sparQ HiFi PCR Master Mix

High-fidelity, high-efficiency library amplification while maintaining even coverage

Features & Benefits

  • HiFi DNA polymerase engineered to minimize amplification bias
  • Increased amplification efficiency resulting in higher yields
  • Uniform coverage across challenging AT- and GC-rich regions
  • Robust amplification from input DNA as low as 250 pg
  • Cost-effective alternative to KAPA HiFi with improved performance

 

sparQ HiFi PCR Master Mix is intended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of a disease.

Description

sparQ HiFi PCR Master Mix is a high-fidelity, high-efficiency PCR master mix for NGS workflows requiring DNA library amplification prior to sequencing. The included primer mix allows amplification of DNA libraries flanked by adapters containing the P5 and P7 sequence elements required for Illumina® sequencing platforms. The hot-start, proofreading DNA polymerase used in the sparQ HiFi PCR Mater Mix is specifically engineered to improve library amplification efficiency while reducing PCR-derived artifacts, resulting in higher library yields and better coverage uniformity. This kit supports low DNA input from 250 pg and efficient amplification of AT- and GC rich regions with minimal bias.


Higher NGS library amplification efficiency

Library yields and fragment size distribution


Library yields and fragment size distribution

Library amplification with sparQ HiFi PCR Master Mix resulted in higher yields. A) Libraries were prepared from Covaris-sheared human genomic DNA with sparQ DNA library prep kit prior to library amplification. Pre-amplified libraries were then amplified using sparQ HiFi PCR Master Mix (orange) or equivalent kit from Supplier K (blue) and Supplier N (red) with identical PCR cycle numbers (6 cycles for 100 ng input DNA, 14 cycles for 1 ng input DNA, and 16 cycles for 250 pg input DNA). Amplified libraries were quantified with Qubit fluorometric method and qPCR-based quantification method (data not shown). B) The fragment size distribution and the quality of the amplified DNA libraries from 250 pg input DNA were analyzed using a High Sensitivity DNA Kit on the Agilent BioAnalyzer.


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  • Engineered hot-start, proofreading DNA polymerase and optimized buffer formulated to ensure sensitive, efficient library amplification
  • Superior amplification efficiency resulted in >45% higher library yields from a broad range of inputs


Create amplified libraries with minimal bias

Genome coverage analysis


Genome coverage analysis

Consistent coverage over a broad range of GC-content with sparQ HiFi PCR Master Mix. DNA libraries were prepared from 100 ng of microbial genomic DNA of different species using sparQ DNA Library Prep Kit. Amplified libraries (orange) underwent 6 cycles of library amplification using sparQ HiFi PCR Master Mix. Libraries were sequenced on Illumina MiSeq and 2 millions of pair-end sequencing reads (2 x 150 bp) from each library were analyzed. Coverage uniformity was examined by plotting normalized coverages of the library against GC-content of the targeted genome. GC-content distribution of targeted genomes is indicated by gray line. Libraries amplified by sparQ HiFi PCR Master Mix (orange) provide uniform genome coverage, similar to corresponding libraries without amplification (blue).


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  • Uniform coverage across AT- and GC- rich regions
  • Similar coverage pattern between amplified and unamplified libraries

Performance Data

sparQ HiFi PCR Master Mix Library Yield Analysis


sparQ HiFi PCR Master Mix Library Yield Analysis

Libraries were prepared from Covaris-sheared DNA with sparQ DNA library prep kit prior to library amplification. Pre-amplified libraries were then amplified using sparQ HiFi PCR Master Mix (red), KAPA Library Amplification kit (blue), or NEBNext Ultra II Q5 Master Mix (green) with the identical amplification cycle numbers (6 cycles for 100 ng input DNA, 14 cycles for 1 ng input DNA, and 16 cycles for 250 pg input DNA). Amplified libraries were quantified with Qubit fluorometric quantitation method and qPCR-based quantification method (data not shown).


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Uniform coverage across the wide range of GC-content


sparQ HiFi PCR Master Mix coverage

Library amplification with sparQ HiFi PCR Master Mix resulted in uniform coverage across the wide range of GC-content. Libraries were prepared by using sparQ DNA Library Prep Kit with 100 ng input DNA.
Coverage depth against GC-content of libraries amplified by sparQ HiFi PCR Master Mix (red) were compared to corresponding libraries without amplification (dark blue: PCR-free library). GC content distribution of targeted genomes is indicated by gray line.


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Fragment size distribution and the quality of the amplified DNA


sparQ HiFi PCR Master Mix fragment size distribution and the quality of the amplified DNA

The fragment size distribution and the quality of the amplified DNA libraries from 250 pg input DNA were analyzed using a high sensitivity DNA analysis kit on a Bioanalyzer. Libraries were amplified using sparQ HiFi PCR Master Mix (red), KAPA Library Amplification kit (blue), or NEBNext Ultra II Q5 Master Mix (green) with identical amplification cycle numbers (16 cycles for 250 pg input DNA).


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Details

  • Contents
    • 2X HiFi PCR Master Mix
    • Primer Mix
    • HiFi Enhancer
  • Storage & Handling

    Store components in a constant temperature freezer at -25°C to -15°C upon receipt.
    Refer to the product label for applicable expiration date.

  • Related Resources
    Product Flyers
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    Publications
    Environmental DNA metabarcoding as a tool for biodiversity assessment and monitoring: reconstructing established fish communities of north-temperate lakes and rivers
    Peter T. Euclide, Diversity and Distributions - 2021
    Abstract
    Aim To evaluate the ability of precipitation-based environmental DNA (eDNA) sample collection and mitochondrial 12S metabarcoding sequencing to reconstruct well-studied fish communities in lakes and rivers. Specific objectives were to 1) determine correlations between eDNA species detections and known community composition based on conventional field sampling, 2) compare efficiency of eDNA to detect fish biodiversity among systems with variable morphologies and trophic states, and 3) determine if species habitat preferences predict eDNA detection. Location Upper Great Lakes Region, North America. Methods Fish community composition was estimated for seven lakes and two Mississippi River navigation pools using sequence data from the mitochondrial 12S gene amplified from 10 to 50 water samples per waterbody collected in 50-mL centrifuge tubes at a single time point. Environmental DNA (eDNA) was concentrated without filtration by centrifuging samples to reduce per-sample handling time. Taxonomic detections from eDNA were compared to established community monitoring databases containing up to 40 years of sampling and a detailed habitat/substrate preference matrix to identify patterns of bias. Results Mitochondrial 12S gene metabarcoding detected 15%–47% of the known species at each waterbody and 30%–76% of known genera. Non-metric multidimensional scaling (NMDS) assessment of the community structure indicated that eDNA-detected communities grouped in a similar pattern as known communities. Discriminant analysis of principal components indicated that there was a high degree of overlap in habitat/substrate preference of eDNA-detected and eDNA-undetected species suggesting limited habitat bias for eDNA sampling. Main conclusions Large numbers of small volume samples sequenced at the mitochondrial 12S gene can describe the coarse community structure of freshwater systems. However, additional conventional sampling and environmental DNA sampling may be necessary for a complete diversity census.
    DNA metabarcoding to unravel plant species composition in selected herbal medicines on the National List of Essential Medicines (NLEM) of Thailand
    Santhosh Kumar J. Urumarudappa, Scientific Reports - 2020
    Abstract
    Traditional medicines are widely traded across the globe and have received considerable attention in the recent past, with expectations of heightened demand in the future. However, there are increasing global concerns over admixture, which can affect the quality, safety, and efficacy of herbal medicinal products. In this study, we aimed to use DNA metabarcoding to identify 39 Thai herbal products on the Thai National List of Essential Medicines (NLEM) and assess species composition and admixture. Among the products, 24 samples were in-house-prepared formulations, and 15 samples were registered formulations. In our study, DNA metabarcoding analysis using ITS2 and rbcL barcode regions were employed to identify herbal ingredients mentioned in the products. The nuclear region, ITS2, was able to identify herbal ingredients in the products at the genus- and family-levels in 55% and 63% of cases, respectively. The chloroplast gene, rbcL, enabled genus- and family-level identifications in 58% and 73% of cases, respectively. In addition, plant species were detected in larger numbers (Family identified, absolute %) in registered herbal products than in in-house-prepared formulations. The level of fidelity increases concerns about the reliability of the products. This study highlights that DNA metabarcoding is a useful analytical tool when combined with advanced chemical techniques for the identification of plant species in highly processed, multi-ingredient herbal products.
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