Kapa biosystems/KAPA Library Quantification Kits/KK4943/500 x 20 µL reactions

Description:

qPCRMasterMix(Bio-Rad)andPrimerPremixonly

KAPALibraryQuantificationKits

forNext-GenerationSequencing

Ourstandardsdon’tchange.Neithershouldyours.

KAPALibraryQuantificationKitscontainallthereagentsneededfortheaccurate,reliableandreproducibleqPCR-basedquantificationofNGSlibrariespriortopoolingforcaptureorflowcellamplification.KitscontainKAPASYBR^®FASTqPCRMasterMix,optimizedforhigh-performanceSYBRGreenI-basedqPCR.TheengineeredKAPASYBRFASTDNAPolymerasecontainedintheMasterMixamplifiesGC-andAT-richDNAfragmentsofdifferentlengthswithsimilarefficiency,makingittheonlyqPCRMasterMixcapableofaccurateqPCR-basedquantificationofNGSlibraries.*Thepre-dilutedsetofDNAStandardscontainedineachkitiscarefullyquality-controlledtoensurelot-to-lotconsistencyandavoiddatadriftovertime.OptimizedPrimerPremixesensureoptimalPCRefficiency.

KitswithDNAStandardsandPrimerPremixesforIllumina^® andIonTorrent™sequencers areavailable.Pleasereferencethe InstrumentCompatibilityChart forguidanceoncompatibleplatforms.

NEW!ImproveexperimentaldesignswithourTechnicalGuideforIlluminaPlatforms

_*Dataonfile.

ForResearchUseOnly.Notforuseindiagnosticprocedures.

ProductHighlights

qPCR-based KAPA Library Quantification Kits reduce variability in cluster density compared to Agilent Bioanalyzer. Human exome samples were prepared using Nimblegen target capture. All preparations were performed in a 96-well plate format on a liquid handling system. Average number of clusters per tile are shown for paired-end, 76-bp Illumina GAIIx runs. Data courtesy of the University of Washington. Data on file.

Compared to spectrophotometric, fluorimetric, and electrophoretic methods, qPCR only quantifies adaptor-ligated and amplifiable library molecules. The ligation of adaptor sequences (pink and blue) to library DNA molecules (grey) results in a mixed population of molecules without the correct adaptor configuration. Electrophoresis and spectrophotometry measure total nucleic acid concentrations, whereas optimal cluster density or template-to-bead ratio depends on the appropriate concentration of PCR-amplifiable DNA molecules. Only qPCR-based library quantification methods count library molecules with the correct adaptor sequence configuration. Data on file.

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AccuratelyquantifyPCR-competentsequencingtemplates

qPCRonly“counts”thoselibrarymoleculesthatcanbesequenced
Moreconsistentclusterdensitiesortemplate-to-beadratiosenableoptimalutilizationofsequencingresources

Equimolar pooling of indexed libraries. Eleven indexed Illumina TruSeq libraries were quantified by qPCR using the KAPA Library Quantification Kit, and then combined to achieve equal final concentrations in two separate pools for multiplexed sequencing on different flow-cell lanes. The eleven libraries ranged ~11-fold in concentration from 0.67 pM to 7.65 pM, while representation of each index varied between 90% and 127% of expected assigned reads per lane. Data on file.

Improvepoolingformultiplexedsequencing

LibraryconcentrationsdeterminedbyqPCRaremorereliablecomparedtoothermethods*
TheengineeredKAPASYBRFASTqPCRMasterMixenablesaccuratepoolingoflibrarieswithextremeGC-contentsorlongerinserts*

Nine human DNA libraries and two microbial DNA libraries (Rhodococcus sp. ~70% GC and Staphylococcus sp. ~35% GC) were used to compare quantification results obtained with distinct lots (”Lot 1” and “Lot 2”), and distinct sets of reagents from the same lot (”Set 1” and “Set 2”) of KAPA Library Quantification Kits for Illumina platforms. Data on file.

Eliminatedriftwithcarefullyquality-controlledDNAStandards

PredilutedDNAStandardseliminatequantificationerrorsassociatedwithpreparationofstandardcurvesusingin-housestandards*
KAPADNAStandardsundergostrictqualitycontroltoensurelot-to-lotconsistencyandeliminatedatadriftovertime

KAPA Library Quantification methods (96- and 384-well) format are available for the Sciclone NGS and NGSx workstation (PerkinElmer) and other instruments used in high-throughput NGS sample preparation pipelines.

Improvethroughputwithautomation

Libraryquantificationassayiscompatiblewith96-and384-wellformat
Librarydilution,reactionsetupanddataanalysiscanbeautomatedforHTPpipelines
LearnMore aboutKAPAAutomatedSolutions

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RelatedProducts

Normalizinglibrariesforpoolingandclusteramplification?CheckouttheseKapaNGSproductstoimproveyourworkflowandresults:

KAPAhgDNAQuantificationandQCKit

KAPALibraryAmplificationKits

KAPAHyperPrepKits

KAPAStrandedmRNA-SeqKits

Applications:

Applications

Quantificationoflibrariespriortopoolingformultiplexedsequencing
QuantificationofindividuallibrariesorlibrarypoolspriortoclusteramplificationoremPCR
Quantificationoflibrariespriortoandafterhybridizationcapture
Qualitycontrol,optimizationandtroubleshootingoflibraryconstructionprocessesorworkflows

KitSpecificationsandContents/Storage:

KitSpecificationsandContents/Storage

Kitscanbestoredforupto12months at-20˚C.

CompletekitsincludeKAPASYBRFASTqPCRMasterMix(2X),PrimerPremix(10X)andasetof6DNAStandards.ReagentsforqPCR(KAPASYBRFASTqPCRMasterMixandPrimerPremix)andDNAStandards(1–6)arealsosoldseparately.Wherenoted,MasterMixescontaininstrument-specificreferencedyes,whiletheUniversalkitincludesROXHighandROXLow(both50X)separately.

Components–Illumina^®Platform

Components–IonTorrent™Platform

LQK-Ion_Component Chart

Specifications

CompatiblePlatforms

IlluminaHiSeq,NextSeq,MiSeq,andGAIIxIonTorrentProtonandPGM454GSFLX+andGSJuniorSOLiD5500series

LibraryType

Any

StartingMaterial

AnyNGSlibraryreadyforclusteramplificationoremPCR

Standardcurveconcentrationrange

20–0.0002pMforIlluminalibraries83–0.00083pMforIonTorrentand454libraries10–0.0001pg/µLforSOLiDlibraries

SequencingApplications

WholeGenomeSequencingWholeExomeSequencingTargetedSequencing(custompanels)RNA-SeqChIP-SeqAmpliconSequencingMethyl-Seq

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