Kapa biosystems/KAPA hgDNA Quantification and QC Kit/KK4962/300 x 20 µL reactions

Description:

qPCRMasterMix(Bio-Rad®)

KAPAhgDNAQuantificationandQCKit

Qualitymatters.

KAPAhgDNAQuantificationandQCKitscontainallthereagentsneededfortheqPCR-basedquantificationandqualityassessmentofhumangenomicDNAsamplespriortoNGSlibraryconstruction.

KitscontainKAPASYBR^®FASTqPCRMasterMix,optimizedforhigh-performanceSYBRGreenI-basedqPCR,aswellasapre-dilutedsetofDNAstandardsandprimerpremixestargetingdifferentportionsofahighlyconservedsingle-copyhumanlocus.Absolutequantificationisachievedwiththeprimerpairdefiningtheshortestfragment,whereastheadditionalprimersareusedtoderiveinformationabouttheamountofamplifiabletemplateintheDNAsample.Qualityscores(orQ-ratios)generatedwiththekitmaybeusedtopredicttheoutcomeoflibraryconstruction,ortailorworkflowsforsamplesofvariablequality,particularlyFFPEDNA.

ThemethodiseasytoautomateandcanbeappliedtoanyprocessorworkflowthatrequiresaccuratequantificationofdiluteDNAsamples,orsamplesthatmaycontainahighproportionofDNAthatisrecalcitranttoPCRamplification.

Downloadour KAPAhgDNAQuantificationandQCDataAnalysisTemplate.

ForResearchUseOnly.Notforuseindiagnosticprocedures.

ProductHighlights

Principle of the hgDNA Quantification and QC assay. A single set of DNA standards is used to generate up to three standard curves using three different primer pairs that amplify targets of 41 bp, 129 bp or 305 bp within a conserved, single-copy human locus. The 41 bp assay is used for absolute quantification of DNA samples. For an assessment of DNA quality, standard curves are generated and samples assayed with the 129 bp and/or 305 bp primer premix(es). Since poor DNA quality has a greater impact on the amplification of longer targets, the relative quality of a DNA sample can be inferred by normalizing the concentration obtained using the 129 bp or 305 bp assay against the concentration obtained from the 41 bp assay. This normalization generates a Q-ratio (with a value between 0 and 1) that is indicative of DNA quality, or the amount of amplifiable material in a DNA sample. Data on file.

Obtainconcentrationandqualityinformationwithasingleassay

AllowsforaccuratequantificationofdiluteDNAsamples
QuantificationwithanadditionalprimerpairprovidesaQ-ratiothatisindicativeofsamplequality

Conversion rates (% input DNA converted to adapter-ligated library) for libraries prepared for targeted re-sequencing on the Illumina platform. Libraries were prepared in duplicate from 80 – 100 ng FFPE DNA of variable quality (orange) or a commercial hgDNA preparation (grey), using the KAPA LTP Library Preparation Kit. FFPE DNA samples were assessed with the KAPA hgDNA Quantification and QC assay prior to Covaris shearing. Q-ratios were as indicated. The amount of fragmented DNA (average size ~180 bp) used for library construction was determined using the Qubit HS dsDNA assay (Life Technologies), whereas adapter-ligated libraries were quantified using the qPCR-based KAPA Library Quantification Kit. Results indicated that conversion rates for FFPE samples were significantly lower than for high-quality DNA. This limits the diversity of FFPE libraries, and necessitates more cycles of library amplification to generate sufficient material for target capture. This results in higher duplication rates and lower target coverage for FFPE libraries. Data on file.

Employqualityscores intheanalysisofNGSlibraryconstructionworkflows

Q-ratioscanprovidevaluableinsightsintothebottlenecksinNGSlibraryconstructionworkflows
ForFFPEsamples,libraryandsequencequalityisprimarilylimitedbyinefficientconversionofinputDNAtoadapter-ligatedlibrary

0.4yieldedlibrariesthatmetminimumsequencequalityrequirements.Dataonfile.">

Q(129/41) ratios for correlate with key sequencing metrics, such as duplication rate. Libraries were prepared manually from 250 ng Covaris-sheared FFPE DNA (n=14) or control DNA, extracted from cultured cells, fresh-frozen tissue or blood (n=6). Samples are arranged on the x-axis (left to right) in order of increasing Q(129/41)-ratio (designated by orange bars for FFPE samples and blue bars for control DNAs). Libraries were constructed with NEBNext reagents (New England Biolabs), whereas KAPA HiFi was used for pre- and post-capture amplification (10 cycles). Target capture was performed with a custom Roche NimbleGen SeqCap EZ panel (300 genes, 0.9 MB). Sequencing (2 x 100 bp) was performed on an Illumina HiSeq. The average duplication rate (grey dots) were 2X higher for FFPE (75% vs. 38% for control samples). FFPE samples with a Q(129/41) ratio >0.4yieldedlibrariesthatmetminimumsequencequalityrequirements.Dataonfile.

0.4yieldedlibrariesthatmetminimumsequencequalityrequirements.Dataonfile.">

Q(129/41) ratios for correlate with key sequencing metrics, such as mean target coverage. Libraries were prepared manually from 250 ng Covaris-sheared FFPE DNA (n=14) or control DNA, extracted from cultured cells, fresh-frozen tissue or blood (n=6). Samples are arranged on the x-axis (left to right) in order of increasing Q(129/41)-ratio (designated by orange bars for FFPE samples and blue bars for control DNAs). Libraries were constructed with NEBNext reagents (New England Biolabs), whereas KAPA HiFi was used for pre- and post-capture amplification (10 cycles). Target capture was performed with a custom Roche NimbleGen SeqCap EZ panel (300 genes, 0.9 MB). Sequencing (2 x 100 bp) was performed on an Illumina HiSeq. The average mean target coverage (grey dots) was 4X higher for control DNA (1,247X, vs. 301X for FFPE samples). FFPE samples with a Q(129/41) ratio >0.4yieldedlibrariesthatmetminimumsequencequalityrequirements.Dataonfile.

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Obtainactionabledataforsamplepreparation

Q-ratioscorrelatewithkeysequencingmetricssuchasduplicationratesandmeantargetcoverage
FFPEsampleswithaQscore>0.4yieldlibrariesofacceptablequalitywhenprocessedinstandardsamplepreparationsfortargetcapture

Applications:

Applications

FFPEsamples
Free-circulatingDNAfromplasmaorserum
Samplesobtainedbylaser-capturemicrodissectionoffresh,frozenorFFPEtissue
Forensicsamples
Cellscollectedbyflowcytometry
Anyotherlimitingorpreciousclinicalsample

KitSpecificationsandContents/Storage:

KitSpecificationsandContents/Storage

Kitscanbestoredforupto12months at-20˚C.

Complete(MasterMix)kitsincludeKAPASYBRFASTqPCRMasterMix(2X),PrimerPremix(41bp,129bpand305bp,10X)andasetof5DNAStandards.PrimerPremixesandDNAStandardsarealsosoldseparately.

Components

Specifications

CompatiblePlatform

AllNGSplatforms

CompatibleSamples

HumangenomicDNA

Samplesources

FFPEtissueCellscollectedbylaser-capturemicrodissectionFlow-sortedcellsFree-circulatingDNAfromplasmaorserumForensicsamplesClinicalsamples

Standardcurveconcentrationrange

2.5ng/µL–10pg/µLor3,080–12copies

SequencingApplications

WholeGenomeSequencingWholeExomeSequencingTargetedSequencing(custompanels)AmpliconSequencing

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