Using Illumina Nextera DNA Sample Preparation Kits and Enabling Dual Indexing (15029964 A)

Using Illumina Nextera DNA Sample Preparation Kits and Enabling Dual Indexing (15029964 A)
Using Illumina Nextera™ DNA Sample Preparation Kits and Enabling
Dual Indexing
FOR RESEARCH USE ONLY
The new Illumina Nextera™ DNA Sample Preparation
Kits (Table 1) provide a rapid and streamlined workflow
enabling library preparation in less than 90 minutes from
only 50 ng of input DNA.
In addition, Illumina now supports up to 96 Nextera
indices per lane on all Illumina sequencing systems. If
sequencing an Illumina Nextera prepared library on HiSeq
2000/1000, HiScanSQ, or GAIIx an accessory sequencing
primer kit is required to enable dual indexing. The MiSeq
System reagent kits already include the appropriate
primers to enable dual indexing and these accessory
sequencing primer kits are not required. Additional details
on completing a successful dual indexing sequencing run
with Illumina Nextera libraries can be found below.
Table 1 Illumina Nextera DNA Sample Preparation Kits
Kit Name
Catalog #
Nextera DNA Sample Preparation Kit FC-121-1031
(96 Samples)
Nextera DNA Sample Preparation Kit FC-121-1030
(24 Samples)
Nextera Index Kit (96 Indices, 384
FC-121-1012
Samples)
Nextera Index Kit (24 Indices, 96
FC-121-1011
Samples)
Table 2 Sequencing Primer Kits for Sequencing Illumina
Nextera Libraries on HiSeq 2000/1000, HiScanSQ, and GAIIx
Kit Name
Catalog #
TruSeq Dual Index Sequencing Primer FC-121-1003
Kit, Single Read (single use kit)
TruSeq Dual Index Sequencing Primer PE-121-1003
Kit, Paired End (single use kit)
Sample Sheet Preparation
To ensure proper index combinations for pooled libraries
with dual index experiments, please use the new Illumina
Experiment Manager (IEM) prior to performing the
Illumina Nextera sample prep. IEM is a tool that helps
create the required sample sheet for use with HiSeq
2000/1000, HiScanSQ, GAIIx or MiSeq, and is available for
download on iCom. IEM also notifies the user whether the
indices chosen provide appropriate sequence diversity to
avoid registration failures during sequencing.
Part # 15029964 Rev A
Pooling Guidelines
When using less than the full complement of indices in the
kit, refer to the Illumina Nextera DNA Sample Preparation
Guide for a compatibility chart for pooling guidelines. To
assist in correctly arranging index primers during the PCR
Amplification steps, a TruSeq Index Plate Fixture Kit is
available to order (Catalog # FC-130-1005).
Using Nextera on MiSeq
If preparing libraries to run on MiSeq, the appropriate
primers are included in the reagent cartridge and the oninstrument analysis software (MiSeq Reporter)
automatically performs dual index processing. When using
IEM for sample sheet generation, select Adapter Trimming
when you create your sample sheet. Shorter inserts may
lead to sequencing into the adapter, and this feature helps
filter out adapter sequence from the final sequence data.
Using Nextera on other Illumina Sequencers
When using the Illumina Nextera kit for sequencing on
HiSeq 2000/1000, HiScanSQ or GAIIx, there are workflow
changes in clustering, sequencing and analysis that require
ordering additional kits.
NOTE
Please note that new primers are required for
cluster generation and sequencing when using
the Illumina Nextera sample prep kit, whether
performing indexing or not.
Please order the TruSeq Dual Index Sequencing Primer
Boxes (Table 2); these kits contain the new primers for both
clustering and sequencing.
cBot or Cluster Station Workflow
} Primers: If preparing Illumina Nextera libraries for
cluster generation on HiSeq, HiScanSQ, or GAIIx, you
must use the TruSeq Dual Index Sequencing Primer
Boxes for all sequencing run types: non-indexed,
single-indexed, and dual-indexed. The TruSeq Dual
Index Sequencing primers will be used in place of the
existing sequencing primers (HP6, HP7, and HP8).
Please see table 2 for catalog numbers.
} Workflow Updates: Dual indexing now requires
aliquotting the new primer mixes into strip tubes on
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Using Illumina Nextera™ DNA Sample Preparation Kits and Enabling Dual Indexing
cBot. Because of this, the appropriate single read or
paired end “Amp_Lin_Block_TubeStripHyb” recipe is
required. Please refer to the updated cBot
documentation for additional information.
} Software: Please upgrade to cBot Software version 1.4
if this has not been done already. Additionally, please
upgrade to or install the latest, dual-indexingcompatible recipes using cBot Recipe Installer version
1.0.13 or later. Both the software installer and the
recipe installer can be found on iCom.
Sequencing Workflow
} Primers: If sequencing Illumina Nextera libraries with
HiSeq, HiScanSQ, or GAIIx, you must be sure to use
the TruSeq Dual Index Sequencing Primer Boxes (Table
2) for all sequencing run types: non-indexed, singleindexed, and dual-indexed. These add on kits are not
required if sequencing an Illumina Nextera prepared
library with the MiSeq System.
} Workflow Updates: Dual indexing now requires 23
additional cycles of sequencing – eight cycles for the
Index 1 (i7) Read, eight cycles for the Index 2 (i5) Read,
plus seven non-imaging, chemistry-only cycles at the
beginning of the Index 2 (i5) Read. Due to these
additional cycles, there are new recommendations for
SBS reagent preparation.
NOTE
A 200-cycle kit will not contain sufficient overfill
for a 2x101-cycle dual indexed run on HiSeq
(now 225 cycles). Instead, it is recommended to
combine four 50-cycle kits.
} Software: Sequencing Illumina Nextera prepared
libraries on HiSeq/HiScanSQ requires an upgrade to
HCS 1.5/RTA 1.13 to enable the dual indexing
workflow. GAIIx requires an upgrade to SCS 2.10/RTA
1.13 as it contains new v10 recipes for dual indexed
sequencing. Software is available for download from
iCom.
Analysis Workflow
CASAVA 1.8.2 is required to analyze dual index data
generated on the HiSeq 2000/1000, HiScanSQ or GAIIx
instruments. A sample sheet in the new dual index format
is also required, and can be generated using IEM. Please
refer to the updated CASAVA User Guide and Release
Notes for more information.
Software Requirements
Software upgrades are required to run Illumina Nextera
prepared libraries. To download the updated software
2 of 2
versions, go to https://icom.illumina.com/Downloads.
Under “Download Categories,” choose “Software” and
scroll down to the following locations:
} Illumina Experiment Manager sample sheet creator:
Illumina Experiment Manager 1.0
} cBot software (if needed): cBot 1.4 Software; the recipe
installer can also be found under this listing.
} HCS software for HiSeq/HiScanSQ: HCS 1.5/RTA
1.13 Software
} SCS software for Genome Analyzer: Genome
Analyzer SCS 2.10/RTA 1.13 Software
} CASAVA: CASAVA 1.8.2 Software
Supporting Documentation
Title
Illumina Nextera DNA Sample
Preparation Guide
Illumina Nextera DNA Sample
Preparation Experienced User Card
MiSeq System User Guide
cBot User Guide
HiSeq 2000 User Guide
HiSeq 1000 User Guide
HiScanSQ User Guide
GAIIx Quick Reference Guide
CASAVA 1.8 User Guide
Part #
15027987 Rev. A
15028285 Rev. A
15027617 Rev. A
15006165 Rev. G
15011190 Rev. K
15023355 Rev. E
15015392 Rev. G
15018815 Rev. C
15011196 Rev. C
To download documentation, go to
http://www.illumina.com/support/documentation.ilmn.
Technical Assistance
For questions, go to the Support tab on illumina.com. If
you do not find the information you need there, contact
Illumina Technical Support by email or phone.
NOTE
The information in this document is only applicable
for Illumina Nextera DNA Sample Preparation Kits
(see Table 1). For legacy Nextera kits provided by
Epicentre (single-indexing only), refer to the
appropriate user documentation on the Epicentre
website (www.epibio.com).
Copyright and Trademarks
© 2011 Illumina, Inc. All rights reserved.
Illumina, illuminaDx, BeadArray, BeadXpress, cBot, CSPro, DASL,
DesignStudio, Eco, GAIIx, Genetic Energy, Genome Analyzer,
GenomeStudio, GoldenGate, HiScan, HiSeq, Infinium, iSelect,
MiSeq, Nextera, Sentrix, Solexa, TruSeq, VeraCode, the pumpkin
orange color, and the Genetic Energy streaming bases design are
trademarks or registered trademarks of Illumina, Inc. All other brands and
names contained herein are the property of their respective owners.
Part # 15029964 Rev A
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