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蚂蚁淘在线 / 品牌中心 / Kapa biosystems / Kapa biosystems/KAPA hgDNA Quantification and QC Kit/KK4969/300 x 20 µL reactions

Kapa biosystems/KAPA hgDNA Quantification and QC Kit/KK4969/300 x 20 µL reactions

价格
￥12080.00
货号：KK4969
浏览量：127
品牌：Kapa biosystems
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商品描述

Description:

qPCRMasterMix(ROXLow)

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

Spec
Description
CompatIBLePlatform
AllNGSplatforms
CompatibleSamples
HumangenomicDNA
Samplesources
FFPEtissueCellscollectedbylaser-capturemicrodissectionFlow-sortedcellsFree-circulatingDNAfromplasmaorserumForensicsamplesClinicalsamples
Standardcurveconcentrationrange
2.5ng/µL–10pg/µLor3,080–12copies
SequencingApplications
WholeGenomeSequencingWholeExomeSequencingTargetedSequencing(custompanels)AmpliconSequencing
Kapa biosystems表观遗传分析基于阵列的方法和基于下一代测序（NGS）的方法都用于研究表观遗传修饰。有三种基于NGS的常见方法可用于表观遗传分析：甲基序列，ChIP序列和ATAC序列。Methyl-seq   用单核苷酸分辨率研究基因组的甲基化状态。该方法采用亚硫酸氢盐处理，可将胞嘧啶残基转化为尿嘧啶，而甲基化残基则保持不变。已经开发了几种甲基序列策略，包括全基因组亚硫酸氢盐测序（WGBS）和简化表示的亚硫酸氢盐测序（RRBS），这丰富了CpG岛。ChIP-seq将染色质免疫沉淀（ChIP）与NGS结合使用，以鉴定整个基因组中与DNA相关的蛋白质的结合位点，通常用于绘制组蛋白修饰和转录因子。该方法依靠靶向抗体的选择来富集与特定蛋白质结合的目标DNA片段。ATAC-seq是用于转座酶可及的染色质测序的一种测定方法，可确定染色质可及性的区域并绘制DNA结合蛋白的图谱，以鉴定活性启动子，增强子和其他顺式调控元件。该方法通过生成具有50,000个细胞的测序文库，已经改变了基因调控的分析方法。由于表观遗传学分析通常涉及超低输入DNA，因此从有限的材料构建高质量文库至关重要。罗氏测序解决方案提供了许多用于表观遗传工作流程的目标富集，文库制备和优化文库质量的解决方案。SeqCap®Epi靶标富集试剂盒可通过单碱基分辨率富集用于DNA甲基化评估的靶标。的  KAPA HyperPrep套件  理想地适合于这两个芯片起和甲基SEQ应用，因为它使接头连接的文库和扩增低偏压的更高的产率。这意味着更高的文库多样性，更低的重复率和更统一的覆盖率，尤其是对于低输入样本而言。对于甲基序列研究，  KAPA HiFi Uracil + 由于对尿嘧啶残基具有耐受性，HotStart DNA聚合酶对于亚硫酸氢盐转化的文库的扩增至关重要。KAPA HiFi HotStart ReadyMix可用于扩增ATAC-seq和ChIP-seq库，从而改善序列覆盖范围并减少偏差。