Protocol for nextera™ dna sample prep kit (illumina-compatible)

Nextera™ DNA Sample Prep Kit
(Illumina-compatible)
Cat. Nos. GA09115, GA091120, GA0911-50, GA0911-96, and GABC0950
The Nextera™ DNA Sample Prep Kit is designed Product Specifications
to prepare genomic DNA libraries compatible with the Illumina® Genome Analyzer I and II and Hi- Storage: Store the Nextera DNA Sample Prep Kit
Seq™ 2000 sequencers. Nextera technology† at –20oC in a freezer without a defrost cycle. employs in vitro transposition to simultaneously Quality Control: Nextera DNA Sample Prep Kit is
fragment and tag DNA in a single-tube reaction, function-tested by tagmenting control DNA (lamb- and prepare sequencer-ready libraries in under 2 da) with both Low-Molecular-Weight (LMW) and hours. The Nextera library preparation procedure High-Molecular-Weight (HMW) Buffers. Size dis- is a significant improvement upon current proce- tribution following tagmentation and PCR amplifi- dures, which generally consist of distinct DNA cation is confirmed by Bioanalyzer profiling. fragmentation, end-polishing, and adaptor-ligation steps. The Nextera library preparation procedure Contaminating Activity Assays: All components
combines these steps into one (tagmentation), of the Nextera DNA Sample Prep Kit are free of uses only 50 ng of starting DNA, and allows incor- poration of platform-specific tags and optional bar- Nextera Control DNA: The Nextera Control DNA
is unmethylated cl857 Sam7 Lambda DNA, iso- lated from infected GM119, an E. coli strain lack- ing both the dam and dcm methylase activities. † Covered by patents issued and pending. Nextera™ DNA Sample Prep Kits (Illumina-compatible) Contents
The Nextera DNA Sample Prep Kit (Illumina-compatible) is available in four sizes (5, 20, 50, and 96 reac-tions). All reagents in these kits are in blue-capped tubes. GA091120
GA0911-50
GA0911-96
Component Name
Reactions
Reactions
Reactions
Reactions
Nextera™ Enzyme Mix (Illumina-compatible). 5 μl 5X Nextera™ Reaction Buffer (LMW). 50 μl 5X Nextera™ Reaction Buffer (HMW) . 50 μl 50X Nextera™ Primer Cocktail (Illumina-compatible) . 5 μl 50X Nextera™ Adaptor 2 (Illumina-compatible) . 5 μl 200X Nextera™ Index Read Primer* . 30 μl * The 5-, 20-, 50-, and 96-reaction kits contain sufficient sequencing primers (Read 1, Read 2, and Index Read) for 3, 10, 15, and 30 flow cells, respectively. If needed, sequencing primers for additional flow cells can be provided upon request.
Additional Required Components (Not Provided)
– Zymo DNA Clean & Concentrator™-5 (Cat. No. D4013) or equivalent.
– Nextera™ PCR Enzyme (Cat. Nos. EM091120, EM091150, EM0911-96)
726 Post Road, Madison, WI 53713 • www.EpiBio.com • (800) 284-8474 • (608) 258-3080 • Fax (608) 258-3088 EPICENTRE
Nextera™ DNA Sample Prep Kit
(Illumina-compatible)

Figure 1. Generating Illumina-compatible libraries.
Add bPCR-compatible sites by PCR (+/– bar codes) Target DNA is fragmented and tagged with Nextera Enzyme Mix containing transposon ends appended with sequencing primer sites (blue and orange). Limited-cycle PCR with a four-primer reaction adds bridge PCR (bPCR)-compatible adaptors (purple and pink) to the core sequencing library. Optional bar codes (triangle) can be added between the downstream bPCR adaptor (pink) and the core sequencing library adaptor (orange). Alternative sequencing primers are required for the Il umina/Solexa®-compatible libraries: Read 1 Primer (blue/gray arrow); Read 2 Primer (orange/gray arrow); Index Read Primer (gray/
Sequences:
Transposon End Sequence: 5'-AGATGTGTATAAGAGACAG-3'
Adaptor 1*: 5'-AATGATACGGCGACCACCGAGATCTACACGCCTCCCTCGCGCCATCAG-3' Adaptor 2 (minus bar code)*: 5'-CAAGCAGAAGACGGCATACGAGATCGGTCTGCCTTGCCAGCCCGCTCAG-3' Nextera Read 1 Primer: 5'-GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG-3' Nextera Read 2 Primer: 5'-GCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG-3' Nextera Index Read Primer: 5'-CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGCAGACCG-3'
Note: The kit contains a 50X Nextera Primer Cocktail, which consists of Primer 1 (10 μM), Primer 2
(10 μM), and Adaptor 1 (0.5 μM). A single primer, 50X Nextera Adaptor 2 (which does not contain a bar code), is also included in the kit. The 50X Nextera Adaptor 2 (0.5 μM) can be replaced with one of the Bar Coding Primers from the Il umina-compatible Bar Codes Kit (optional). Nextera Read 1, Read 2, and Index Read Primers are provided at a concentration of 100 μM (200X). * The sequences of Primer 1 and Primer 2 and portions of Adaptor 1 and Adaptor 2 correspond to Il u- mina bPCR sequences and are copyrighted to Il umina, Inc. Oligonucleotide sequences 2006-2010 EPICENTRE
Nextera™ DNA Sample Prep Kit
(Illumina-compatible)
Important Considerations

1. Fragment Size Distribution: The Nextera DNA Sample Prep Kit contains two buffers, Low-Molecular-
Weight Buffer (LMW) and High-Molecular-Weight Buffer (HMW). Refer to table below for approximate fragment size distribution using each buffer. 40-265 bp
40-565 bp
*Fragment Size (approx.) includes the 135-bp adaptor sequences (67-bp Adaptor 1 + 68-bp Adaptor 2) without bar coding. For all paired-end sequencing, we recommend using HMW Buffer only.
Note: These are approximations only, as the actual fragment size distribution will depend on a number
of factors including the type and quality of the starting DNA.
Figure 2. Fragment Size Distribution (approx.) using LMW Buffer (A) and HMW Buffer (B),
per standard protocol.

2. DNA Quality: The quality of the starting DNA is critical. Contaminants such as protein and DNA may
inhibit the Nextera reaction if present in the DNA preparation. If DNA purity is in question, the DNA should be cleaned using Zymo Genomic DNA Clean & Concentrator, Cat. No. D4010 (or equivalent).
3. Transposon End Sequence: The 19-bp transposon DNA sequence is present at the 5' end of all
Illumina-compatible libraries. However, the 19-bp transposon DNA sequence is NOT sequenced on the Illumina platform. The Nextera Read 1 and Read 2 primers anneal to this sequence so that the first nucleotide sequenced is target DNA.
4. Input DNA: The kit has been optimized to process 50 ng of DNA to the target MW distribution. MW
distribution will be lower if using less than 50 ng of DNA.
5. Amplicons: The Nextera DNA Sample Prep Kit can also make libraries from amplicons. Amplicons
as small as ~2 kb have been successfully sequenced. However, the distal ~50-100 bp of linear frag-ments may exhibit a decrease in coverage. Nextera kits can also be used to make libraries from circu-lar DNA samples. EPICENTRE
Nextera™ DNA Sample Prep Kit
(Illumina-compatible)

6. Bias: The transposase used in the Nextera system carries mutations and is used under conditions that
result in near-random integration. As with any enzymatic system, there is a slight bias in the reaction. However, since the reaction is driven to completion with an excess of the Nextera Enzyme, we have not seen any impact on the distribution of coverage. The coverage across assemblies is comparable to those seen with mechanical methods of shearing.
7. Bar Codes: Platform-specific Bar Coding kits are available to prepare up to 12 bar coded libraries. If
additional or alternate Bar Codes are needed, the following template design can be used: Adaptor 2 (plus Bar Code-optional)
5'-CAAGCAGAAGACGGCATACGAGAT-[BAR CODE]*-CGGTCTGCCTTGCCAGCCCGCTCAG-3' *Reverse complement of sequencing read.
8. Sequencing in the Same Channel: The Nextera sequencing primers are compatible with the Illumina
sequencing primers, and can be used together.
9. Nextera PCR Enzyme: Use only Nextera PCR Enzyme for limited-cycle PCR (Step B, page 7). Other
PCR systems have been tested and do not perform as well. EPICENTRE
Nextera™ DNA Sample Prep Kit
(Illumina-compatible)
Flowchart for Nextera™ DNA Sample Preparation
Nextera Tagmentation Reaction
• 50 ng DNA
• 4 μl LMW or HMW Buffer
• 1 μl Nextera Enzyme Mix
• x μl Nuclease-Free Water
55oC Incubation.5 min.
Zymo Cleanup
• Elute with 11 μl Nuclease-Free Water
Limited-Cycle PCR
• 5 μl Zymo-purified Tagmentation Reaction
• 17 μl Nuclease-Free Water9 cycles PCR Zymo Cleanup
Ampure™ XP Purification (0.7X)
Use recovered DNA as input for bPCR and cluster
generation per standard Illumina protocol
Dilute Nextera Sequencing Primers for single or
paired-end sequencing (pp. 6-7)
EPICENTRE
Nextera™ DNA Sample Prep Kit
(Illumina-compatible)
Nextera DNA Sample Prep Kit (Illumina-compatible) Protocols
A. Tagmentation Reaction

1. Prior to assembling the reaction, briefly centrifuge the 5X Nextera Reaction Buffer and Nextera En-
zyme Mix tubes to assure that the reagents are at the bottom of the tubes. 2. Assemble the following reaction components on ice, in the order listed: 50 ng Target DNA (in T10E1 Buffer [10 mM Tris-HCl (pH 7.5), 1 mM EDTA]) 4 µl 5X Nextera Reaction Buffer LMW or HMW (see Important Considerations, no. 1, p. 3) 1 µl Nextera Enzyme Mix (Illumina-compatible) 3. Mix briefly by vortexing, and incubate at 55oC for 5 minutes. Notes: To prevent evaporation, the reaction should be carried out in a thermocycler with a heated lid
or the reaction should be overlaid with mineral oil. The tagmentation reaction does occur, although very slowly, at room temperature. We recommend assembling the components on ice and proceeding immediately to the 55oC incubation 4. Purify the tagmented DNA using a Zymo DNA Clean & Concentrator-5 Kit (or equivalent). Brief Zymo Protocol (perform at room temperature): – Add 100 µl of DNA Binding Buffer to the 20 µl Tagmentation Reaction from step 2 (above). – Mix briefly by vortexing, and transfer the mixture to a Zymo-Spin™ Column in a Collection Tube. – Centrifuge at 10,000 x g for 60 seconds. Discard the flow-through. – Add 250 µl of Wash Buffer to the column. Centrifuge at 10,000 x g for 60 seconds. Discard the – Centrifuge the column at 10,000 x g for 60 seconds to eliminate any residual Wash Buffer. – Transfer the column to a clean and sterile 1.5-ml microcentrifuge tube – Add 11 µl of Nuclease-Free Water directly to the column and incubate at room temperature for 1-2 minutes. Centrifuge at 10,000 x g for 60 seconds to elute the DNA. – The final eluted volume should be ~10 µl. Use 5 µl as DNA template in Step B1 (p. 6). Note: Nextera technology has been validated with the Zymo DNA Clean & Concentrator-5 and
Qiagen MinElute® DNA Purification Kits. Equivalent kits can also be used; however, care must be taken when eluting the DNA from the spin columns. EPICENTRE
Nextera™ DNA Sample Prep Kit
(Illumina-compatible)
B. Addition of bPCR-Compatible Sites and Library Enrichment

Add bPCR-compatible sites and optional bar coding by PCR.
1. Assemble the following reaction components at room temperature: 5 µl Recovered DNA Fragment Library (from Step A3) 1 µl 50X Nextera Primer Cocktail (Illumina-compatible) 1 µl Nextera PCR Enzyme (sold separately, see Related Products p. 8) * Note: For a bar coded library, replace 50X Nextera Adaptor 2 with a bar coded Illumina-compatible
Adaptor 2 from the Nextera Bar Codes (Illumina-compatible) kit (e.g., GA Adaptor 2 [IDX1]). 2. Cycle the samples in a thermocycler under the following conditions: **Note: It is critical to perform the 72oC extension step before denaturing the DNA templates.
3. Purify the tagged DNA fragments using a Zymo DNA Clean & Concentrator-5 kit, or equivalent.
Note: The anticipated yield is ~300 ng of amplified DNA. To remove fragments below 300 bp, we
strongly recommend using Agencourt AMPure™ XP beads (0.7X) instead of the Zymo DNA Clean and Concentrator-5™. 4. Use the recovered DNA as input for bPCR and cluster generation per the standard Illumina protocol. Note: FOR CLUSTER SEQUENCING, IT IS CRITICAL TO USE THE PROVIDED NEXTERA
SEQUENCING PRIMERS.
• Use the Nextera Read 1 Primer for Paired-End Read 1 Sequencing or for Single-Read
• Use the Nextera Index Read Primer for Index Read Sequencing.
• Use the Nextera Read 2 Primer for Paired-End Read 2 Sequencing.
Note: The 5-, 20-, 50-, and 96-reaction kits contain sufficient sequencing primers (Read 1, Read 2,
and Index Read) for 3, 10, 15, and 30 flow cells, respectively. If needed, sequencing primers for additional flow cells can be provided upon request. Illumina paired-end sequencing mixes (HP1 and HP2) contain the Illumina sequencing primers. These primers do not interfere with the Nextera sequencing primers and sequencing can be per-formed in the presence of HP1 and HP2 primers. • Paired-End Read 1: Dilute Nextera Read 1 Primer 1:200 into Sequencing Mix HP1.
• Index Read: Dilute Nextera Index Read Primer 1:200 into Hybridization Buffer.
• Paired-End Read 2: Dilute Nextera Read 2 Primer 1:200 into Sequencing Mix HP2.
Alternatively, if it is required to perform sequencing in the presence of only the Nextera primers, the 200X Nextera sequencing primers can be diluted 1:200 into Hybridization Buffer (GA0084204-HT1 or 5X SSC, 0.05% Tween®-20). EPICENTRE
Nextera™ DNA Sample Prep Kit
(Illumina-compatible)
• Paired-End Read 1: Dilute Nextera Read 1 Primer 1:200 into Sequencing Mix HP4.
• Index Read: Dilute Nextera Index Read Primer 1:200 into Hybridization Buffer.
Appendix A
The Illumina-compatible Bar Codes Kit contains 12 bar codes. A 50-µl aliquot of each is provided at a concentration of 0.5 µM. This is sufficient for 50 bar coded libraries. All reagents in this kit are in blue-capped tubes. For bar coding, one of these bar codes can be substituted with 50X Nextera Adaptor 2 (Illumina-compat-ible) from the Nextera DNA Library Prep Kit (Illumina-compatible). Use 1 µl in the reaction. GA Adaptor 2 (IDX1) 5'-CAAGCAGAAGACGGCATACGAGATCGTGATCGGTCTGCCTTGCCAGCCCGCTCAG-3'
5'-CAAGCAGAAGACGGCATACGAGATACATCGCGGTCTGCCTTGCCAGCCCGCTCAG-3'
5'-CAAGCAGAAGACGGCATACGAGATGCCTAACGGTCTGCCTTGCCAGCCCGCTCAG-3'
5'-CAAGCAGAAGACGGCATACGAGATTGGTCACGGTCTGCCTTGCCAGCCCGCTCAG-3'
5'-CAAGCAGAAGACGGCATACGAGATCACTGTCGGTCTGCCTTGCCAGCCCGCTCAG-3'
5'-CAAGCAGAAGACGGCATACGAGATATTGGCCGGTCTGCCTTGCCAGCCCGCTCAG-3'
5'-CAAGCAGAAGACGGCATACGAGATGATCTGCGGTCTGCCTTGCCAGCCCGCTCAG-3'
5'-CAAGCAGAAGACGGCATACGAGATTCAAGTCGGTCTGCCTTGCCAGCCCGCTCAG-3'
5'-CAAGCAGAAGACGGCATACGAGATCTGATCCGGTCTGCCTTGCCAGCCCGCTCAG-3'
5'-CAAGCAGAAGACGGCATACGAGATAAGCTACGGTCTGCCTTGCCAGCCCGCTCAG-3'
5'-CAAGCAGAAGACGGCATACGAGATGTAGCCCGGTCTGCCTTGCCAGCCCGCTCAG-3'
5'-CAAGCAGAAGACGGCATACGAGATTACAAGCGGTCTGCCTTGCCAGCCCGCTCAG-3'

Note: Bar Code sequence as read from the Index Read Primer (reverse complement).
IDX1: ATCACG
EPICENTRE
Nextera™ DNA Sample Prep Kit
(Illumina-compatible)

Related Products: The following products are also available:
– Nextera™ Bar Codes (Illumina-compatible) – Nextera™ PCR Enzyme Nextera™ Products are covered by patent applications assigned to EPICENTRE and by U.S. Patent
Nos. 5,965,443, and 6,437,109; European Patent No. 0927258, and related patents and patent applica-
tions, exclusively licensed to EPICENTRE. These products are accompanied by a limited nonexclusive li-
cense for the purchaser to use the purchased product(s) solely for life science research.
Illumina and Solexa are registered trademarks of, and HiSeq is a trademark of Tween is a registered trademark of ICI Americas Inc., Wilmington, Delaware. 454 and GS FLX are trademarks of Roche, Nutley, New Jersey. Genbank is a registered trademark of the United States Department of Health and Human Services, Bethesda, Maryland. MinElute is a registered trademarks of Qiagen Inc., Valencia, California. DNA Clean & Concentrator and Zymo-Spin are trademark of Zymo Research., Orange, California. AMPure is a registered trademark of Beckman Coulter, Inc. Nextera is a trademark of EPICENTRE, Madison, Wisconsin.

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