
Patterned Flow Cell (Box of 20)
1
cBot Manifold
20
SBS Kit v2 (Box A)
1
SBS Kit v2 (Box B)
2
SBS Kit v2 (Box C)
6
SBS Kit v2 (Box D)
2
SBS Kit v2 (Box E)
2
SBS Kit v2 (Box F)
2
Accessory Kit v2 (Box A)
1
Accessory Kit v2 (Box B)
1
To help in unpacking the 20-pack kit, see the HiSeq X HD Reagent Kit (20 Pack) Checklist
(part # 15061331). The checklist is available for download from the HiSeq X support page
on the Illumina website.
HiSeq X HD Reagent Kit v2 Reference Guide
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PE Cluster Kit Box A
Box A contains the cBot reagent plate.
Quantity
Component
cBot Reagent Plate
20
Storage
Description
-25°C to -15°C
cBot reagent plate, PE
PE Cluster Kit Box B
Box B contains RSB used to dilute libraries and reagents used make the ExAmp reaction
master mix used on the cBot.
The ExAmp reagents provided in the 20-pack kit include M in the reagent name, which
indicates that each tube supports multiple flow cells.
} One tube of EPX1M, EPX2M, and EPX3M from the 20-pack kit contains sufficient
reagent for 4 flow cells.
} One tube of the same reagents from the single-pack kit contains sufficient reagent for
only 1 flow cell.
Reagent
Quantity
Storage
Description
RSB
5
-25°C to -15°C
Resuspension Buffer
EPX1M
5
-25°C to -15°C
Enhanced Patterned Cluster Mix 1
EPX2M
5
-25°C to -15°C
Enhanced Patterned Cluster Mix 2
EPX3M
5
-25°C to -15°C
Enhanced Patterned Cluster Mix 3
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Part # 15058861 Rev. A
Kit Contents and Storage (20-Pack Kit)
PE Cluster Kit Box C
Box C contains reagents and sequencing primers used for indexing and paired-end
resynthesis on the HiSeq X.
Quantity
Reagent
Storage
Description
PRM
20
-25°C to -15°C
Patterned Resynthesis Mix
PLM2
20
-25°C to -15°C
Patterned Linearization Mix 2
PAM
20
-25°C to -15°C
Patterned Amplification Mix
PPM
20
-25°C to -15°C
Patterned Amp Premix
PDR
20
-25°C to -15°C
Patterned Denaturation Mix
(contains formamide)
HP11
20
-25°C to -15°C
Read 2 Primer Mix
HP12
20
-25°C to -15°C
Index i7 Primer Mix
SBS Kit Boxes A–F
SBS boxes A–F contain sequencing buffers and reagents used on the HiSeq X.
Box
Reagent
SBS Box A
PW1
SBS Box B
Quantity
Storage
Description
20
2°C to 8°C
Wash Buffer
PB1
10
2°C to 8°C
Patterned SBS Buffer 1
SBS Box C
PB2
10
2°C to 8°C
Patterned SBS Buffer 2
SBS Box D
PCM
10
-25°C to -15°C
Patterned Cleavage Mix
SBS Box E
PIM
10
-25°C to -15°C
Patterned Incorporation Mix
SBS Box F
PSM
10
-25°C to -15°C
Patterned Scan Mix
* PB1 and PIM are sensitive to light. Store PB1 and PIM in a dark location away from light.
HiSeq X HD Reagent Kit v2 Reference Guide
9
Accessory Kit Boxes A and B
The accessory kits contain components used for setting up a sequencing run on the
HiSeq X.
Quantity
Box
Accessory
Box A
Flow cell storage tube
with storage buffer, 50 ml
20
Use to store the flow cell after
clustering and before sequencing.
Box B
Flow cell gaskets
(package of 4)
20
Use to replace the flow cell gaskets
between runs; replace before a
maintenance wash.
Funnel bottle caps
(package of 8)
20
Use to replace SBS reagent bottle caps
before loading reagents onto the
instrument.
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Purpose
Part # 15058861 Rev. A
To help identify compatible components in storage, kit components that contain flow
cells and reagents are labeled with color-coded indicators that show kit version, number
of cycles, and type of reagents.
Always check the label markings when you prepare consumables for a run.
Component
Marking on Label
PE cluster kit boxes
Flow cells
SBS kit boxes
HiSeq X HD Reagent Kit v2 Reference Guide
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Kit Compatibility Labeling
Kit Compatibility Labeling
cBot Reagent Plate Contents
Figure 1 cBot Reagent Plate
The cBot reagent plate contains 12 rows with 8 deep wells each. Each reagent occupies 1
row of 8 wells. Not all rows of the reagent plate contain reagent.
Row
1
2
3
4
5
Reagent
HT1
Empty
LPM1
BMS
LDR1
6
7
8
9
10
11
12
Empty
HT2
LMX1
Empty
Empty
HP10
Empty
Description
Hybridization Buffer
Empty
Low Bias Amplification Premix
Amplification Mix
Low Bias Denaturation Reagent
(contains formamide)
Empty
Wash Buffer
Read 1 Linearization Mix
Empty
Empty
Sequencing Primer Mix 1
Empty
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 # 15058861 Rev. A
The following user-supplied consumables are used for preparation and loading of
reagents. For a complete list of user-supplied consumables, see the HiSeq X Lab Setup and
Site Prep Guide (part # 15050093).
Component
Supplier
Purpose
1 N NaOH
General lab supplier
Use for library denaturation step
after diluting to 0.1 N NaOH.
200 mM Tris-HCl, pH 8.0
General lab supplier
Use for library denaturation step.
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.
Conical tubes, 15 ml
Corning,
catalog # 430052*
Use for PW1 in PE positions #12, 18,
and 19.
Centrifuge tube, 250 ml
Corning,
catalog # 430776*
Use for PW1 in SBS position #2 and
for instrument washes.
Laboratory-grade water
Millipore or
General lab supplier
Optionally used for PE positions
#12, 18, and 19, instead of PW1.
Optionally used for SBS position #2,
instead of PW1.
*or equivalent
HiSeq X HD Reagent Kit v2 Reference Guide
13
User-Supplied Consumables
User-Supplied Consumables
HiSeq X Patterned Flow Cell
The HiSeq X flow cell is a patterned flow cell with billions of ordered nano-wells. 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
HiSeq X patterned flow cell contains 8 lanes with 2 swaths per lane, and has the same
general dimensions as a HiSeq high-output flow cell.
The patterned flow cell is provided in the HiSeq X HD Reagent Kit v2, and 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 # 15058861 Rev. A
If you are using the 20-pack kit, prepare 4 flow cells.
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.
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
5
Remove the clear clamshell package from the foil packaging. After the foil packaging
has been opened, use the flow cell within the next 12 hours.
HiSeq X HD Reagent Kit v2 Reference Guide
15
Prepare the Flow Cell
Prepare the Flow Cell
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 # 15058861 Rev. A
Clustering reagents provided in the HiSeq X HD Reagent Kit v2 are used on the cBot. To
prepare reagents, thaw the cBot reagent plate and prepare the ExAmp master mix.
NOTE
If you are using the 20-pack kit to sequence 4 flow cells, thaw 4 cBot reagent plates. Make
sure that 4 cBot instruments are available. Reagents cannot be stored after preparation.
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.
Do not to puncture the foil seals.
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.
HiSeq X HD Reagent Kit v2 Reference Guide
17
Prepare Clustering Reagents
Prepare Clustering Reagents
Thaw EPX1, EPX2, EPX3, and RSB
The following reagents are used to create the ExAmp master mix and dilute libraries.
1
Remove 1 tube each of the following reagents from -25°C to -15°C storage.
• Single-pack kit—EPX1, EPX2, EPX3, and RSB. Each tube contains sufficient
reagent for 1 flow cell.
• 20-pack kit—EPX1M, EPX2M, EPX3M, and RSB. Each tube contains sufficient
reagent for 4 flow cells.
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.
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 the 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 either TruSeq Nano DNA (350 bp) or TruSeq DNA PCRFree libraries. 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.
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Part # 15058861 Rev. A
1
Dilute the library or pooled libraries to one of the following concentrations:
• TruSeq Nano DNA libraries—Dilute to 2–3 nM in RSB.
• TruSeq DNA PCR-Free libraries—Dilute to 1–2 nM in RSB.
2
[Optional] Spike-in 1% non-denatured Illumina PhiX control to non-denatured libraries:
• TruSeq Nano DNA libraries—Add 0.5 µl 2–3 nM PhiX to 50 µl 2–3 nM library.
• TruSeq DNA PCR-Free libraries—Add 0.5 µl 1–2 nM PhiX to 50 µl 1–2 nM
library.
3
Label a new 8-tube strip #1 through #8.
NOTE
If using the 20-pack kit to prepare 4 flow cells, consider adding another designator on
the 8-tube strip for proper sample tracking.
4
To denature the library, add the following volumes to the labeled 8-tube strip in the
order listed:
a Add 5 µl of non-denatured 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 ExAmp Reaction
Prepare the ExAmp reaction master mix immediately before use.
} Single-pack kit—Each tube of ExAmp reagent is sufficient for preparing 1 flow cell.
} 20-pack kit—Each tube of ExAmp reagent is sufficient for preparing 4 flow cells.
CAUTION
Do not refreeze ExAmp reagents after thawing.
Follow the appropriate instructions for the number of flow cells you are preparing.
HiSeq X HD Reagent Kit v2 Reference Guide
19
Prepare Clustering Reagents
Repeat the following instructions for each flow cell to be sequenced.
ExAmp Reaction for 1 Flow Cell (Single-Pack Kit)
Prepare the ExAmp reaction master mix immediately before use.
1
Invert EPX1 and EPX2 five times each to make sure that the tubes are thoroughly
mixed. Then, briefly centrifuge EPX1, EPX2, and EPX3. Do not vortex.
NOTE
EPX3 does not move when inverted. This condition is normal due to the viscosity of
EPX3.
2
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. Do not vortex.
Make sure that there are no air bubbles at the bottom of the tube.
NOTE
The solution can be cloudy, which is normal.
The mixture separates into a cloudy portion and a transparent portion after sitting for
a minute or more. If you see a separation, repeat the pipette mixing step to make
sure that the solution is uniform.
3
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.
4
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.
5
Make sure that the tubes are capped, and then briefly centrifuge the 8-tube strip.
6
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 contain the ExAmp master
mix and library solution onto the cBot.
20
Part # 15058861 Rev. A
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.
ExAmp Reaction for 4 Flow Cells (20-Pack Kit)
Prepare the ExAmp reaction master mix immediately before use.
1
Invert EPX1M and EPX2M five times each to make sure that the tubes are
thoroughly mixed. Then, briefly centrifuge EPX1M, EPX2M, and EPX3M. Do not
vortex.
NOTE
EPX does not move when inverted. This condition is normal due to the viscosity of
EPX3M.
2
Add the following volumes to a 1.5 ml tube in the order listed:
a Add 756 µl EPX1M.
b Add 108 µl EPX2M. Using a P1000 pipette set to 600 µl, slowly pipette up and
down 10 times to mix the reaction. Do not vortex.
NOTE
Due to the viscosity of ExAmp reagents, aspirate and dispense reagents slowly to
ensure accurate pipetting.
c
d
Add 396 µl EPX3M. Using a P1000 pipette set to 1000 µl, slowly pipette up and
down 10 times to mix the reaction. Do not vortex.
Briefly centrifuge the tube of ExAmp reagents. Make sure that there are no air
bubbles at the bottom of the tube.
NOTE
The solution can be cloudy, which is normal.
The mixture separates into a cloudy portion and a transparent portion after sitting for
a minute or more. If you see a separation, repeat the pipette mixing step to make
sure that the solution is uniform.
3
Add 35 µl of the master mix into the bottom of each well of the 8-tube strips
containing denatured and neutralized libraries. Change tips between samples.
4
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.
5
Make sure that the tubes are capped, and then briefly centrifuge the 8-tube strip.
6
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.
HiSeq X HD Reagent Kit v2 Reference Guide
21
Prepare Clustering Reagents
7
CAUTION
Do not exceed 15 minutes before loading the 8-tube strip contain the ExAmp master
mix and library solution onto the cBot.
7
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.
Prepare the cBot Reagent Plate
1
Invert the reagent plate five 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.
4
Remove the clear plastic cover from the cBot reagent plate.
NOTE
Do not puncture the foil seals.
5
Promptly proceed to setting up the cBot.
22
Part # 15058861 Rev. A
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.
If you prepared reagents for 4 flow cells using the 20-pack kit, load reagents and flow
cells on 4 cBot instruments.
Clustering takes approximately 3 hours. After clustering, the patterned flow cell can be
stored in the flow cell storage buffer provided in the kit up to 48 hours at 2°C to 8°C. For
workflow instructions on the cBot, see the cBot User Guide (part # 15006165).
During clustering on the cBot, prepare SBS and paired-end reagents for the HiSeq X.
HiSeq X HD Reagent Kit v2 Reference Guide
23
Next Steps
Next Steps
Prepare SBS Reagents
SBS reagents are loaded onto the instrument at the beginning of the run. To prepare
reagents, use the following instructions to thaw and inspect PSM, PIM, and PCM. Use
PB1 and PB2 directly from 2°C to 8°C storage.
If you are using the 20-pack kit for 4 sequencing runs, make sure that you prepare the
appropriate number of reagent bottles for each SBS reagent.
Reagent
PB1
PB2
PCM
PIM
PSM
Quantity for 1 Flow Cell
Quantity for 4 Flow Cells
1
3
1
1
1
4
12
4
4
4
Thaw SBS Reagents
1
Remove PSM, PIM, and PCM from -25°C to -15°C storage and thaw at 2°C to 8°C for
about 16 hours.
Alternatively, thaw PSM and PIM in a water bath filled with room temperature
deionized water for about 90 minutes. Thaw PCM in a separate water bath. Replace
your gloves after handling PCM.
2
Invert each bottle several times to make sure that the reagents are mixed thoroughly.
NOTE
PSM requires thorough mixing. Visibly inspect the solution to make sure that it is
homogeneous and that no swirling patterns are visible before proceeding.
3
Inspect the reagents to make sure that the reagents are thawed completely and free of
ice crystals.
4
When thawed, set PSM and PIM aside on ice until you are ready to load reagents.
Do not set PSM and PIM on ice with PCM.
5
When thawed, set PCM aside on ice separately to prevent cross-contamination.
Replace your gloves after handling PCM.
NOTE
Remember to invert each bottle several times before loading SBS reagents onto the
instrument.
24
Part # 15058861 Rev. A
Use the following information for reference. For reagent loading instructions, see the
HiSeq X System User Guide (part # 15050091).
Figure 6 SBS Reagent Rack
Table 1 SBS Reagent Positions
Position Reagent
Description
PB2
Patterned
SBS Buffer 2
8
PCM
Patterned Cleavage Mix
7
PB2
Patterned SBS Buffer 2
6
PB2
Patterned SBS Buffer 2
5
PB1
Patterned SBS Buffer 1
4
PSM
Patterned Scan Mix
3
PW1
25 ml PW1 or laboratory-grade water
2
PIM
Patterned Incorporation Mix
1
HiSeq X HD Reagent Kit v2 Reference Guide
25
SBS Reagent Positions
SBS Reagent Positions
Prepare Indexing and Paired-End Reagents
Reagent
Quantity
Storage
Description
PRM
1
-25°C to -15°C
Patterned Resynthesis Mix
PLM2
1
-25°C to -15°C
Patterned Linearization Mix 2
PAM
1
-25°C to -15°C
Patterned Amplification Mix
PPM
1
-25°C to -15°C
Patterned Amp Premix
PDR
1
-25°C to -15°C
Patterned Denaturation Mix
(contains formamide)
HP11
1
-25°C to -15°C
Read 2 Primer Mix
HP12
1
-25°C to -15°C
Index i7 Primer Mix
Indexing and paired-end reagents are loaded onto the instrument at the beginning of the
run and used during the Read 2 resynthesis step of a paired-end sequencing run.
If you are using the 20-pack kit for 4 sequencing runs, make sure that you prepare the
appropriate number of reagent tubes for each indexing and paired-end reagent.
Reagent
PRM
PLM2
PAM
PPM
PDR
HP11
HP12
Quantity
4
4
4
4
4
4
4
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.
26
Part # 15058861 Rev. A
1
Remove the following reagents from -25°C to -15°C storage: PRM, PLM2, PAM,
PPM, PDR, HP11, and HP12.
NOTE
For non-indexed libraries, HP12 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 PRM, PLM2, and PAM on ice.
NOTE
PDR solidifies on ice.
Prepare PRM, PLM2, PAM, PPM, PDR, HP11, and HP12
1
Invert each tube five times to mix the reagent.
2
Centrifuge the reagent at 1000 rpm for 1 minute.
3
For PRM, PLM2, and PAM—Set aside on ice.
For PPM, PDR, HP11, and HP12—Set aside at room temperature.
HiSeq X HD Reagent Kit v2 Reference Guide
27
Prepare Indexing and Paired-End Reagents
Thaw Indexing and Paired-End Reagents
Paired-End and Indexing Reagent Positions
Use the following information for reference. For reagent loading instructions, see the
HiSeq X System User Guide (part # 15050091).
Figure 7 Paired-End Reagent Rack
Table 2 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
Index 1 (i7) Primer Mix
17
HP12
Read 2 Primer Mix
16
HP11
Patterned Denaturation Reagent
15
PDR
Patterned Amplification Premix
14
PPM
Patterned Amplification Mix
13
PAM
10 ml PW1 or laboratory-grade water
12
PW1
Patterned Linearization Mix 2
11
PLM2
Patterned Resynthesis Mix
10
PRM
28
Part # 15058861 Rev. A
After you have prepared SBS and paired-end reagents, you are ready to load them onto
the HiSeq X. For reagent loading and sequencing workflow instructions, see the HiSeq X
System User Guide (part # 15050091).
HiSeq X HD Reagent Kit v2 Reference Guide
29
Next Steps
Next Steps
Notes
For technical assistance, contact Illumina Technical Support.
Table 3 Illumina General Contact Information
Address
5200 Illumina Way
San Diego, CA 92122 USA
Website
www.illumina.com
Email
techsupport@illumina.com
Table 4 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.
HiSeq X HD Reagent Kit v2 Reference Guide
Technical Assistance
Technical Assistance
*15058861*
Part # 15058861 Rev. A
Illumina
San Diego, California 92122 U.S.A.
+1.800.809.ILMN (4566)
+1.858.202.4566 (outside North America)
techsupport@illumina.com
www.illumina.com