KASP Manual

KASP Genotyping Quick Start Guide

KBioscience Ltd. Unit 7 Maple Park

Essex Road Hoddesdon

EN11 0EX UK Tel: +44 (0) 1992 470757 Fax: +44 (0) 8700 511302 [email protected] www.kbioscience.co.uk

Introduction

Welcome to the KBioscience KASP system for the genotyping of single nucleotide polymorphisms (SNPs) or insertion / deletions. This Quick Start Guide contains the basic information required to carry out KASP genotyping. For more detailed information, please refer to the KASP SNP Genotyping Manual.

KASP genotyping process overview

1. Sample arraying DNA samples should be arrayed into a PCR plate and either dried down or stored in hydrated form. No‐template controls (NTCs) should be included on each plate.

2. Assay The assay should either be assembled or KASP‐on‐Demand (KOD) / KASP‐by‐Design (KBD) Assays used. 3. Make up KASP genotyping Mix

Table 1. Constituent reagent volumes for making KASP genotyping mix in 96‐well reaction volume (5ul final volume) or 384‐well reaction volume (10ul final volume). *DNA samples diluted to final concentration of 0.5‐20ng per 5µl reaction.

Due to the inability to pipette accurately at very low volumes, multiple reaction mixtures must be assembled as master mixes. 4 Dispense genotyping mix The KASP genotyping mix should then be dispensed on to the DNA samples. 5 Seal plate Once dispensed, the plate should be sealed with an optically‐clear seal. 6 Thermocycle KASP genotyping reactions may be carried out on either a standard, Peltier block‐based thermocycler or on a waterbath‐based system such as one of the KBioscience HydroCycler instruments.

1. 94 °C for 15 minutes Hot‐start enzyme activation

2. 94 °C for 20 seconds

Touchdown over 65‐57°C for 60 seconds 10 cycles (dropping 0.8°C per cycle)

3. 94°C for 20 seconds 57°C for 60 seconds 26 cycles

7 Read plate The plate should be read on a suitable fluorescent plate

reader. NB. Reading temperature should be 40°C or below (preferably ambient).

Table 1 KASP Genotyping mix assembly

Component Wet DNA method

(µl) Dry DNA method

(µl)

DNA* 2.5 5 N/A N/A

KASP 2X Reaction Mix 2.5 5 2.5 5

Assay 0.07 0.14 0.07 0.14

H20 N/A N/A 2.5 5

Total reaction volume 5µl 10µl 5µl 10µl

KASP Genotyping Quick Start Guide

KBioscience Ltd. Unit 7 Maple Park

Essex Road Hoddesdon

EN11 0EX UK Tel: +44 (0) 1992 470757 Fax: +44 (0) 8700 511302 [email protected] www.kbioscience.co.uk

8 Analyse data After thermal cycling, genotyping data can be analysed using most FRET‐capable readers and viewed graphically as shown below.

The KASP SNP Genotyping Manual v 3.0

KBioscience Ltd. Unit 7 Maple Park Essex Road Hoddesdon EN11 0EX UK Tel: +44 (0) 1992 470757 Fax: +44(0) 8700 511302 [email protected] www.kbioscience.co.uk

The KASP SNP Genotyping Manual

Introduction The KBioscience Competitive Allele‐Specific PCR genotyping system (KASP) is a homogeneous, fluorescent, endpoint‐ genotyping technology. The technology was initially developed for use at the KBioscience in‐house genotyping facility but gradually evolved into a global benchmark technology. KASP offers the simplest, most cost‐effective and flexible way to determine both SNP and insertion / deletion genotypes. Analysis can be carried out in 48, 96, 384 and 1536‐well plate formats.

The KASP genotyping system is comprised of two components (1) The SNP‐specific assay (a combination of three unlabelled primers) (2) The universal Reaction Mix, which contains all other required components including the universal fluorescent reporting system and a specially‐developed Taq polymerase.

The KASP genotyping system has been used successfully in a wide variety of organisms, achieving around 90% SNP‐to‐assay conversion rate.

Principal of the KASP genotyping assay Please go to the following URL to view an animation describing the mechanism of action of the KASP chemistry, http://www.kbioscience.co.uk/reagents/KASP.html

The mechanism of action behind KASP is novel; to elucidate, it is necessary to first consider the constituent oligonucleotides:

• Two allele‐specific primers (one for each SNP allele). Each primer contains a unique unlabelled tail sequence at the 5' end.

• One common (reverse) primer.

• Two 5’ fluor‐labelled oligos, one labelled with FAM, one with CAL Fluor Orange 560. These oligo sequences are designed to interact with the sequences of the tails of the allele‐specific primers.

• Two oligos, with quenchers bound at the 3' ends. These oligo sequences are complementary to those of the fluor‐labelled oligos (and therefore also complementary to the tails of the allele‐specific primers). These quenched oligos therefore bind their fluor‐labelled complements and all fluorescent signal is quenched until required.

In the initial stage of PCR, the appropriate allele‐specific primer binds to its complementary region directly upstream of the SNP (with the 3' end of the primer positioned at the SNP nucleotide). The common reverse primer also binds and PCR proceeds, with the allele‐specific primer becoming incorporated into the template. During this phase, the fluor‐ labelled oligos remain bound to their quencher‐bound complementary oligos, and no fluorescent signal is generated.

As PCR proceeds further, one of the fluor‐labelled oligos, corresponding to the amplified allele, also gets incorporated into the template, and is hence no longer bound to its quencher‐bound complement. As the fluor is no longer quenched, the appropriate fluorescent signal is generated and detected by the usual means.

If the genotype at a given SNP is homozygous, only one or other of the possible fluorescent signals will be generated. If the individual is heterozygous, the result will be a mixed fluorescent signal.

Kit Contents:

• Reaction Mix (supplied at 2X concentration, containing Taq polymerase enzyme and the passive reference dye, 5‐carboxy‐X‐rhodamine, succinimidyl ester (ROX)

• MgCl2 (50mM; for A/T‐rich DNA regions)

• DMSO (for G/C‐rich DNA regions)

Customer Requirements: 1. FRET‐capable plate reader 1

2. PCR microtitre plate 3. DNA samples (dissolved in Tris‐HCl buffer (10mM;

pH 8.3) or PCR grade H20) 4. 10mM Tris‐HCl pH 8.3 or PCR grade H20 5. Optical plate seal

1 KBioscience are happy to advise on the choice of plate reader.

Storage and Shelf Life

Reaction Mix can be safely stored for one week at 4°C, one year at ‐20°C or indefinitely at ‐80°C. If the reaction mix is divided into aliquots, it is recommended that the tubes used are light‐ protective; assays may also be divided into convenient aliquots. Frequent freeze / thawing of both KASP reaction mix and assays will adversely affect performance, although up to 10 freeze thaws has been shown to not affect performance.

Important notes

DNA quantity / quality Most KASP assays will function well with 3‐10ng of high quality DNA per reaction. However genome size is also an important consideration as a greater mass of DNA per reaction will be required if genotyping a larger genome; conversely, a smaller DNA mass per reaction will be required for smaller genomes.

The purity of DNA is important when using KASP, but no more so than for standard PCR. However, when DNA is crudely extracted, inhibitors of PCR can potentially remain, causing a greater or lesser issue depending on the source of the DNA (and hence the nature of the potential contaminants). Where the DNA has been extracted from plant material, polysaccharides and polyphenols can co‐purify with the DNA and interfere with PCR. Addition of polyvinylpyrrolidone (PVP)



at around 3% to the DNA samples can bind polyphenols and allow better PCR. If the extracted DNA is impure but of high DNA concentration, it should be possible to dilute such that DNA concentration remains sufficiently high, whilst effectively diluting‐out the impurity. Whilst it is feasible to use KASP with DNA derived from such sources, KBiosciences recommends use of the KlearGene family of DNA extraction and purification kits to provide very high quality / quantity, contaminant‐free DNA (see Related Products).

KASP can be used in conjunction with a variety of DNA sources: genomic DNA, mitochondrial / bacterial (haploid) DNA, nested PCR amplicons and whole genome amplified (WGA) DNA (by either Phi 29 or DOP‐based methods).

Assay mix Ignore this section if using KASP‐on‐Demand (KOD) or KASP‐by‐ Design (KBD) assays and proceed to sample arraying.

In the KASP system, an assay mix refers to the combination of the three unlabelled primers required for the assaying of a SNP with the KASP system. By utilising the assay design algorithm of Kraken (KBioscience’s Workflow Manager; see Related Products), the user can design KASP genotyping assays.

Preparation of a KASP assay from the three Kraken‐designed primers is described in Table 1. Standard, desalted primers should be used. The oligonucleotide quality is critical and as such KBioscience recommends purchasing from IDT.

Table 1 Final

concentration in assay mix (µM)

Final volume in assay mix (µl)

Allele‐specific primer 1 (100µM)

12 12

Allele‐specific primer 2 (100µM)

12 12

Common (reverse) Primer (100µM)

30 30

H20 / Tris‐HCl (10mM, pH 8.3)

‐ 46

TOTAL 100

Table 1. Preparation of a KASP genotyping assay from the constituent allele‐specific and common primers.

100µl of assay is sufficient to carry out at least 650 genotypes in 96‐well format or at least 1300 genotypes in 384‐well format (based on 10µl and 5µl reaction volumes, respectively with plate type). The assay mix is combined with the Reaction Mix (see below) and added to the DNA samples to be genotyped.

Sample Arraying DNA samples may be arrayed in any microtitre PCR plate though typically 96, 384 or 1536‐well plates are used. The

recommended amounts of DNA to use are: 5µl of DNA for 96‐ well plates and 2.5µl of DNA (1‐40ng/ul) for 384‐ and 1536‐well plates. Genotyping should be carried out on at least 24 samples to ensure there are sufficient genotypes to show clustering. DNA may be either dried in the plate, or genotyped as liquid samples.

Negative controls It is recommended that two no‐template controls (NTCs) are included on each genotyping plate. A difference in fluorescent signal intensity between the presence and absence of template DNA allows improved confidence in the validity of the genotyping results.

Positive controls When validating an assay, and particularly when using an assay with low allele frequency, it is advisable to include positive controls i.e. DNA samples of known genotype. If such samples are not available, it is possible to synthesise an alternative positive control comprised of an oligonucleotide representing the amplicon region of the assay. The nucleotide representing the desired SNP allele should be specified in the synthesised oligonucleotide. Please contact KBioscience for more information.

DNA Drying ‐ the DNA samples can be dried into the wells of the PCR plate whereupon the reaction mix must be diluted to 1X concentration (rather than 2X for liquid samples) to compensate for the absence of liquid in the well (see Table 2). Drying the DNA samples in the plate wells is a useful technique when performing large‐scale genotyping, as it allows many plates of DNA to be prepared in advance without the concern of sample evaporation which would otherwise alter the final reagent concentrations.

The dried DNA samples are stable at room temperature for at least 3 months track changes turned on if protected from moisture. To dry the DNA samples, after arraying, the plates should be briefly centrifuged and placed in a drying oven at 60°C for 1‐2hours. Faster Drying will occur if the oven is fan assisted. A quick visual check is all that is required to ensure the samples are dry.

Liquid DNA‐ It is not necessary to dry the DNA samples in the PCR plate. Samples can simply be arrayed, the plate centrifuged, and genotyping of the samples carried out. However, the arrayed samples should be stored such that any evaporation is minimised, for the reason stated above. Sealing

the plate and storing at 4°C (short term) or ‐20°C (long term) is recommended if genotyping is not to be carried out straight away.

KASP Reaction Mix (in 96‐ and 384‐well plates) KBioscience recommends carrying out SNP genotyping using

total reaction volumes of 5µl for 384‐well or 10µl for 96‐well genotyping (see Table 2; these volumes can be reduced but the overall robustness of the data quality may also be reduced).



The volumes in Table 2 must be proportionally scaled‐up depending on the number of reactions required. The final MgCl2 concentration of KASP reaction mix at 1X concentration is 1.8mM, as this is optimal for the large majority of assays. However assays in especially A/T‐rich regions may require more MgCl2, which should be added to a final concentration of 2.2mM before use (i.e. an increase of 0.4mM, to increase the concentration from 1.8mM to 2.2mM). A simple to use spreadsheet that calculates these volumes can be downloaded from http://www.kbioscience.co.uk/download/index.html

Where the users prefers to dry down the arrayed DNA samples, the reaction mix must be diluted by the addition of water, to bring the overall final mix concentration to 1X. NB. Do not use the KASP chemistry at higher final concentrations than 1X as the concentrations of the PCR reagents is critical. Where a solution of DNA is used, the addition of water is not required, as the DNA volume itself will bring the final KASP reagent concentration to 1X.

Table 2. Constituent reagent volumes for making KASP genotyping mix in 96‐well reaction volume (5ul final volume) or 384‐well reaction volume (10ul final volume). *DNA samples diluted to final concentration of 0.5‐20ng per 5µl reaction.

All reagents should be briefly vortex‐mixed prior to use.

KASP Reaction Mix (in 1536‐well plates) Where genotyping is carried out in 1536 well plates, KASP 1536 Reaction Mix should be used in place of the standard mix. KASP 1536 Reaction Mix is specifically‐optimised for use with the very low well volumes in 1536 plates (see Related Products).

Dispensing the KASP Genotyping Mix samples Dispensing can be carried out robotically or manually with a suitable pipette, depending on plate type and sample number. KBioscience is happy to advise on liquid dispensing systems.

Plates and plate sealing KASP genotyping can be carried out in any plate format. Most commonly however, 96‐ and 384‐well formats are used. For 96‐ and 384‐well plate formats, KBioscience recommends use of the Flexiseal heat‐based plate sealer, however plate sealing can be achieved with any optically‐clear seal. For black 384‐ and especially 1536‐well plates, KBioscience recommends the Fusion Laser welding system. For further information on these and other KBiosciences products (see Related Products).

Thermocycling conditions The KASP chemistry can be used with any standard thermocycler. Similar results have been obtained on Peltier block‐based thermocyclers and the KBioscience Hydrocycler waterbath‐based thermal cyclers (see Related Products). KASP uses the following PCR thermal‐cycling program:

1. 94 °C for 15 minutes Hot‐start enzyme activation

2. 94 °C for 20 seconds Touchdown over 65‐57°C for 60 seconds 10 cycles (dropping 0.8°C per cycle)

3. 94°C for 20 seconds 57°C for 60 seconds 26 cycles

If using a Peltier block‐based thermocycler, ensure that the PCR plate type is correct for the block being used, as an incorrect fit can cause uneven PCR and variation of resultant data quality across the plate.

Plate Reading Most FRET‐capable plate readers (with the relevant filter sets) can be used in conjunction with KASP. Some plate readers can be set to read at a range of temperatures but elevated

temperatures (above 40°C), will lead to poor / no data (see the troubleshooting guide). Because of the underlying mechanism of the KASP chemistry, care should be taken that KASP genotypes are only analysed at (or around) ambient temperature. Whilst using real time PCR machines, plates should be read at ambient temperature after the completion of the PCR, run rather than using the real time data to generate end point curves.

Table 3. Excitation and Emission values for the fluors used in the KASP chemistry. *The excitation and emission values for CAL Fluor Orange 560 are the same as those of VIC / JOE.

KASP uses the fluorophores FAM and CAL Fluor Orange 560 for distinguishing genotypes. The passive reference dye ROX is also used to allow normalisation of variations in signal caused by differences in well‐to‐well liquid volume (see Figure 1). The relevant excitation and emission wavelengths are shown in Table 3 above.

If using a plate reader optimised for use with the dye VIC (e.g. Applied Biosystems), no modification of settings will be necessary as the excitation and emission values for VIC and CAL Fluor Orange 560 are extremely close. However, if required,

Table 2 KASP Genotyping mix assembly

Component Wet DNA method (µl)

Dry DNA method (µl)

DNA* 2.5 5 N/A N/A

KASP 2x Reaction Mix 2.5 5 2.5 5

Assay 0.07 0.14 0.07 0.14

H20 N/A N/A 2.5 5

Total reaction volume 5µl 10µl 5µl 10µl

Table 3 Excitation

(nm) Emission (nm)

FAM 485 520

CAL Fluor Orange 560* 534 556

ROX 575 610



KASP fluorescently‐labelled oligos can be made with most fluorescent dyes to customer specification.

Graphical viewing of genotyping data KBiosciences offer a data analysis software package either as part of a full Workflow Manager (Kraken) or a version with reduced functionality (KlusterKaller) (see Related Products). In KBioscience’s software, the FAM and Cal Fluor 560 data are plotted on the x‐ and y‐ axes, respectively. Inclusion of a passive reference dye (ROX) allows data to be normalised by dividing FAM and Cal Fluor 560 values by the passive reference value for that particular well, thus removing the variable of liquid volume and plate reader variation. Genotypes can then be determined according to sample clusters (Figure 1). The inclusion of a passive reference leads to tighter clustering and, as a result, more accurate calling of data.

Figure 1. Genotyped samples marked red are homozygous for the allele reported with CAL Fluor Orange 560, those marked blue are homozygous for the FAM allele; those marked green are heterozygous. The data shown in two cluster plots are the same but are shown without ROX normalisation (left) and with ROX normalisation (right).

Validating the genotyping process Before using the KASP genotyping chemistry for the first time, it is recommended (though not necessary) to contact KBiosciences for a free‐of‐charge validation kit. Using the validation kit, the following procedures will determine whether the plate reader and the PCR machine are functioning correctly for the KASP chemistry.

Determining correct functionality of the plate reader for the KASP chemistry The KASP validation kit contains three separate tubes of amplicons which have been labelled with the fluors used in KASP and therefore represent the three observable genotyping groups. The tubes are labelled ‘Fluor 1’ (FAM), ‘Fluor 2’ (CAL Fluor Orange 560) and ‘Fluor 1 / 2’ (FAM and CAL Fluor Orange 560). The appropriate volume (for the plate type) of each of these samples should simply be dispensed into the required PCR plate (several wells for each of the three samples) and the plate sealed, centrifuged and read on the plate reader (no thermocycling required). When viewed graphically, the reader output should reveal three tightly clustered, well‐separated groups of genotypes, demonstrating that the plate reader is correctly set up to read genotyped KASP samples correctly.

Determining correct functionality of the thermocycler for the KASP chemistry Please note these reagents require that they are thermal cycled. The KASP validation kit also contains three tubes labelled ‘test DNA samples’ along with another, small, 2‐D barcoded tube labelled ‘test assay’. When genotyped with the test assay, the three DNA samples (each dispensed into multiple wells), labelled ‘Genotype 1’, ‘Genotype 2’ and ‘Genotype 1 / 2’, will give rise to the three observable genotyping groups, respectively FAM (homozygotes), FAM / CAL Fluor Orange 560 (heterozygotes) and CAL Fluor Orange 560 (homozygotes).

Successful completion of these tests demonstrates that the plate reader and thermocycler are correctly set up and suitable for use with the KASP chemistry.

Troubleshooting guide

KBioscience Ltd. Unit 7 Maple Park Essex Road Hoddesdon EN11 0EX UK Tel: +44 (0) 1992 470757 Fax: +44 (0) 8700 511302 [email protected] www.kbioscience.co.uk

Description of problem Likely cause(s) and suggested solutions

Insufficient amplification

Poor amplification is characterised by genotyping groups moving more slowly than expected away from the origin, before resolving into separate clusters.

PCR cycle number If, after the recommended cycling protocol, the signature genotyping groups have not yet formed (i.e. amplification is incomplete), it is advisable to thermocycle the samples further and re‐read on the fluorescent plate reader. Three further PCR

cycles should be used (of the type of the last 26 cycles in section 7 i.e. 94°C for 20 seconds and 55°C for 60 seconds) followed by re‐reading on the plate reader. This re‐cycling process can be repeated until the desired grouping is achieved. The re‐ thermocycling / re‐reading process can be carried out on a plate that has been stored at room temperature for as long as one week.

G/C percentage Low G/C (G/C <30%) Where the G/C percentage of the assay oligos is low (G/C <30%) slow amplification can result. Increasing the final MgCl2 concentration in the reaction mix from 1.8mM to 2.2mM or even 2.5mM can compensate for this.

High G/C (G/C >70%) Assay oligos with a high G/C percentage (G/C >70%) can also be problematic. Where this situation is encountered, running the assay at the standard MgCl2 concentration (1.8mM) with the addition of 5‐10% DMSO to the final volume of the assay will usually provide a solution. When adding DMSO to the reaction, there is no need for a concomitant reduction in water volume to compensate for the disturbed reaction volume.

Low DNA concentration DNA concentration may be lower than expected causing samples to take longer to amplify. This can sometimes be addressed by simply cycling the samples further (see section above), though too many extra cycles may result in amplification of the negative control samples. It is preferable to repeat the genotyping with DNA in the recommended concentration range (1‐40ng/µl). It is recommended that the Picogreen dsDNA fluorescent quantification system be used in preference to spectrophotometric methods, as the latter tends to overestimate DNA concentration in this range. Alternatively, a more pragmatic approach can be used: representative test samples can be genotyped at a variety of dilutions and the optimal dilution selected.

High DNA concentration Conversely, samples of very high DNA concentration can also cause poor / no amplification. Where this is the case, it is because there is also a high concentration of PCR‐inhibiting contaminants present. The solution to this is simply to dilute the samples such that the contaminants are diluted to non‐inhibitory levels.



DNA samples dissolved in a buffer that contains EDTA After purification, DNA samples are often eluted / diluted in TE buffer, which contains EDTA. EDTA chelates Mg 2+ ions leading to insufficient Mg 2+ for the reaction to proceed. However this can be overcome by increasing the MgCl2 to compensate. For example, if the DNA samples contain 1mM EDTA, in a 4µl reaction, where the DNA samples account for 50% of the reaction volume, addition of extra Mg 2+ to the amount of 0.5mM will resolve the issue. A greater problem is when the DNA samples have been diluted such that they contain non‐uniform concentrations of EDTA. In such a case, EDTA should be added to the NTC wells.

Assay mix storage conditions Assays that have been subjected to multiple freeze/thaw cycles or otherwise stored incorrectly can lead to poor amplification in the subsequent reaction.

It is advisable to divide the mix into aliquots to avoid multiple freeze/thaw cycles. Incorrect storage, or multiple free/thaw cycles will negatively affect the performance of the assay.

Aliquots of assay can be safely stored at 4°C for 1‐2 weeks, approximately 1 year at

‐20°C or indefinitely at ‐80°C.

Reaction Mix storage conditions As with assay mix storage, it is essential that the Reaction Mix is stored as directed.

Amplification too fast High DNA concentration If the reaction is proceeding too quickly, it may be an indication of too much DNA in the reaction, causing samples to amplify more quickly than expected. To resolve this, dilute the DNA samples to the recommended range of 1‐40ng/µl (as determined by Picogreen).

Magnesium concentration Alternatively, it may be that there is too much magnesium in the reaction for the G/C percentage of the assay being genotyped. Inclusion of 5 ‐10% of DMSO will generally rectify this. Alternatively reduce the amount of DNA in the reaction or reduce the number of PCR cycles in the second phase from 26 to 20 (see the KASP genotyping manual), such that the total number of cycles becomes 30.



Scattered grouping of genotyping calls Scattering of the genotyping groups can be caused by a variety of factors:

Cross‐contamination between the DNA samples on the plate can cause genotypes to cluster outside the expected genotyping groups.

Too much magnesium for the SNP being assayed is another factor that can lead to scattered grouping. A reductive magnesium titration may resolve the issue.

Poor DNA quality (and inconsistent quality across the plate) can lead to this effect.

TempliPhi WGA DNA used as the template DNA. In some cases scattered heterozygous groups can be seen with WGA amplified DNA. Re‐WGA with a DOP based method may well rectify this, such as the KlearAmp product available from KBioscience.

Little / no separation of the hetero‐ and a homozygote group

An imbalance in the allele‐specific primers can result in the heterozygous group migrating towards one of the homozygote groups, making genotype scoring difficult.

Such an imbalance can occur for a variety of reasons: poor synthesis of one of the allele‐specific primers, accidental addition of an insufficient quantity of the allele‐ specific primers when making the assay, or more efficient amplification of one allele‐specific primer compared to the other. Regardless of cause, the problem can be ameliorated with an increase in the concentration of the alternate allele‐specific primer. In the example shown on the left, allele‐specific primer 1 leads to the blue group, allele‐specific primer 2 giving the red group. In increase of around 40% of the AL‐spec 1 primer would cause the green heterozygous group to move to a more central location on the plot, allowing more confident scoring of the genotypes.

Heterozygous group migrating to a much lower position (with respect to the homozygous groups) on

the x‐ or y‐axis than expected

The cluster plot shows an example of the effect that occurs when the assay aliquots are thawed without subsequent mixing (or not mixed before they are aliquoted) causing the forward primers to saturate the fluorescent quenching system. The effect is characterised by the genotyping clusters seemingly adopting incorrect positions on the plot (despite often clustering well into groups).

Whilst mixing the assay aliquots after thawing and before use is strongly recommended for the reasons described, it is worth noting that the problem could also have occurred when assembling the assay if the constituent primers were hydrated and then frozen /thawed without proper mixing.

The solution to the problem is to remake the assay with sufficient mixing of oligonucleotides



Heterozygous cluster too close to the origin A related problem to that described above is encountered when the homozygous cluster positions appear to amplify correctly but the heterozygous group amplifies less than might be expected, remaining close to the origin. This is also caused by the forward primers saturating the quenching system and can be resolved by remaking the assay with a reduced concentration of both forward primers (instead of 12µM, try 8µM).

Too many genotyping groups

It is possible to observe more than three clusters in a genotyping plot.

Multiple genotyping clusters caused by the presence of polymorphism(s) within primer binding site(s). In diploid organisms, more genotyping clusters can be observed when primers (forward or reverse) are designed such that they bind to a region containing other polymorphism(s). Such non‐target polymorphism causes differential PCR amplification efficiencies, making confident allocation of the genotypes less reliable. In the example shown, the reverse primer was designed in a region containing another polymorphism. The issue can be resolved by re‐locating the primer(s) to a region containing no polymorphisms, if one is available. Alternatively, a wobble base can be inserted in the primer sequence at the site of the neighbouring (non‐assayed) SNP.

Duplicated diploid or polyploid genomes However, when assaying duplicated‐diploid plant genomes, up to five genotyping clusters are potentially possible and does not mean that the system is misreporting.

Fewer genotyping groups than expected Some assays will report just one genotyping cluster (monomorphic genotyping), which can be a genuine result if the population being analysed contains only one genotype with respect to the (probably low frequency) SNP being studied. However, monomorphic data can also occur because the SNP is not real.

Another reason for one group appearing can be that the primers are hybridising to a homologous region in the genome. Redesigning the assay to the opposite strand can often resolve this.



Some samples not amplifying at all

This is characterised by some samples amplifying as expected, but some not amplifying at all, clustering instead around the origin.

Inconsistent DNA quality / quantity

If some of the samples on the plate are of a much lower concentration and / or purity that the other samples, the observed effect may be seen due to some of the samples amplifying and some not. This is more likely if the DNA samples being analysed on the same plate are from different sources.

Arraying / dispensing

This pattern can be seen if the arraying of the DNA into the PCR plates was done carried out correctly (such that there is little / no DNA in some wells supposed to contain it.

Alternatively, poor dispensing of reaction mix into the wells could lead to the same effect. This possibility can be checked by viewing the ROX levels in each of the wells across the plate as this relates directly to the volume of the assay / reaction mix combination that was dispensed into the plate.

Misreading of data Even if the KASP genotyping reaction has proceeded correctly, the resultant data can still appear incorrect. Possible causes are:

Plate reader fault The plate reader may have developed a fault

Incorrect plate positioning Check that the plate is inserted correctly into the plate reader (or if not correctly placed on a plate holder).

Temperature of plate reading The plate reader could be reading at an elevated temperature. Because of the mechanism of action of KASP (see the KASP genotyping manual), KASP data must be read at 40degC or below (ambient temp is preferable). Reading data at temperatures higher than this will dissociate the quencher‐bound oligo from any remaining unincorporated fluor‐bound oligo and cause a meaningless signal.

Genotyping groups too close together / merging Specificity I Genotyping groups close together indicate that the specificity of the KASP reaction is not sufficient. In this instance, the two allele specific primers are, to some extent, able to bind to each others’ sites and hence the genotyping cluster positions are closer to each other than they should be. Experimentally increasing in the annealing / extension temperature in the thermo‐cycling program will solve this problem as it will force improved specificity.



One genotyping group in the top right hand area of the plot

Specificity II Where only one the observed genotyping group appears, the problem is related to specificity. In the case shown in the diagram, the assay is completely non‐specific, i.e. the allele‐specific primers are not able to bind specifically, causing a completely mixed fluorescent signal. This situation requires a re‐design of the assay. For complicated homologous genomic sequence please contact KBioscience as we are often able (with some work) to produce a working assay.

Skewed positioning of genotyping groups Homology The skewing effect is caused by binding of the KASP primers to a sequence homologous to the one intended for analysis. If the KASP primers are able to bind elsewhere in the genome, amplification will occur there as well. However, the SNP may well not exist at the alternate binding site(s) so the eventual signal output of the assay will be influenced, causing the skewed grouping effect.

KASP primers binding in identical regions The problem occurs because the region being assayed is not unique in the genome. However, small sequence differences between the homologous sites often exist. The solution therefore is to redesign the assay taking advantage of any sequence differences between the desired region and the others, such that the 3’ end of the common (reverse primer) is unique to the region being analysed. KASP primers binding in similar regions The KASP primers may bind in very similar regions in the genome (rather than identical regions) in which case the issue is specificity and it is possible that the situation could also be resolved by simply increasing the annealing / extension temperature.

Alternatively, a two‐step solution can be used whereby an initial PCR is carried out to specifically amplify the region of interest, prior to genotyping. Providing the primers chosen for the initial PCR are known to uniquely amplify on the region of interest, the resultant amplicon from the initial PCR can then be used as the template DNA. A variety of experimental dilutions of the amplicon DNA may be required to find the best concentration to use.

Appendix ‐ Related Products

Ordering information can be

found on our website


Reagents KASP Reaction Mix The patented KASP SNP genotyping system is a cost‐effective, homogeneous, FRET‐based genotyping system. Coupled with the power of competitive, allele‐specific PCR, KASP offers a truly superior system for determination of SNP (or insertion / deletion) genotypes in your laboratory. KASP 1536 Reaction Mix KBioscience has developed a version of KASP Reaction Mix optimised for use with ultra low volume 1536‐well plates. KlearKall KlearKall mastermixes contain KlearTaq DNA polymerase in a highly optimised reaction buffer, along with dNTPs required to perform both general and real‐time PCR reactions. KlearKall mastermix compared favourably to Applied Biosystems’ TaqMan Mastermixes. KlearTaq KlearTaq Hot Start DNA polymerase is a highly specific robust enzyme that can be used for many PCR applications. KlearTaq is suitable for all PCR applications and is a direct replacement for all leading commercial Hot Start Taq polymerases. KlearGene KlearGene is KBioscience’s proven, proprietary DNA purification chemistry, available now in kit form. KlearGene Blood XL KlearGene Blood XL is a DNA purification kit for use with blood volumes of 1‐10ml. The kit gives the highest DNA yield and purity on the market, whilst being quick, convenient and cost effective. KlearGene Miniplant KlearGene Miniplant has been developed for the extraction and purification of DNA from plant leaves or seeds, amenable to high throughput / automation. The system can be specified in either

magnetic bead (96‐well) or spin‐through plate (96‐ or 384‐well) formats.

KlearAMP KlearAMP is KBiosience’s chemistry for the whole genome amplification (WGA) of DNA, using the primer extension pre‐amplification (PEP). This robust system allows approximately 500‐1000‐fold amplification of DNA mass.

Instrumentation SNPLine This is a replicate of KBioscience’s SNP genotyping pipeline in a customer’s lab to enable high throughput, flexible SNP genotyping. SNPLine can be specified to your laboratory environment and in its basic form can generate around 50,000 genotypes per day. It is fully configurable to allow throughputs as high as 500,000 genotypes a day.

Plate sealers Flexi‐seal The Flexi‐seal is our next‐generation thermal plate sealer. We have taken a fresh look at the design of thermal plate sealers and re‐designed the instrument completely, improving on pre‐existing designs. K‐Seal The K‐Seal plate sealer is a low‐cost thermal plate sealer and KBioscience’s second generation product for this application. The K‐Seal, seals plates repeatably by electronically controlling the heat, time and pressure at which the plate is sealed (unlike standard manual plate sealers where these parameters are user‐controlled, often resulting in imperfect seals). Fusion Laser Sealer KBioscience has developed an automated laser plate sealer that utilises the process of transmission diode laser welding. It is able to seal virtually any plate density from single tubes to 3456‐well plates.





Hydrocycler – Thermal Cycler The HydroCyclers are water bath based thermal cyclers for high‐throughput PCR applications. HydroCycler 16 / 32 ‐ plate The HydroCycler ensures both rapid thermal transfer and the best possible well‐to‐well / plate‐to‐plate uniformity. The Hydrocycler 16‐ and 32‐plate instruments are tried and tested, having been placed in many labs throughout the world. Hydrocycler 4 The HydroCycler 4 represents a cost‐effective alternative to the Biorad Tetrad four‐block thermal cycler, for both microtitre plates and large volume Emulsion PCR.

Software

Kraken Kraken is KBioscience’s dedicated Workflow Manager for all genotyping, sequencing & DNA extraction work. Kraken has been developed by scientists for scientists and coded accordingly. KlusterKaller KlusterKaller is a semi automated calling software package for cluster‐based genotyping data. It has the functionality of the genotype calling module built in to our Kraken package. It may be used with customer genotyping projects or with KBioscience genotyping data. SNPViewer SNPViewer is a free‐of‐charge tool for visualising your genotyping data. This allows graphical viewing of the clusters that group the allele calls.

Consumables Microtitre plates for PCR KBioscience offers a range of 384‐ and 1536‐well PCR plates. As with the majority of our products the KBioscience range of PCR plates are in routine daily use in our Laboratory Services division. Seals KBioscience offers foils and films for thermal, laser and adhesive sealers.

Laboratory Services Genotyping KBioscience offer a very competitively priced, in‐house genotyping service based on the KASP chemistry. Assay design KBioscience offers a genotyping assay design service (drawing on its long experience). The service is available at two levels, depending on cost: KASP‐on‐Demand (KOD) KBioscience will design, validate and (where necessary) optimise assays for the customer. KASP‐by‐Design (KBD) Same service as KOD but reduced cost as the assay is assembled without validation. DNA extraction/ purification KBioscience offers a highly‐competitive DNA extraction and purification service based on our proprietary KlearGene chemistry. The KlearGene‐purified DNA is of the highest quantity / quality and is rigorously quality‐control checked.





Whole Genome Amplification (WGA) KBioscience offers a service to carry out whole genome amplification of customers’ DNA samples using a combination of our proprietary KlearAmp WGA chemistry and HydroCycler high‐throughput amplification. Contact For more details or for technical assistance, please contact: [email protected] www.kbioscience.co.uk

KASP Manual

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