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

Description:

qPCR Master Mix (ROX Low)

KAPA hgDNA Quantification and QC Kit

Quality matters.

KAPA hgDNA Quantification and QC Kits contain all the reagents needed for the qPCR-based quantification and quality assessment of human genomic DNA samples prior to NGS library construction.

Kits contain KAPA SYBR^® FAST qPCR Master Mix, optimized for high-performance SYBR Green I-based qPCR, as well as a pre-diluted set of DNA standards and primer premixes targeting different portions of a highly conserved single-copy human locus. Absolute quantification is achieved with the primer pair defining the shortest fragment, whereas the additional primers are used to derive information about the amount of amplifiable template in the DNA sample. Quality scores (or Q-ratios) generated with the kit may be used to predict the outcome of library construction, or tailor workflows for samples of variable quality, particularly FFPE DNA.

The method is easy to automate and can be applied to any process or workflow that requires accurate quantification of dilute DNA samples, or samples that may contain a high proportion of DNA that is recalcitrant to PCR amplification.

Download our KAPA hgDNA Quantification and QC Data Analysis Template.

For Research Use Only. Not for use in diagnostic procedures.

Product Highlights

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.

Obtain concentration and quality information with a single assay

Allows for accurate quantification of dilute DNA samples
Quantification with an additional primer pair provides a Q-ratio that is indicative of sample quality

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.

Employ quality scores in the analysis of NGS library construction workflows

Q-ratios can provide valuable insights into the bottlenecks in NGS library construction workflows
For FFPE samples, library and sequence quality is primarily limited by inefficient conversion of input DNA to adapter-ligated library

0.4 yielded libraries that met minimum sequence quality requirements. Data on file.">

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 >0.4 yielded libraries that met minimum sequence quality requirements. Data on file.

0.4 yielded libraries that met minimum sequence quality requirements. Data on file.">

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 >0.4 yielded libraries that met minimum sequence quality requirements. Data on file.

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Obtain actionable data for sample preparation

Q-ratios correlate with key sequencing metrics such as duplication rates and mean target coverage
FFPE samples with a Q score >0.4 yield libraries of acceptable quality when processed in standard sample preparations for target capture

Applications:

Applications

FFPE samples
Free-circulating DNA from plasma or serum
Samples obtained by laser-capture microdissection of fresh, frozen or FFPE tissue
Forensic samples
Cells collected by flow cytometry
Any other limiting or precious clinical sample

Kit Specifications and Contents / Storage:

Kit Specifications and Contents / Storage

Kits can be stored for up to 12 months at -20˚C.

Complete (Master Mix) kits include KAPA SYBR FAST qPCR Master Mix (2X), Primer Premix (41 bp, 129 bp and 305 bp, 10X) and a set of 5 DNA Standards. Primer Premixes and DNA Standards are also sold separately.

Components

Specifications

Compatible Platform

All NGS platforms

Compatible Samples

Human genomic DNA

Sample sources

FFPE tissueCells collected by laser-capture microdissectionFlow-sorted cellsFree-circulating DNA from plasma or serumForensic samplesClinical samples

Standard curve concentration range

2.5 ng/µL–10 pg/µL or 3,080–12 copies

Sequencing Applications

Whole Genome Sequencing Whole Exome Sequencing Targeted Sequencing (custom panels)Amplicon Sequencing

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