Kapa biosystems/KAPA Library Amplification Kits/KK2702/250 x 50 µL reactions

Description:

Real-timePCRlibraryamplification,withfluorescentstandards

KAPALibraryAmplificationKits

ThegoldstandardforNGSlibraryamplification.

KAPALibraryAmplificationKitscontainKAPAHiFiDNAPolymerase,anovelenzymeengineeredforultra-highfidelityandrobustnessusingKapa’sdirectedevolutiontechnologyplatform.

KAPAHiFihasbecometheenzymeofchoiceforNGSlibraryamplificationduetoitsabilitytoamplifycomplexDNApopulationswithhighfidelity,highefficiency,decreasedPCRduplicationratesandverylowbias.*ThisresultsinlowerduplicationratesandimprovedcoverageofGC-andATrichregions,promoters,lowcomplexityandotherchallengingregionsinallNGSlibraryconstructionworkflowsrequiringlibraryamplification.

Inadditiontothestandardlibraryamplificationformulation,KAPAHiFiisavailableinauniquereal-timeformulation,forapplicationsthatdemandprecisecontroloverlibraryamplification.Auracil-tolerantvariant,KAPAHiFiUracil+isalsoavailableforthehigh-efficiency,high-fidelity,low-biasamplificationoflibrariesconstructedfrombisulfite-treatedDNA.

KAPALibraryAmplificationKitsforIllumina^® platformsnowalsoincludeanoptimallyformulatedLibraryAmplificationPrimerMix.

_*Dataonfile.

ForResearchUseOnly.Notforuseindiagnosticprocedures.

ProductHighlights

Coverage depth vs GC content for indexed Illumina TruSeq libraries prepared from 1 µg M. tuberculosis (65% GC) gDNA. Libraries amplified with KAPA HiFi HotStart or the Illumina TruSeq PCR Master Mix are compared to an equivalent unamplified library. Data was generated by paired-end sequencing (2 x 75 bp). After filtering and aligning read pairs to reference sequences, 250 000 read pairs were randomly sampled for each genome, and scatter plots of mean sequence coverage depth vs. GC content were generated by analyzing 250 bp windows. GC-rich sequences were under-represented in libraries amplified with the TruSeq PCR Master Mix. In contrast, library amplification with KAPA HiFi resulted in coverage distribution across the range of GC-content that is almost indistinguishable from that of the unamplified control. Data on file.

Key sequencing metrics for libraries prepared from 1 µg human gDNA with the KAPA HTP Library Preparation Kit (green) or TruSeq DNA Sample Prep Kit (orange), for multiplexed exome capture using the SeqCap EZ Human Exome Library v3.0 (Roche Nimblegen). KAPA HiFi only required fewer cycles of pre- and post-capture amplification (7 and 14, respectively), as compared to the 8 cycles and 18 cycles performed libraries generated and amplified with Illumina enzymes. The PCR duplication rate for libraries amplified KAPA libraries were significantly lower, leading to improved coverage metrics. Data on file.

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Achievethelowestamplificationbiasandduplicationrates

Loweramplificationbiasresultinimprovedrepresentationofalllibraryfragmentsandsequenceregions
High-efficiencyamplificationleadstofeweramplificationcyclesandlowerPCRduplicates
Lessadditionalnext-generationorSangersequencingneededtocompletegenomes

The first exons of many genes have a high GC content, and are therefore difficult to amplify. Regions of these exons are therefore typically underrepresented in whole exome sequence data. Pre-capture amplification with the evolved, low-bias KAPA HiFi DNA Polymerase results in significantly improved coverage of GC- and AT-rich regions, and other sequence elements that are difficult to amplify. Data courtesy of The Broad Institute. Data on file.

ImprovecoverageofGC-andAT-richregions

LoweramplificationbiasimprovescoverageuniformityofGC-andAT-richregions,promoters,lowcomplexityandotherchallengingregions
ImprovedoverallcoverageandcoverageuniformityobservedonbothIllumina andIonTorrent™ sequencingplatforms

Real-time, high fidelity amplification of multiple libraries with the KAPA Real-Time Library Amplification Kit. Twenty libraries, spanning a ~64-fold concentration range (equivalent to 6 cycles), were simultaneously amplified and terminated after 14 cycles. Fourteen of the twenty libraries fall within the targeted amplification range (the fluorescence range defined by the Real-Time Amplification Standards 1¬–3). The remaining six libraries could either be used as is, noting that they may be outside the optimal concentration range, or they could be re-amplified individually or in high- or low-concentration groups. Data on file.

Exerciseprecisecontroloverlibraryamplification

KAPAReal-TimeAmplificationKitsallowforlibraryamplificationtobeobservedinrealtime
Terminateamplificationattheoptimalpointforindividualsamples
Optimizelibraryamplificationparametersforhigherthroughputworkflows

Error rates of Taq DNA polymerase and polymerases typically used in NGS library amplification. The error rates of proofreading DNA polymerases such as Q5 (New England Biolabs), Phusion (Thermo Scientific) and the evolved KAPA HiFi DNA Polymerase is 50 – 100 times lower than that of Taq. The error rate of KAPA HiFi was confirmed by deep sequencing on the 454 platform, and is 1 error per 3.54 x 106 nucleotides incorporated. Data on file.

AmplifyNGSlibrarieswithindustry-leadingfidelity

KAPAHiFiwasengineeredusingdirectedevolutiontohaveenhancedproofreading(3′-5′exonuclease)activity
Industry-leadingfidelityconfirmedby454sequencing

Applications:

Applications

AmplificationofIllumina libraries(DNAandRNAsequencing)
Real-timeamplificationoflibrariespreparedfromlowinput,ChIPDNAandotherprecioussamples
Pre-andpost-captureamplificationintargetedcaptureworkflows

KitSpecificationsandContents/Storage:

KitSpecificationsandContents/Storage

Kitscanbestoredforupto12months at-20˚C.

KitsincludeKAPAHiFiHotStartReadyMix(2X),aconvenientPCRmastermixcontainingKAPAHiFiHotStartDNAPolymerase,KAPAdNTPs,reactionbuffer,andMgCl₂atafinalconcentrationof2.5mM.Kitswithprimersarealsoavailable.

Kitsforreal-timelibraryamplificationincludeareal-timeversionoftheKAPAHiFiHotStartReadyMix,aswellasfluorescentstandardstomonitorlibraryamplification.

Components

LAK_Component Chart

Specifications

CompatiblePlatform

IlluminaHiSeq,MiSeq,NextSeq,andGAIIx

LibraryType

DNA,RNA,andbisulfite-treatedDNA

StartingMaterial

AnyNGSlibrarythatrequiresamplification

SequencingApplications

WholeGenomeSequencingWholeExomeSequencingTargetedSequencing(custompanels)RNA-SeqChIP-SeqAmpliconSequencing

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