PerfeCTa NGS Quantification Kit

Features & Benefits

  • Precise quantification of adapter-tagged library molecules
  • Accurate and sensitive method for NGS library quantification
  • Stabilized, prediluted standards for convenient use
  • Consistency across a broad range of samples

 

PerfeCTa NGS Quantification Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of a disease.

Description

Accurate quantification of the number of amplifiable library molecules is the most critical step in the NGS workflow in obtaining high quality read data with next-generation sequencing technologies. The PerfeCTa NGS Library Quantification Kit uses real-time PCR to specifically quantify library molecules that possess the appropriate adapter tag at each end. These are the suitable template molecules for either emulsion PCR used for the Ion Torrent platform or Bridge PCR used for Illumina NGS platforms. PerfeCTa NGS Quantification Kits simplify the library quantification process by providing stabilized, pre-diluted standards, pre-qualified primer sets, and an optimized dilution buffer for your NGS library samples. This minimizes pipetting errors and ensures reproducible and precise qPCR results, even with dilute samples. The robust qPCR performance of PerfeCTa SYBR Green SuperMix provides accurate quantification of NGS libraries with varying fragment sizes or GC content. A common problem with some NGS library quantification protocols is the use of DNA standards that are too concentrated and generate qPCR data that are outside of the linear dynamic range for many qPCR instruments. Improper baseline settings result in compressions between the highest concentrated DNA standards that in turn give rise to inflated PCR efficiencies and inaccurate library quantification results. The NGS DNA standards supplied with the PerfeCTa NGS Library Quantification Kits have been carefully selected to avoid these artifacts and produce NGS library standard curves with exceptionally high linear regression correlation coefficients. The DNA standard for Illumina NGS platforms generates a 426-bp amplicon (48.8% GC).

Primer sequences correspond to the "P5" and “"P7" primer sequences for Illumina sequencing libraries:
Illumina forward: 5’-AATGATACGGCGACCACCGA-3’
Illumina reverse: 5’-CAAGCAGAAGACGGCATACGA-3’

Performance Data

qPCR amplification of the supplied DNA standards


PerfeCTa NGS Library Quantification Kit Performance

qPCR amplification of each of the five supplied DNA standards for Illumina NGS libraries (panel A) or Ion Torrent libraries (panel C) were carried out with the supplied primer sets (300 nM final concentration) and PerfeCta SYBR Green SuperMix in 20-uL reaction volumes on a Bio-Rad CFX-96. Reactions were incubated for 5 min at 95°C followed by 35 cycles of: 95°C, 10s; 60°C, 20s; 45s, 72°C. Real-time fluorescence data was collected and analyzed at completion of the 72°C extension step. After completion of PCR, a dissociation (melt) curve was performed to verify amplification of a single specific product (panels B and D). 


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Details

  • Contents
    • Library dilution buffer (10X)
    • Illumina primer mix
    • Illumina Standards 1-5
    • qPCR reagent (either standard, ROX, or Low-ROX)
  • Storage & Handling
    Kit components are stable for 2 years when stored in a constant temperature freezer at or below -20°C, protected from light. For convenience, they may be stored unfrozen at 4ºC for up to 6 months. Reagent performance is unaffected by repetitive (>20X) freeze thaw. Prevent photobleaching of the SYBR Green I dye by avoiding prolonged exposure to light.
  • Instrument Capability
    ROX
    • Applied Biosystems 5700
    • Applied Biosystems 7000
    • Applied Biosystems 7300
    • Applied Biosystems 7700
    • Applied Biosystems 7900
    • Applied Biosystems 7900HT
    • Applied Biosystems 7900 HT Fast
    • Applied Biosystems StepOne™
    • Applied Biosystems StepOnePlus™
    Low ROX
    • Applied Biosystems 7500
    • Applied Biosystems 7500 Fast
    • Stratagene Mx3000P®
    • Stratagene Mx3005P™
    • Stratagene Mx4000™
    • Applied Biosystems ViiA 7
    • Applied Biosystems QuantStudio™
    • Agilent AriaMx
    • Douglas Scientific IntelliQube®
    • QIAGEN Rotor-Gene Q
    No ROX
    • Quantabio Q
    • BioRad CFX
    • Roche LightCycler 480
    • Other
    Bio-Rad iCycler iQ systems
    • BioRad iCycler iQ™
    • BioRad MyiQ™
    • BioRad iQ™5
  • Related Resources
    Product Manuals
    Technical Notes
    Publications
    The Potential of Class II Bacteriocins to Modify Gut Microbiota to Improve Host Health
    Özgün C. O. Umu, PLOS ONE - 2016
    Abstract
    Production of bacteriocins is a potential probiotic feature of many lactic acid bacteria (LAB) as it can help prevent the growth of pathogens in gut environments. However, knowledge on bacteriocin producers in situ and their function in the gut of healthy animals is still limited. In this study, we investigated five bacteriocin-producing strains of LAB and their isogenic non-producing mutants for probiotic values. The LAB bacteriocins, sakacin A (SakA), pediocin PA-1 (PedPA-1), enterocins P, Q and L50 (enterocins), plantaricins EF and JK (plantaricins) and garvicin ML (GarML), are all class II bacteriocins, but they differ greatly from each other in terms of inhibition spectrum and physicochemical properties. The strains were supplemented to mice through drinking water and changes on the gut microbiota composition were interpreted using 16S rRNA gene analysis. In general, we observed that overall structure of the gut microbiota remained largely unaffected by the treatments. However, at lower taxonomic levels, some transient but advantageous changes were observed. Some potentially problematic bacteria were inhibited (e.g., Staphylococcus by enterocins, Enterococcaceae by GarML, and Clostridium by plantaricins) and the proportion of LAB was increased in the presence of SakA-, plantaricins- and GarML-producing bacteria. Moreover, the treatment with GarML-producing bacteria co-occurred with decreased triglyceride levels in the host mice. Taken together, our results indicate that several of these bacteriocin producers have potential probiotic properties at diverse levels as they promote favorable changes in the host without major disturbance in gut microbiota, which is important for normal gut functioning.
    Microbial Composition and Diversity Patterns in Deep Hyperthermal Aquifers from the Western Plain of Romania
    Cecilia M. Chiriac, Microbial Ecology - 2017
    Abstract
    A limited number of studies have investigated the biodiversity in deep continental hyperthermal aquifers and its influencing factors. Here, we present the first description of microbial communities inhabiting the Pannonian and Triassic hyperthermal aquifers from the Western Plain of Romania, the first one being considered a deposit of “fossilized waters,” while the latter is embedded in the hydrological cycle due to natural refilling. The 11 investigated drillings have an open interval between 952 and 3432 m below the surface, with collected water temperatures ranging between 47 and 104 °C, these being the first microbial communities characterized in deep continental water deposits with outflow temperatures exceeding 80 °C. The abundances of bacterial 16S rRNA genes varied from approximately 105–106 mL−1 in the Pannonian to about 102–104 mL−1 in the Triassic aquifer. A 16S rRNA gene metabarcoding analysis revealed distinct microbial communities in the two water deposits, especially in the rare taxa composition. The Pannonian aquifer was dominated by the bacterial genera Hydrogenophilus and Thermodesulfobacterium, together with archaeal methanogens from the Methanosaeta and Methanothermobacter groups. Firmicutes was prevalent in the Triassic deposit with a large number of OTUs affiliated to Thermoanaerobacteriaceae, Thermacetogenium, and Desulfotomaculum. Species richness, evenness, and phylogenetic diversity increased alongside with the abundance of mesophiles, their presence in the Triassic aquifer being most probably caused by the refilling with large quantities of meteoric water in the Carpathian Mountains. Altogether, our results show that the particular physico-cheminal characteristics of each aquifer, together with the water refilling possibilities, seem to determine the microbial community structure.
    Circadian rhythm disruption impairs tissue homeostasis and exacerbates chronic inflammation in the intestine
    René Pagel, The FASEB Journal - 2017
    Abstract
    Endogenous circadian clocks regulate 24 h rhythms of physiology and behavior. Circadian rhythm disruption (CRD) is suggested as a risk factor for inflammatory bowel disease. However, the underlying molecular mechanisms remain unknown. Intestinal biopsies from Per1/2 mutant and wild-type (WT) mice were investigated by electron microscopy, immunohistochemistry, and bromodeoxyuridine pulse–chase experiments. TNF-α was injected intraperitoneally, with or without necrostatin-1, into Per1/2 mice or rhythmic and externally desynchronized WT mice to study intestinal epithelial cell death. Experimental chronic colitis was induced by oral administration of dextran sodium sulfate. In vitro, caspase activity was assayed in Per1/2-specific small interfering RNA–transfected cells. Wee1 was overexpressed to study antiapoptosis and the cell cycle. Genetic ablation of circadian clock function or environmental CRD in mice increased susceptibility to severe intestinal inflammation and epithelial dysregulation, accompanied by excessive necroptotic cell death and a reduced number of secretory epithelial cells. Receptor-interacting serine/threonine-protein kinase (RIP)-3-mediated intestinal necroptosis was linked to increased mitotic cell cycle arrest via Per1/2-controlled Wee1, resulting in increased antiapoptosis via cellular inhibitor of apoptosis-2. Together, our data suggest that circadian rhythm stability is pivotal for the maintenance of mucosal barrier function. CRD increases intestinal necroptosis, thus rendering the gut epithelium more susceptible to inflammatory processes.—Pagel, R., Bär, F., Schröder, T., Sünderhauf, A., Künstner, A., Ibrahim, S. M., Autenrieth, S. E., Kalies, K., König, P., Tsang, A. H., Bettenworth, D., Divanovic, S., Lehnert, H., Fellermann, K., Oster, H., Derer, S., Sina, C. Circadian rhythm disruption impairs tissue homeostasis and exacerbates chronic inflammation in the intestine.
    A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice
    Chrstine Bäuerl, JoVE (Journal of Visualized Experiments) - 2017
    Abstract
    Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.
    RNA‐seq: Applications and Best Practices
    Michele Araújo Pereira, Intech - 2017
    Abstract
    RNA‐sequencing (RNA‐seq) is the state‐of‐the‐art technique for transcriptome analysis that takes advantage of high‐throughput next‐generation sequencing. Although being a powerful approach, RNA‐seq imposes major challenges throughout its steps with numerous caveats. There are currently many experimental options available, and a complete comprehension of each step is critical to make right decisions and avoid getting into inconclusive results. A complete workflow consists of: (1) experimental design; (2) sample and library preparation; (3) sequencing; and (4) data analysis. RNA‐seq enables a wide range of applications such as the discovery of novel genes, gene/transcript quantification, and differential expression and functional analysis. This chapter will encompass the main aspects from sample preparation to downstream data analysis. It will be discussed how to obtain high‐quality samples, replicates amount, library preparation, sequencing platforms and coverage, focusing on best recommended practices based on specialized literature. Basic techniques and well‐known algorithms are presented and discussed, guiding both beginners and experienced users in the implementation of reliable experiments.
    Click here to see all Publications

Ordering Information

    Product
    Kit Size
    Order Info
  • PerfeCTa NGS Quantification Kit - Illumina
    500 x 20 μL rxns
  • PerfeCTa NGS Quantification Kit - Illumina/ROX
    500 x 20 μL rxns
  • PerfeCTa NGS Quantification Kit - Illumina/Low ROX
    500 x 20 μL rxns