KASP Genotyping Assays FAQs

LGC do not have any restrictions on the species for which we can design and run KASP genotyping assays. As long as DNA of a sufficient quality and quantity can be extracted from your species of interest, the DNA can be used in KASP genotyping assays. Standard primer design restrictions will apply; it is not always possible to design working assays in particularly repetitive or G/C rich regions.

Download a partial list of organisms that we have performed KASP genotyping on.

If you are shipping dried-down DNA to LGC Biosearch Technologies, you will need to ensure that you provide a sufficient mass of DNA for the number of SNPs that you wish to run. If you are shipping wet DNA to LGC Biosearch Technologies, you will need to provide a sufficient volume of DNA at (or preferably above) the minimum accepted concentration for the number of SNPs that you wish to run.

Please note: it is always advisable to send DNA at a higher concentration or mass than the minimum requirement. This allows for a range of dilutions to be tested in-house and, in the case of wet DNA, reduces the requirement for dead volumes. Any unused DNA can be stored at LGC for future genotyping projects, or arrangements can be made to return surplus DNA to the customer.

This manual explains how to calculate the required DNA quantity for both dry and wet DNA samples.

The first step is to calculate the quantity of DNA that you need to send – this varies depending on the number of SNPs that you wish to run across your samples and the genome size of your study organism. This factsheet explains how to calculate the required DNA quantity for both dry and wet DNA samples.

The next step is to prepare your sample plate(s) and complete a sample submission form in your preferred format. This completed form will enable us to import your sample identifiers directly into Kraken™, our information and laboratory workflow management software. If you wish to keep your sample identifiers confidential, just use the well location as the identifier.

If you have been assigned a project manager, please e-mail your data files directly to them. Alternatively, e-mail the information to genomics@lgcgroup.com. Please ensure that your plates are labelled and packaged correctly. It is important that you plan when you are going to send your samples – please do not send them on a Friday as the laboratory will not be open over the weekend to receive them.

LGC prefer DNA samples to be eluted in 10mM Tris buffer. If your elution buffer contains EDTA, please contact us prior to shipping your samples and ensure that your project manager is aware.

DNA samples are usually shipped to LGC in 96-well or 384-well plate formats as these are compatible with our robotics systems. Please ensure that you complete the appropriate DNA sample submission form prior to sending in your samples. DNA plates should be securely sealed to prevent sample loss and contamination, and plates should be clearly labelled.

The minimum number of DNA samples that we accept is 22, plus two no-template controls (NTCs). There is no maximum number of DNA samples. As a general rule, the larger the project, the better the value in terms of cost per datapoint.

Two no template control (NTC) wells should be included on each DNA sample plate that you submit.

If you have positive control DNA samples for one or more of the possible genotypes, it is recommended to include these when submitting your DNA samples for genotyping. You should identify these samples in your sample submission file. Positive controls are particularly important if one of the alleles in your assay is a low-frequency or rare allele.

Once you know the DNA sequences for your SNPs, you should complete a sequence submission form. LGC requires sequence data supplied by you to avoid any ambiguities caused by different databases and to ensure that your SNP assays are designed for the correct sequence. The SNP should be marked by a forward slash (e.g. G/T) or by IUPAC codes. Each SNP that you submit will require a unique SNP ID. If you have been assigned a project manager, please e-mail your data files directly to them. Alternatively, e-mail the information to genomics@lgcgroup.com.

Please ensure that you supply sequence for at least 50 bases (bp) either side of the SNP site. If you have more sequence available, please include up to 100 bp either side of the SNP site as this will assist with optimal assay design.

If you require an assay for an indel, please submit these using the SNP submission file. Please see below for examples of how to format the sequence: One bp insertion/deletion: GCATTTCATCCTC[/T]TGCATAATGACA Insertion/deletion larger than 1 bp: GCATTTCATCCTC[/ATTCTCTCCCAAGTCTCC]TGCATAATGACA Square brackets should be used to identify the polymorphism.

If there is more than one SNP present in your sequence, please mark the SNP of interest with square brackets e.g. [G/T]. The additional SNPs should be marked using IUPAC codes (no brackets).

For KASP assay design, you can submit the DNA sequence in either orientation. Our Kraken™ software is used for primer design, and will choose the most suitable DNA orientation based on the putative primer properties. The SNP will always be reported to you in the same orientation that you submitted the sequence in.

One KASP assay aliquot can be used to perform 2500 genotyping reactions. When an assay is first ordered for an in-house genotyping project, you will be charged a new assay setup fee. If more than 2500 reactions are required, you will be charged an assay re-stock fee.

If you have submitted your samples for a genotyping project in our service laboratory, you should expect a call rate of >95% in all genomes, and a near perfect call rate (typically >99%) in well-defined genomes (e.g. human). If you are running KASP in your own laboratory, the expected call rate will depend upon the type of assay that you ordered and the quality of your DNA samples:

• KBD assays – these are non-validated assays, and the call rate will therefore depend on the assay design and the quality of your DNA samples.
• KOD assays – these are validated in-house prior to being shipped out to you, and, unless specifically requested, an assay will not be classed as validated unless call rates of >95% are achieved in-house.

If the initial assay design does not yield high quality data, the primers are re-designed using different parameters or designed to the opposite DNA strand to ensure that the assay is working optimally when you receive it in your laboratory. As for KBD assays, the call rate achieved will be dependent upon the quality of your DNA samples.

To perform KASP genotyping of 100 SNPs on your extracted DNA, we would need approximately 200 µL of DNA at 5ng / µL (based on human genome size). This factsheet explains how to calculate the required DNA quantity according to the number of SNPs that you wish to genotype and according to the genome size of your study species.

KASP™ chemistry utilises a two-step touchdown PCR method, with the elongation and annealing steps incorporated into a single step. The temperature used for the annealing stage determines the specificity of the reaction and hence the ability of the primers to anneal to the DNA template. A touchdown PCR involves starting with a high annealing temperature and incrementally decreasing the annealing temperature each PCR cycle. The higher annealing temperatures in the early cycles of a touchdown ensure that only very specific base pairing will occur between the DNA and the primer, hence the first sequence to be amplified is most likely to be the sequence of interest. The annealing temperature is gradually decreased to increase the efficiency of the reaction. The regions that were originally amplified during the highly specific early touchdown cycles will be further amplified and outcompete any non-specific amplification that may occur at the lower temperatures. The standard KASP thermal cycling protocol has 10 cycles of touchdown PCR (annealing 61°C to 55°C, decreasing 0.6°C per cycle), then 26 cycles of standard 2-step PCR at the lower annealing temperature (55°C).

KASP™ chemistry utilises a two-step touchdown PCR method, with the elongation and annealing steps incorporated into a single step. A 15-minute activation is required (94°C), followed by 10 cycles of touchdown PCR and 26 cycles of standard 2-step PCR. The standard KASP thermal cycle conditions are detailed in the table below.

Please ensure that you check the cycling conditions in your assay information pack to ensure that your assay does not have any specific cycling conditions. Some assays may require alternative cycling conditions due to high or low %GC content.

It is straightforward to perform KASP™ genotyping in your own laboratory. Aside from standard laboratory equipment such as pipettes, all that is additionally required is a thermal cycler and a FRET-capable plate reader, or a qPCR instrument.

The plate reader or qPCR instrument must be capable of reading FAM and HEX fluorophores; the relevant excitation and emission values are detailed in the table below. KASP Master mix also contains ROX, a passive reference dye. Although it is not essential to read the ROX levels in your reactions, it is advisable to do so as normalisation will improve the quality of the data obtained.

LGC can supply you with a free-of-charge genotyping trial kit to enable you to try KASP chemistry in your laboratory.

KASP chemistry reports with two dyes (FAM and HEX) and is a singleplex chemistry.

KASP chemistry is an endpoint chemistry and cannot be used in real time.

As the qPCR instruments available on the market have differing requirements for ROX, LGC manufacture KASP Master mix with varying levels of ROX. KASP Master mix is available in Low, Standard, and High ROX formulations.

These formulations only differ in the level of ROX that they contain and are otherwise identical. To determine the correct version of KASP Master mix for your machine, please click here.

The qPCR instrument or plate reader must be capable of reading FAM and HEX fluorophores; the relevant excitation and emission values are detailed in the table below. KASP Master mix also contains ROX, a passive reference dye. Although it is not essential to read the ROX levels in your reactions, it is advisable to do so as normalisation will improve the quality of the data obtained.

To view a list of the qPCR instruments that we know work well with KASP, please click here.

To read KASP chemistry, it is essential that your plate reader is capable of reading FAM and HEX fluorophores. It is not essential to read the ROX levels in your reactions, but it is advisable to do so as normalisation will improve the quality of the data obtained. Many of the commonly used qPCR instruments are capable of reading ROX but in some cases the software linked to the instrument does not utilise ROX in the genotyping calculations; it is still possible to obtain good quality genotyping data with these systems.

KASP™ chemistry is supplied with the FAM and HEX fluorophores for allelic discrimination. All of the ABI qPCR instruments (e.g. 7500, 7900, Viia7) have the ability to read VIC by default instead of HEX. VIC does, however, have the same excitation and emission spectra as HEX. When reading KASP genotyping reactions on one of these machines, ensure that you select VIC as a substitute for HEX.

The KASP exome sample tracking panel of assays provides 24 bi-allelic SNPs with 48 points of allelic comparison. The panel of alleles were chosen for their commonality between the enrichment kits selected (3 kits), with the targets being reproducibly enriched. The optimised panel provides 18 differential alleles between any two samples enabling discrimination. Caution should however be taken when sampling includes the same individual or monozygotic twins.

The reproducibility of the data was tested on sampling of 10,000 individuals from HAPMAP populations (CEU, CHB, JPT and YRI) for duplicate profiles being produced. The worst performing simulation based on population data for Southern Han Chinese indicated one duplicate dataset in 85000 samples. For further information see: Pengelly et al. Genome Medicine 2013, 5:89

The panel was designed with datasets derived from three exome enrichment kits, all of which are therefore compatible with the KASP exome sample tracking panel: 1. Agilent SureSelect Human All Exon V4; 2. Illumina TruSeq Exome Enrichment; and 3. Nimblegen SeqCap EZ Human Exome Library V3.0. Higher version number kits of the same providers usually target larger portions of the exome (and can include intronic regions) in addition to the same targets as the earlier version numbers and hence should also be compatible with the panel.

The available range of in-solution enrichment kits differ in terms of what they specifically target within the exome. All kits will target the human exome, but some will target slightly larger regions or have slightly different target regions than others. Other exome enrichment kits and enrichment methods may also be applicable to the KASP exome sample tracking panel; please contact our technical support team if you require further information.

The sequence for each target allele is available to download from NCBI. Sample tracking genotyping data should be compared with exome mapping data for confirmation of genotype match.

When submitting your plate layout file containing sample identifiers, please identify which wells correspond to your no template control samples. To ensure that your files are processed as efficiently as possible, please label no template controls as ‘NTC’; our Kraken software can identify wells labelled in this way and automatically assign them as controls.

KASP assays can include up to two other polymorphisms in the surrounding sequence (within 30 base pairs of the sequence of interest). These additional SNPs should be marked with their IUPAC codes, whilst the SNP of interest should be marked with square brackets surrounding it e.g. [A/G] or [R]. The assay design function in our Kraken software will design KASP primers that either avoid the surrounding polymorphisms or that incorporate degenerate / wobble bases into the primers to ensure that the primers are still able to bind efficiently to the DNA template.

If your KBD assay does not work as expected on your DNA samples, there are several possible causes including DNA quantity and quality, incorrect thermal cycling program, homology of the region of interest with other regions of the genome, and plate reader / qPCR instrument set-up incorrectly. Our technical support team are available to help with troubleshooting of problematic assays; please visit our support centre to raise a request within our ticketing system.

As KBD assays are design-only and have not been validated in our laboratory, the extent of support that can be offered is limited. If the reason for the unexpected results cannot be resolved through our troubleshooting procedures, the only available option is to upgrade the KBD assay to a KOD assay that LGC can validate and optimise in our laboratory. If you choose this option, you will only be charged the difference in price between the KBD and the KOD.

KASP Master mix is stable for 1 week at 4ºC, and 1 year at -20ºC / -80ºC. KASP Master mix should be aliquoted upon receipt to minimise the need for repeated freeze-thaw cycles. KASP Master mix should be stored in light-protective tubes and should not be exposed to greater than three freeze-thaw cycles.

KASP genotyping reactions consist of three components: universal KASP Master mix, SNP-specific KASP Assay mix, and template DNA. The reactions can be prepared using either wet DNA (Table 1) or dried down DNA (Table 2), depending on your preferred laboratory workflow.

Table 1: Wet DNA reaction assembly

Table 2: Dry DNA reaction assembly

The volumes outlined in the tables above give the exact ratios of the reaction components that should be used when preparing KASP reactions. It is expected that reactions will not be assembled individually, but that larger volumes (sufficient for all planned reactions plus an additional percentage to account for pipetting error) will be prepared prior to dispensing into the reaction plate.

Please note that it is possible to round up the volumes of DNA / water used in the reactions if preferred (e.g. from 2.43 µL to 2.5 µL for 5 µL reactions). The below example details how to prepare reactions in this way, if setting up a full 96-well plate of reactions using wet DNA.

1. Dispense 5 µL of template DNA at the appropriate concentration into each well of the 96-well plate. Please note that at least two wells of the plate should contain water instead of DNA – these wells will act as the no template controls (NTCs).
2. Combine KASP Master mix and KASP Assay to create KASP genotyping mix. The table below details how to prepare sufficient KASP genotyping mix for 96 reactions plus 10% excess.

3. Pipette 5 µL of KASP genotyping mix (see table above) into each well of the reaction plate. Combined with the template DNA, this gives a final reaction volume of 10 µL per well.

The minimum final DNA concentration that LGC recommend in KASP genotyping reactions is 2.5 ng / µL. For example, if you were preparing a 10 µL reaction consisting of 5 µL of DNA and 5 µL of genotyping mix (KASP Master mix + KASP Assay mix) then the input DNA would need to be at 5 ng / µL to ensure a final concentration of 2.5 ng / µL.

This value is based on the human genome size (~3000 Mbp). If the genome size of your study organism is larger than human, you will need to adjust final DNA concentration (and hence input concentration) accordingly. (Please note that we do not recommend reducing the input DNA concentration for genomes smaller than human).

For genomes larger than human, a higher concentration of DNA is required. To calculate this, divide the genome size of your organism by the size of the human genome (3000 Mbp), and use the resulting number to multiply the final concentration of DNA that should be used in your KASP reactions.

e.g. Triticum aestivum (wheat): 15966 Mbp

15966 Mbp / 3000 Mbp = 5.3

You will need a final DNA concentration that is 5.3 times more concentrated = 2.5 ng / µL DNA x 5.3 = 13.25 ng / µL final concentration.

Completed KASP genotyping reactions should be cooled to below 40ºC before performing the plate read. KASP chemistry cannot be read above 40ºC, and reading completed reactions above this temperature will not generate any meaningful data.

If you are using a qPCR instrument, many of the software packages that are supplied with the instruments have the ability to analyse endpoint genotyping data. For a number of the most commonly used instruments, LGC have prepared manuals that detail how to program the instrument-specific software to run and analyse KASP genotyping reactions – these can be accessed at here.

For some qPCR instruments, and for plate readers, the supplied software cannot perform endpoint genotyping analysis directly. Raw data can be exported to alternative software packages for analysis. It is possible to use MS Excel or similar for analysis, but this is will not be efficient for large datasets. LGC can provide KlusterCaller (http://www.lgcgroup.com/products/genotyping-software/klustercaller/), a software package that enables genotyping data analysis and reporting.

The minimum number of DNA samples recommended for cluster analysis is 22 – this is to ensure that there are sufficient data points for reliable clusters to form. If you only have a few different DNA samples available, LGC would recommend running the samples in duplicate or triplicate to increase the number of data points in the analysis. It may also be beneficial to run positive controls of known genotypes in this situation. At least two no template control should be run for each KASP genotyping assay, giving a minimum of 24 data points per cluster plot.

A no template control, typically referred to as an NTC, is a well that is set up in the same way as all DNA sample wells but does not contain any template DNA. Sterile water must be used to replace the volume that would be occupied by the wet DNA, as this ensures that the KASP Master mix in the NTC well is diluted from 2X to 1X concentration. As an example, a 10 µL NTC well would typically contain 5 µL 2X KASP Master mix, 0.14 µL KASP Assay mix and 5 µL sterile water. NTC wells are essential for detecting contamination or non-specific amplification, and a minimum of two NTC wells should be included per KASP assay per reaction plate.

As analysis of KASP genotyping data is performed using cluster plots, individual DNA samples do not need to be run in duplicate or triplicate – a single data point per DNA sample per KASP assay (SNP / indel) is sufficient. It is, however, important to meet or exceed the required minimum of 22 DNA samples per KASP assay to ensure that there are sufficient data points for reliable clusters to form.

It is possible to run a number of different KASP assays (i.e. for different SNPs / indels) on one reaction plate, as long as there are sufficient data points for each assay (minimum 22 plus 2 no template controls). For example, if performing KASP genotyping in 96-well plates, the same 22 DNA samples plus 2 NTCs could be represented up to four times on the plate. Each set of samples could be genotyped with a separate KASP Assay mix, thus enabling four different KASP assays to be run on the plate. The resultant fluorescent data should be analysed assay by assay – data points generated from different KASP assays should not be plotted on the same cluster plot.

KASP reactions should be run in PCR-suitable reaction plates. Both clear and white plates work well with the majority of qPCR instruments and plate readers. Ensure that you select fully-skirted, semi-skirted, or non-skirted plates according to the specific requirements of your platform.

For 96-well plates, a reaction volume of 10 µL should be used. For 384-well plates, a reaction volume of 5 µL should be used. Ensure that the appropriate total reaction volume is used for the plate type.

A PCR-suitable optically clear seal must be used to enable fluorescent signal to be read properly. The reaction plate must also be sealed sufficiently to prevent evaporation as evaporation will affect efficiency of the reaction and the signal that is generated.

The standard cycling conditions for KASP are the 61-55 ºC touchdown protocol (click for protocol). There are two additional KASP thermal cycling protocols that may either be recommended as optimal (for laboratory-validated and Assay Search Tool assays) or may be worth trying (for design-only assays) if non-optimal results are obtained with the standard 61-55 ºC touchdown protocol.

The ‘68-62 ºC touchdown protocol’ (click for protocol) may be beneficial for assays that have a high %GC content (greater than 65%).

The ‘2-step 57 ºC protocol’ (click for protocol) may be beneficial for assays that have a low %GC content (less than 40%).

Full details of all KASP thermal cycling conditions can be found in the ‘KASP thermal cycling conditions’ (click for document).

Yes - if you have previously ordered and run KASP Assay mix with KASP Master mix v3, these assays are also compatible with KASP Master mix v4.

Please note that 2X KASP Master mix v3 is provided at a magnesium concentration of 3.6 mM to give a final concentration of 1.8mM, and 2X KASP Master mix v4 is provided at a concentration of 5 mM to give a final concentration of 2.5mM. Please contact tech.support@lgcgroup.com if you require advice regarding reaction optimisations.

Once you have completed the initial KASP thermal cycle and have read your reaction plate, the plate can be stored at 4ºC. Prior to storage, ensure that the plate is suitably sealed to prevent evaporation. Completed reaction plates are stable for at least 1 week. This means that KASP reactions can be further cycled (recycled) up to 7 days after initial cycling. If you are contacting LGC for technical support with your assays, please ensure that you retain the reaction plate (stored as outlined above) as you may be asked to re-read and / or recycle the reaction plate as part of the troubleshooting procedure.

On June 01, 2020, the part numbers for KASP™ by Design (KBD) and KASP on Demand (KOD) assays were updated as shown in the table below. This was a necessary step to accommodate critical updates in our internal systems.

Please note there was no change in the formulation or manufacture of these products.

We appreciate your understanding, and want to affirm our commitment to you as a reliable source for your SNP genotyping products. Please feel free to contact us should you have any further questions or require additional information.

KASP Master Mix formulations have been updated to remove Triton™ X-100 in compliance with the REACH Regulation for Registration, Evaluation, Authorization and Restriction of Chemicals ((EC) No 1907/2016), which was implemented by the European Commission to protect human health and the environment through the safe use of chemicals. As of January 04, 2021, use of nonylphenol ethoxylate (NPE) and octylphenol ethoxylate (OPE) based surfactants such as Triton X-100 are not permitted in the EU, unless an authorization is granted or the use is exempted from authorization. The new KASP-TF Master Mixes are free of Triton X-100, and provide the same performance and quality you have always expected from your KASP genotyping mix.

Find your recommended KASP-TF Master Mix part number in the table below. You can also refer to the KASP genotyping protocols for your PCR instrument to select the correct KASP-TF Master Mix for your instrument.