The NGSBIO Library Quant Kit contains all the components required for accurate and sensitive quantification of libraries prepared for Illumina® NGS systems.
The kit uses qPCR to specifically quantify adapter-ligated DNA molecules, ensuring optimal cluster densities for improved sequencing efficiency and quality of data.
- Uses qPCR to accurately and rapidly quantify a library prior to sequencing
- Gives consistent library quantification across a wide range of sample types, concentrations, fragment sizes and GC content
- Uses a single extension time for all libraries
- Allows specific quantification of only DNA molecules that can be sequenced by NGS
- Uses antibody-mediated hot start technology to ensure all reactions start simultaneously
- Compatible with all Illumina® instruments and qPCR platforms
- Suitable for manual and automated workflows
- Available as an easy-to-see blue qPCR mix
- Easily calculate library concentration with the online NGSBIO Library Quantification Tool
The NGSBIO Library Quant Kit offers a reliable qPCR-based method for the quantification of libraries prepared for Illumina® NGS systems. The kit includes 5 DNA standards, primers specific to the P5 and P7 Illumina® adapter sequences and qPCRBIO SyGreen Mix. The advanced qPCR buffer system has been developed using our high-throughput smart screen technology to ensure efficient amplification of all your libraries, including those that are GC or AT-rich. The kit is also supplied with a convenient library dilution buffer.
Accurate Library Quantification
qPCR is considered the best method for quantifying NGS libraries as it only measures adapter-bound molecules that can be used as templates for library amplification and cluster generation. The NGSBIO Library Quant Kit enables highly accurate quantification crucial for optimal cluster densities and greater sequencing efficiency.
Library quantification can be used at any time after adapter ligation and should always be used prior to cluster generation. For easy and accurate data analysis we recommend using the NGSBIO Library Quantification Tool. Overestimating library concentration can result in insufficient cluster density and underestimating library concentration can result in a high, saturating cluster density. To learn more about optimal cluster density, consult the instructions of your Illumina® machine.
Wide Dynamic Range
The DNA standards supplied are precisely measured and ready-to-use, covering 5 orders of magnitude from 2pM to 0.2fM. The kit is suitable for quantification of even low concentration libraries including libraries constructed without a PCR amplification step.
The NGSBIO Library Quant Kit is compatible with all qPCR platforms and is optimised to give consistent and reproducible library quantification across a wide range of sample types, fragment sizes (up to 1000bp), concentrations and GC content. Use our qPCR Selection Tool to find out which ROX variant is compatible with your instrument.
For easy and accurate data analysis we recommend using the NGSBIO Library Quantification Tool.
- NGS library quantification for Illumina® systems
The kit should be stored between -30°C and -15°C. Avoid prolonged exposure to light. If stored correctly the kit will retain full activity for 12 months. The kit can be stored at 4°C for 1 month. The kit can go through 30 freeze/thaw cycles with no loss of activity.
Figure 1. Amplification Curves
An adapter-ligated library sample (purple) is run alongside six standard templates (grey) provided in the NGSBIO Library Quant Kit.
Figure 2. Standard Curve
The Cts of the amplification curves are plotted against the log of the concentration of the standard templates. A linear curve is fitted through the standards. The concentration of the unknown sample is then calculated from its position on the curve.
Figure 3. High consistency and reproducibility of quantification
Quantification of 16 adapter-ligated libraries using NGSBIO Library Quant Kit (purple), NEBNext® Library Quant Kit
(orange) and KAPA Library Quantification Kit (green).
The NGSBIO Library Quant Kit shows less spread and greater consistency among replicates. The quantification results are within those obtained by two leading manufacturers of NGS library quantification kits. The number on the bottom right corner of each graph represents the concentration of dsDNA obtained using a Qubit Fluorometer from Invitrogen.
ROX (6-carboxy-X-rhodamine) is used as a passive reference dye in ROX-dependent real-time PCR instruments to normalize for variations of fluorescence levels that can arise mainly due to optical path variations among wells. Normalisation of the fluorescence intensity (Rn) is done in real-time PCR software by dividing the emission intensity of the specific signal by the emission intensity of ROX.
ROX does not take part in the PCR reaction and its fluorescence levels are not proportional to the quantity of DNA in each well, so the addition of this fluorophore to a mix provides a constant fluorescent signal during amplification.
Different types of real-time PCR instruments requiring a passive reference standard have different optimal concentrations of ROX, mainly due to the different optical configurations of each system (i.e. the different type of excitation source and optics used).
The addition of either too little or too much ROX would result in a very noisy signal impacting on the results of the reaction. Therefore, it is extremely important for the user to:
- Determine the correct ROX concentration to optimise real-time PCR results, and
- Check the ROX settings on the software used to set up the reaction
ROX concentrations in NGSBIO Library Quant Kit:
The NGSBIO Library Quant Kits come in 3 different formulations, each with a different concentration of ROX:
- NGSBIO Library Quant Kit Lo-ROX (PB71.11) contains 112 nM ROX
- NGSBIO Library Quant Kit Hi-ROX (PB71.12) contains 1.12 µM ROX
- NGSBIO Library Quant Kit Separate ROX (PB71.14) contains no ROX and include a separate tube of 50 µM ROX additive. This enables you to choose what concentration of ROX you’d like to use.
|PB71.12-01||100 x 20μL Reactions|
|PB71.12-05||500 x 20μL Reactions|
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|2x qPCRBIO SyGreen Mix Hi-ROX||
1 x 1mL | 100 Reactions
5 x 1mL | 500 Reactions
|DNA Standards 1-5||
30μL each | 100 Reactions
85μL each | 500 Reactions
|10x Illumina® Primers||
1 x 0.2mL | 100 Reactions
1 x 1mL | 500 Reactions
|10x Dilution Buffer||
1 x 0.6mL | 100 Reactions
2 x 1.5mL | 500 Reactions