TruSeq Rapid Duo Sample
Loading Kit
Reagent Preparation Guide
(for cBot - HiSeq 1500/2500)
FOR RESEARCH USE ONLY
Introduction
3
Kit Contents
5
Preparing the Reagent Plate
7
About Template DNA
9
What’s Next
Technical Assistance
ILLUMINA PROPRIETARY
Part # 15036504 Rev. A
October 2012
10
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The TruSeq Rapid Duo Sample Loading Kit enables you to hybridize different library
samples in different lanes. Only template hybridization and first extension will occur
on the cBot. Cluster generation will occur on the HiSeq 2500.
This guide includes a description of the TruSeq Duo cBot Sample Loading Kit and
explains how to prepare reagents for template hybridization on the cBot.
For a description of the workflow and cluster generation protocols, see the cBot User
Guide.
For a description of the sequencing workflow using multiplexing and paired-end
reagents, see the user guide for your sequencing instrument.
TruSeq Rapid Duo Template Hybridization Reagents
The TruSeq family of reagents is the newest in a long line of Illumina sequencing
chemistry advances focused on improved workflow, ease of use, and data quality.
TruSeq Rapid Duo cBot template hybridization kits fully support the following
sequencing applications:
Reagents
Single-read cluster generation reagents
Paired-end cluster generation reagents
Multiplexing sequencing reagents
Paired-end sequencing reagents
Sequencing Application
Single-read sequencing
ChIP-sequencing
Small RNA sequencing
mRNA sequencing
Paired-end sequencing
Mate pair sequencing
Single-read multiplexed sequencing
Paired-end multiplexed sequencing
Paired-end sequencing
Mate pair sequencing
Reagents and materials provided in the Template Hybridization Kit are sufficient for
processing one flow cell. All materials are single-use.
TruSeq PE Rapid Cluster Kit Reagent Preparation Guide (cBot-HS)
3
Introduction
Introduction
What’s New
TruSeq Rapid Duo template hybridization reagents enable individually addressable
lanes with different samples in different lanes. They provide an alternative to HiSeq
on-board template hybridization.
Rapid Flow Cell
The TruSeq Rapid Duo cBot template hybridization kit should be used with the new
Rapid flow cell, which features two lanes and the same imaging area as v3 flow
cells.
NOTE
You must run HCS v2.0 or later on your sequencing instrument when using a
Rapid flow cell.
Documentation
Additional documentation is available for download from the Illumina website. Refer
to the inside back cover of this guide for more information.
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Part # 15036504 Rev. A
Kit Contents
Kit Contents
It is very important to store the reagents at the temperature specified on the box to
ensure that they perform correctly.
Generation Components
Component
Rapid Duo cBot reagent plate
Storage Specification
-15° to -25°C
Kit Reagent Plate
The Rapid Duo cBot template hybridization reagent plate contains 3 rows of foilsealed eight-tube strips filled with template hybridization and first extension
reagents. Rows 4 through 12 of the reagent plate are empty.
Figure 1 cBot Template Hybridization Reagent Plate
A
B
Row 1
Row 3
Each reagent tube strip is labeled with the reagent name followed by a number. The
number indicates the row it occupies on the reagent plate. Should a tube strip become
dislodged from the reagent plate holder, use the row number on the label to return
the tube strip to the appropriate position on the plate.
Reagent Plate Contents
} Row 1–HT1 (Hybridization Buffer)
TruSeq PE Rapid Cluster Kit Reagent Preparation Guide (cBot-HS)
5
} Row 2–AMS (Fast Amplification mix)
} Row 3–USB (Universal Sequencing Buffer)
} Row 4–12 Empty
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Part # 15036504 Rev. A
Reagents take a minimum of one hour to thaw using a room temperature water bath.
Alternatively, you can thaw reagents at 2° to 8°C overnight or a maximum of
16 hours. After thawing, carefully follow the steps to prepare reagents for use on the
cBot.
Best Practices
} Wear a fresh pair of gloves when setting up a run.
} Hold the 96-well reagent plate by the plate base to avoid dislodging any reagent
tubes.
} Always check that the tubes are securely seated in the reagent plate before and
after vortexing or inverting reagents. Loose reagent tubes can damage the cBot
manifold and stop the run.
NOTE
The clear plastic lid on the reagent plate protects the foil seals from being
damaged or punctured during thawing. Remove the protective lid only when
necessary, such as when you are checking that tubes are securely seated.
} After completing the reagent preparation steps, promptly load them onto the cBot
and begin your run.
Thaw Reagents
1
Remove the reagent plate from -15° to -25°C storage.
2
Press down on the outside edge of each row of tubes in the reagent plate to make
sure that they are securely seated in the plate. Take care not puncture the foil
seals.
3
Place the reagent plate in a water bath containing only enough room temperature
deionized water to submerge the reagent plate base. To prevent contamination,
do not allow the water to select the foil seals. Allow the reagents to thaw in the
water bath for at least 60 minutes.
NOTE
Reagent tubes with greater volumes may take longer to thaw. Inspect the
reagents to make sure that they have completely thawed.
TruSeq PE Rapid Cluster Kit Reagent Preparation Guide (cBot-HS)
7
Preparing the Reagent Plate
Preparing the Reagent Plate
Prepare Reagents
NOTE
To ensure proper run performance, it is important that you thoroughly mix
reagents and collect reagent droplets as described in the following steps.
1
Hold the reagent plate by the base and place your other hand on top of the tubes,
then invert the reagent plate five times to mix the thawed reagents.
2
While holding your hand on top of the tubes, vortex the plate for approximately
10 seconds to dislodge any trapped air bubbles.
3
Tap the reagent plate on a hard surface 5–10 times to collect any reagent droplets
to the bottom of the tubes. Alternatively, pulse centrifuge the reagent plate to
collect reagent droplets.
4
Visually inspect the reagent plate to make sure that no air bubbles exist at the
bottom of the tubes.
5
Visually inspect the numbering on the tube labels to make sure that they are in
the correct order.
6
Promptly proceed to setting up the run. Do not return reagents to 2° to 8°C
storage or allow reagents to sit at room temperature.
For more information, see the cBot User Guide.
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Part # 15036504 Rev. A
To achieve the highest quality data on Illumina sequencing platforms, it is important
to create optimum cluster densities across every lane of every flow cell. This requires
accurate quantification of template DNA libraries. For recommended quantification
methods, see the sample prep documentation for the kit that you used to create your
libraries.
For Rapid Run flow cells, use the same loading concentration as you would for onboard cluster generation on the HiSeq 2500 and HiSeq 1500.
TruSeq PE Rapid Cluster Kit Reagent Preparation Guide (cBot-HS)
9
About Template DNA
About Template DNA
What’s Next
Template Hybridization
After you properly thawed reagents, you are ready prepare your libraries for template
hybridization and proceed to the run setup steps using the cBot software interface.
See the cBot User Guide, Part # 15006165.
Make sure that you use the appropriate recipe and cBot software version for the
TruSeq Rapid Duo cBot kit.
Kit Version
cBot Recipe Version
cBot Software
Version
TruSeq Rapid Duo Sample
Loading Kit
TruSeq Rapid PE Cluster Kit - HS
for Rapid flow cell
cBot v1.5 or later
For complete workflow instructions on the cBot, see the cBot User Guide.
Sequencing
Make sure that you use the appropriate version of SBS Kit and HiSeq Control
Software (HCS) with a Rapid flow cell.
Kit Version
SBS Kit Version
HCS/RTA Version
TruSeq Rapid Duo Sample
Loading Kit
TruSeq Rapid PE Cluster Kit HS for Rapid flow cell
HCS v2.0/RTA v1.17
or later
For complete workflow instructions, see the user guide for your sequencing
instrument.
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Part # 15036504 Rev. A
For technical assistance, contact Illumina Technical Support.
Table 1 Illumina General Contact Information
Illumina Website
Email
www.illumina.com
[email protected]
Table 2 Illumina Customer Support Telephone Numbers
Region
Contact Number
Region
Contact Number
North America
1.800.809.4566
Italy
800.874909
Austria
0800.296575
Netherlands
0800.0223859
Belgium
0800.81102
Norway
800.16836
Denmark
80882346
Spain
900.812168
Finland
0800.918363
Sweden
020790181
France
0800.911850
Switzerland
0800.563118
Germany
0800.180.8994
United Kingdom
0800.917.0041
Ireland
1.800.812949
Other countries
+44.1799.534000
MSDSs
Material safety data sheets (MSDSs) are available on the Illumina website at
www.illumina.com/msds.
Product Documentation
You can obtain PDFs of additional product documentation from the Illumina website.
Go to www.illumina.com/support and select a product. To download documentation,
you will be asked to log in to MyIllumina. After you log in, you can view or save the
PDF. To register for a MyIllumina account, please visit
my.illumina.com/Account/Register.
TruSeq PE Rapid Cluster Kit Reagent Preparation Guide (cBot-HS)
Technical Assistance
Technical Assistance
Illumina
Headquartered in San Diego, California, U.S.A.
+1.800.809.ILMN (4566)
+1.858.202.4566 (outside North America)
[email protected]
www.illumina.com