Nextera™ DNA Sample Prep Kit Epicentre

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Nextera™ DNA Sample Prep Kit (Illumina®-Compatible)

Cat. Nos. GA09115, GA091120, GA0911-50, GA0911-96, and GABC0950

TheNextera™DNASamplePrepKitisdesignedtopreparegenomicDNAlibrariescompatiblewiththeIllumina®GenomeAnalyzerIandIIandHiSeq™2000sequencers.Nexteratechnology†employsin vitrotranspositiontosimultaneouslyfragmentandtagDNAinasingle-tubereaction,andpreparesequencer-readylibrariesinunder2hours.TheNexteralibrarypreparationprocedureisasignificantimprovementuponcurrentprocedures,whichgenerallyconsistofdistinctDNAfragmentation,end-polishing,andadaptor-ligationsteps.TheNexteralibrarypreparationprocedurecombinesthesestepsintoone(tagmentation),usesonly50ngofstartingDNA,andallowsincorporationofplatform-specifictagsandoptionalbarcodes.

Product Specifications

Storage:StoretheNexteraDNASamplePrepKitat–20°Cinafreezerwithoutadefrostcycle.

Quality Control:NexteraDNASamplePrepKitisfunction-testedbytagmentingcontrolDNA(lambda)withbothLow-Molecular-Weight(LMW)andHigh-Molecular-Weight(HMW)Buffers.SizedistributionfollowingtagmentationandPCRamplificationisconfirmedbyBioanalyzerprofiling.

Contaminating Activity Assays:AllcomponentsoftheNexteraDNASamplePrepKitarefreeofdetectableRNaseandDNaseactivities.

Nextera Control DNA:TheNexteraControlDNAisunmethylatedc1857Sam7LambdaDNA,isolatedfrominfectedGM119,anE. colistrainlackingboththedamanddcmmethylaseactivities.GenBank®/EMBLAccessionNumberJ02459,Size:48.5kb.

Additional Required Components(NotProvided)–ZymoDNAClean&Concentrator™-5(Cat.No.D4013)orequivalent.–Nextera™PCREnzyme(Cat.Nos.EM091120,EM091150,EM0911-96)

Related Products:Thefollowingproductsarealsoavailable:–Nextera™BarCodes(Illumina®-compatible)–Nextera™PCREnzyme

*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.

TargetDNAisfragmentedandtaggedwithNexteraEnzymeMixcontainingtransposonendsappendedwithsequencingprimersites(blueandorange).Limited-cyclePCRwithafour-primerreactionaddsbridgePCR(bPCR)-compatibleadaptors(purpleandpink)tothecoresequencinglibrary.Optionalbarcodes(triangle)canbeaddedbetweenthe

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Nextera™ DNA Sample Prep Kits (Illumina®-compatible) Contents

TheNexteraDNASamplePrepKit(Illumina®-compatible)isavailableinfoursizes(5,20,50,and96reactions).Allreagentsinthesekitsareinblue-cappedtubes.

GA09115 GA091120 GA0911-50 GA0911-96 5 20 50 96 Component Name Reactions Reactions Reactions Reactions

Nextera™EnzymeMix 5μl 20μl 50μl 96μl(Illumina®-compatible)

5XNextera™ReactionBuffer(LMW) 50μl 200μl 500μl 1.0ml

5XNextera™ReactionBuffer(HMW) 50μl 200μl 500μl 1.0ml

50XNextera™PrimerCocktail 5μl 20μl 50μl 96μl(Illumina®-compatible)

50XNextera™Adaptor2 5μl 20μl 50μl 96μl(Illumina®-compatible)

2XNextera™PCRBuffer 125μl 500μl 1.25ml 2x1.2ml

Nextera™ControlDNA 10μl 10μl 10μl 10μl

200XNextera™Read1Primer* 30μl 100μl 150μl 300μl

200XNextera™Read2Primer* 30μl 100μl 150μl 300μl

200XNextera™IndexReadPrimer* 30μl 100μl 150μl 300μl

downstreambPCRadaptor(pink)andthecoresequencinglibraryadaptor(orange).AlternativesequencingprimersarerequiredfortheIllumina/Solexa®-compatiblelibraries:Read1Primer(blue/grayarrow);Read2Primer(orange/grayarrow);IndexReadPrimer(gray/orangearrow).

Sequences:

TransposonEndSequence:5′-AGATGTGTATAAGAGACAG-3′

Primer1*:5′-AATGATACGGCGACCACCGA-3′

Adaptor1*:5′-AATGATACGGCGACCACCGAGATCTACACGCCTCCCTCGCGCCATCAG-3′

Primer2*:5′-CAAGCAGAAGACGGCATACGA-3′

Adaptor2(minusbarcode)*:5′-CAAGCAGAAGACGGCATACGAGATCGGTCTGCCTTGCCAGCCCGCTCAG-3′

NexteraRead1Primer: 5′-GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG-3′

NexteraRead2Primer: 5′-GCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG-3′

NexteraIndexReadPrimer:5′-CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGCAGACCG-3′

Note: The kit contains a 50X Nextera Primer Cocktail, which consists of Primer 1 (10 μM),

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Nextera™ Enzyme Mix

Add bPCR-compatible sites by PCR (+/– bar codes)

bPCR input

Add genomic DNA

Primer 2

Adaptor 1

Primer 1

Adaptor 2 (+/– Bar Code)Bar Code

Nextera Read 1

Nextera Index Read

Nextera Read 2 Alternate SequencingPrimers

Figure 1. Generating Illumina®-compatible libraries.

*See note, page 4.

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 Illumina®-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 Illumina® bPCR sequences and are copyrighted to Illumina, Inc. Oligonucleotide sequences © 2006-2010 Illumina, Inc. All rights reserved.

Important Considerations

1. Fragment Size Distribution: TheNexteraDNASamplePrepKitcontainstwobuffers,Low-Molecular-WeightBuffer(LMW)andHigh-Molecular-WeightBuffer(HMW).Refertotablebelowforapproximatefragmentsizedistributionusingeachbuffer.

Fragment Size (approx.)* Insert Size (approx.)

LMWBuffer HMWBuffer LMWBuffer HMWBuffer

LambdaDNA(Control)

175-400bp 175-700bp 40-265bp 40-565bp

*FragmentSize(approx.)includesthe135-bpadaptorsequences(67-bpAdaptor1+68-bpAdaptor2)withoutbarcoding.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.

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2. DNA Quality:ThequalityofthestartingDNAiscritical.ContaminantssuchasproteinandDNAmayinhibittheNexterareactionifpresentintheDNApreparation.IfDNApurityisinquestion,theDNAshouldbecleanedusingZymoGenomicDNAClean&Concentrator,Cat.No.D4010(orequivalent).

3. Transposon End Sequence: The19-bptransposonDNAsequenceispresentatthe5′endofallIllumina®-compatiblelibraries.However,the19-bptransposonDNAsequenceisNOTsequencedontheIllumina®platform.TheNexteraRead1andRead2primersannealtothissequencesothatthefirstnucleotidesequencedistargetDNA.

4. Input DNA:Thekithasbeenoptimizedtoprocess50ngofDNAtothetargetMWdistribution.MWdistributionwillbelowerifusinglessthan50ngofDNA.

5. Amplicons:TheNexteraDNASamplePrepKitcanalsomakelibrariesfromamplicons.Ampliconsassmallas~2kbhavebeensuccessfullysequenced.However,thedistal~50-100bpoflinearfragmentsmayexhibitadecreaseincoverage.NexterakitscanalsobeusedtomakelibrariesfromcircularDNAsamples.

6. Bias:ThetransposaseusedintheNexterasystemcarriesmutationsandisusedunderconditionsthatresultinnear-randomintegration.Aswithanyenzymaticsystem,thereisaslightbiasinthereaction.However,sincethereactionisdriventocompletionwithanexcessoftheNexteraEnzyme,wehavenotseenanyimpactonthedistributionofcoverage.Thecoverageacrossassembliesiscomparabletothoseseenwithmechanicalmethodsofshearing.

7. Bar Codes:Platform-specificBarCodingkitsareavailabletoprepareupto12barcodedlibraries.IfadditionaloralternateBarCodesareneeded,thefollowingtemplatedesigncanbeused:

Adaptor 2 (plus Bar Code-optional)

Primer Sequencing Tag

5′-CAAGCAGAAGACGGCATACGAGAT-[BARCODE]*-CGGTCTGCCTTGCCAGCCCGCTCAG-3′*Reverse complement of sequencing read.

8. Sequencing in the Same Channel:TheNexterasequencingprimersarecompatiblewiththeIllumina®sequencingprimers,andcanbeusedtogether.

9. Nextera PCR Enzyme:UseonlyNexteraPCREnzymeforlimited-cyclePCR(PartB).OtherPCRsystemshavebeentestedanddonotperformaswell.

Figure 2. Fragment Size Distribution (approx.) using LMW Buffer (A) and HMW Buffer (B), per standard protocol.

A B



55oC Incubation......5 min.

Limited-Cycle PCR

• 1 µl 50X Nextera Primer Cocktail• 25 µl 2X Nextera PCR Buffer• 5 µl Zymo-purified Tagmentation Reaction

• 1 µl 50X Nextera Adaptor 2

• 17 µl Nuclease-Free Water• 1 µl Nextera PCR Enzyme

9 cycles PCR

Zymo Cleanup

Use recovered DNA as input for bPCR and clustergeneration per standard Illumina protocol

Dilute Nextera Sequencing Primers for single orpaired-end sequencing (pg. 7-9)

Nextera Tagmentation Reaction

• 1 µl Nextera Enzyme Mix• 4 µl LMW or HMW Buffer• 50 ng DNA

• x µl Nuclease-Free Water20 µl Total Reaction Volume

Zymo Cleanup• Elute with 11 µl Nuclease-Free Water

Flowchart for Nextera™ DNA Sample Preparation

AMPure ® XP Purification (0.7X)

Flowchart for Nextera™ DNA Sample Preparation

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Nextera DNA Sample Prep Kit (Illumina®-compatible) Protocols

A. Tagmentation Reaction

1. Priortoassemblingthereaction,brieflycentrifugethe5XNexteraReactionBufferandNexteraEnzymeMixtubestoassurethatthereagentsareatthebottomofthetubes.

2. Assemblethefollowingreactioncomponentsonice,intheorderlisted:

x μl Nuclease-FreeWater 50 ngTargetDNA(inT10E1Buffer[10mMTris-HCl(pH7.5),1mMEDTA]) 4 μl 5XNexteraReactionBufferLMWorHMW (seeImportantConsiderations,no.1,p.4) 1 μl NexteraEnzymeMix(Illumina®-compatible) 20 μl Totalreactionvolume

3. Mixbrieflybyvortexing,andincubateat55°Cfor5minutes.

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 55°C incubation.

4. PurifythetagmentedDNAusingaZymoDNAClean&Concentrator-5Kit(orequivalent).

Brief Zymo Protocol (performatroomtemperature):

– Add100μlofDNABindingBuffertothe20μlTagmentationReactionfromstep2 (above).

– Mixbrieflybyvortexing,andtransferthemixturetoaZymo-Spin™Columnina CollectionTube.

– Centrifugeat10,000xgfor60seconds.Discardtheflow-through.

– Add250μlofWashBuffertothecolumn.Centrifugeat10,000xgfor60seconds. Discardtheflow-through.

– Repeatthewashstep.

– Centrifugethecolumnat10,000xgfor60secondstoeliminateanyresidualWash Buffer.

– Transferthecolumntoacleanandsterile1.5-mlmicrocentrifugetube

– Add11μlofNuclease-FreeWaterdirectlytothecolumnandincubateatroom temperaturefor1-2minutes.Centrifugeat10,000xgfor60secondstoelutethe DNA.

– Thefinalelutedvolumeshouldbe~10μl.Use5μlasDNAtemplateinPartB,Step1.

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.



B. Addition of bPCR-Compatible Sites and Library Enrichment

AddbPCR-compatiblesitesandoptionalbarcodingbyPCR.

1. Assemblethefollowingreactioncomponentsatroomtemperature:

17 μl Nuclease-Freewater 5 μl RecoveredDNAFragmentLibrary(fromPartA,Step3) 25 μl 2XNexteraPCRBuffer 1 μl 50XNexteraPrimerCocktail(Illumina®-compatible) 1 μl 50XNexteraAdaptor2* 1 μl NexteraPCREnzyme(soldseparately,seeRelatedProducts) 50 μl Totalreactionvolume

*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. Cyclethesamplesinathermocyclerunderthefollowingconditions:

followedby9cyclesof:72°Cfor3minutes** 95°Cfor10seconds95°Cfor30seconds62°Cfor30seconds Holdat4°C

72°Cfor3minutes

**Note: It is critical to perform the 72°C extension step before denaturing the DNA templates.

3. PurifythetaggedDNAfragmentsusingaZymoDNAClean&Concentrator-5kit,orequivalent.

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 kit.

4. UsetherecoveredDNAasinputforbPCRandclustergenerationperthestandardIlluminaprotocol.

Note: For cluster sequencing, it is critical to use the provided Nextera sequencing primers.

• UsetheNexteraRead1PrimerforPaired-EndRead1SequencingorforSingle-Read Sequencing.

• UsetheNexteraIndexReadPrimerforIndexReadSequencing. • UsetheNexteraRead2PrimerforPaired-EndRead2Sequencing.

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.

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For Paired-End Reads:

Illuminapaired-endsequencingmixes(HP1andHP2)containtheIllumina®sequencingprimers.TheseprimersdonotinterferewiththeNexterasequencingprimersandsequencingcanbeperformedinthepresenceofHP1andHP2primers.

• Paired-EndRead1:DiluteNexteraRead1Primer1:200intoSequencingMixHP1.• IndexRead:DiluteNexteraIndexReadPrimer1:200intoHybridizationBuffer.• Paired-EndRead2:DiluteNexteraRead2Primer1:200intoSequencingMixHP2.Alternatively,ifitisrequiredtoperformsequencinginthepresenceofonlytheNexteraprimers,the200XNexterasequencingprimerscanbediluted1:200intoHybridizationBuffer(GA0084204-HT1or5XSSC,0.05%Tween®-20).

For Single Reads:

• Paired-EndRead1:DiluteNexteraRead1Primer1:200intoSequencingMixHP4.• IndexRead:DiluteNexteraIndexReadPrimer1:200intoHybridizationBuffer.

Appendix A

TheIllumina®-compatibleBarCodesKitcontains12barcodes.A50-μlaliquotofeachisprovidedataconcentrationof0.5μM.Thisissufficientfor50barcodedlibraries.Allreagentsinthiskitareinblue-cappedtubes.Cat.No.GABC0950

Forbarcoding,oneofthesebarcodescanbesubstitutedwith50XNexteraAdaptor2(Illumina®-compatible)fromtheNexteraDNALibraryPrepKit(Illumina®-compatible).Use1μlinthereaction.

GAAdaptor2(IDX1)5′-CAAGCAGAAGACGGCATACGAGATCGTGATCGGTCTGCCTTGCCAGCCCGCTCAG-3′

GAAdaptor2(IDX2)5′-CAAGCAGAAGACGGCATACGAGATACATCGCGGTCTGCCTTGCCAGCCCGCTCAG-3′

GAAdaptor2(IDX3)5′-CAAGCAGAAGACGGCATACGAGATGCCTAACGGTCTGCCTTGCCAGCCCGCTCAG-3′

GAAdaptor2(IDX4)5′-CAAGCAGAAGACGGCATACGAGATTGGTCACGGTCTGCCTTGCCAGCCCGCTCAG-3′

GAAdaptor2(IDX5)5′-CAAGCAGAAGACGGCATACGAGATCACTGTCGGTCTGCCTTGCCAGCCCGCTCAG-3′

GAAdaptor2(IDX6)5′-CAAGCAGAAGACGGCATACGAGATATTGGCCGGTCTGCCTTGCCAGCCCGCTCAG-3′

GAAdaptor2(IDX7)5′-CAAGCAGAAGACGGCATACGAGATGATCTGCGGTCTGCCTTGCCAGCCCGCTCAG-3′

GAAdaptor2(IDX8)5′-CAAGCAGAAGACGGCATACGAGATTCAAGTCGGTCTGCCTTGCCAGCCCGCTCAG-3′

GAAdaptor2(IDX9)5′-CAAGCAGAAGACGGCATACGAGATCTGATCCGGTCTGCCTTGCCAGCCCGCTCAG-3′



GAAdaptor2(IDX10)5′-CAAGCAGAAGACGGCATACGAGATAAGCTACGGTCTGCCTTGCCAGCCCGCTCAG-3′

GAAdaptor2(IDX11)5′-CAAGCAGAAGACGGCATACGAGATGTAGCCCGGTCTGCCTTGCCAGCCCGCTCAG-3′

GAAdaptor2(IDX12)5′-CAAGCAGAAGACGGCATACGAGATTACAAGCGGTCTGCCTTGCCAGCCCGCTCAG-3′

Note: Bar Code sequence as read from the Index Read Primer (reverse complement).

IDX1:ATCACG IDX4:TGACCA IDX7:CAGATC IDX10:TAGCTT

IDX2:CGATGT IDX5:ACAGTG IDX8:ACTTGA IDX11:GGCTAC

IDX3:TTAGGC IDX6:GCCAAT IDX9:GATCAG IDX12:CTTGTA

†Covered by patents issued and pending.

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 applications, exclusively licensed to Epicentre. These products are accompanied by a limited nonexclusive license for the purchaser to use the purchased product(s) solely for life science research.

Nextera is a trademark of Epicentre, Madison, Wisconsin.

Illumina and Solexa are registered trademarks of, and HiSeq is a trademark of Illumina Inc., San Diego, California.

Agencourt and AMPure are registered trademarks of Beckman Coulter, Inc. Brea, California.

Genbank is a registered trademark of the United States Department of Health and Human Services, Bethesda, Maryland.

MinElute is a registered trademark of Qiagen Inc., Valencia, California.

Tween is a registered trademark of ICI Americas Inc., Wilmington, Delaware.

DNA Clean & Concentrator and Zymo-Spin are trademark of Zymo Research., Orange, California.

454 and GS FLX are trademarks of Roche, Nutley, New Jersey.

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Documents

Nextera™ DNA Sample Prep Kit Epicentre