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KASP Workshop

KASP Workshop. Science for a safer world. Overview of topics to be covered. Sequence submission Running KASP genotyping reactions Different Platforms Troubleshooting KASP data- where support is available Design solutions. Sequence submission

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KASP Workshop

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  1. KASP Workshop Sciencefor a safer world

  2. Overview of topics to be covered • Sequence submission • Running KASP genotyping reactions • Different Platforms • Troubleshooting KASP data- where support is available • Design solutions

  3. Sequence submission • how to submit sequence information for KASP assay design

  4. Sequence submission • For KASP assay design, sequence information should be submitted to LGC using the appropriate SNP submission template. • Please note that we require 50 bases of sequence on each side of the submitted polymorphism. • Assay name field • Use this field to provide a unique identifier for the assay. • Can be up to 20 characters in length • Use alphanumeric and underscore characters only e.g. ABCD1234_ab • Do not begin the name with a hyphen (-)

  5. Formatting guidelines for SNP submission • Sequence information • Sequence orientation • 5’ – 3’ orientation • Upper case and lower case bases • Use upper case characters (ATCG). • Lower case characters denote uncertain bases (atcg) • Identify the target SNP using square brackets • E.g. ATCGATCGATCG[A/G]ATCGATCGATCG • Identify the target InDel using square brackets • E.g. ATCGATCGATCG[A/-]ATCGATCGATCG • Surrounding SNPs and InDels • These should be identified using IUPAC codes • Avoiding bases • Replace bases to be avoided with an N

  6. Acceptable submission formats

  7. Unacceptable submission formats

  8. Marking special requirements

  9. Surrounding polymorphisms Reverse primer: 5’ CAAATATATTCTTTACCTCCATGGYGGTAA 3’

  10. Surrounding polymorphisms • Assay run without degenerate base – split heterozygous group • Assay run with degenerate base included in the reverse common primer – heterozygous group no longer splits

  11. Three adjacent SNPs • If the region of interest has 3 adjacent SNPs, three KASP assays will be required XXXXX [C/A][A/G][G/T] XXXXX • For each assay • submit the SNP of interest in square brackets • submit the surrounding SNPs using IUPAC codes SNP 1 XXXXX [C/A] R K XXXXX SNP 2 XXXXX M [A/G] K XXXXX SNP 3 XXXXX M R [G/T] XXXXX

  12. Running KASP genotyping reactions • How to set-up and run KASP genotyping reactions

  13. Customer requirements • FRET-capable plate reader • PCR microtitre plate (96-, 384- or 1536-well) • DNA samples at an appropriate concentration for KASP reactions • Minimum 22 DNA samples per assay • PCR grade water • Run 2 NTC wells per assay • Optically clear seal

  14. KASP genotyping workflow

  15. Setting up a KASP genotyping reaction • DNA samples at the appropriate concentration for KASP • 5 – 50 ng per reaction (human genome size) • 96- or 384-well PCR plates • Arrayed DNA can be dried down or stored in hydrated form

  16. Setting up a KASP genotyping reaction • All reagents should be vortexed before use • Prepare sufficient mix for the number of reactions you intend to perform, plus additional dead volume • KASP Master mix must have 1x final concentration

  17. Setting up a KASP genotyping reaction • Add the required amount of genotyping mix to each DNA sample in the reaction plate • Pipette • Dispensing robot

  18. Setting up a KASP genotyping reaction • Seal the plate with an optically clear seal • Centrifuge the plate briefly to ensure all reaction volume is at the bottom of the wells

  19. Setting up a KASP genotyping reaction • KASP chemistry can be used with any standard thermal cycler • The KASP thermal cycle is a two-step PCR program • Denaturation • Annealing and extension – all in one step. • 10 cycles of touchdown PCR • Gradually decrease the annealing temperature each cycle to increase specificity of the reaction. • 26 cycles of standard PCR

  20. Setting up a KASP genotyping reaction Stage 1 Stage 2 Stage 3 94oC 94oC 94oC 00:20 15:00 00:20 61-55oC 55oC 1:00 1:00 Enzyme activation Hot Start 1 cycle PCR cycling 26 cycles Touchdown PCR 10 cycles This temperature decreases by 0.6oC each cycle 61-55oC

  21. Setting up a KASP genotyping reaction • Read the plate in a FRET-capable plate reader • All plates should be read below 40ºC • To analyse the data, import it into a genotype cluster analysis software package • Data can be plotted and genotyping clusters analysed

  22. Setting up a KASP genotyping reaction Homozygous HEX • Each data point represents an individual DNA sample • Samples of the same genotype cluster together HEX allele reported on the Y axis Heterozygous Homozygous FAM FAM allele reported on the X axis

  23. Recycling • Efficiency of KASP dependent on a number of factors • DNA concentration • Composition of DNA sequence surrounding polymorphism • Different KASP assays will reach completion at different rates • Can require additional cycles to product clear genotyping clusters • Reaction plate should be thermally cycled further • “Recycling”

  24. Recycling protocol and effects • Two recycling programs result in tight clustering • LGC recommend up to 4 recycling programs

  25. Thermal cycling conditions • Standard KASP thermal cycling protocol • 61-55ºC touchdown protocol (36 cycles) • If recycling improves clustering, add cycles to initial thermal cycle e.g. run 39 cycles each time instead • Alternative KASP thermal cycling protocols • 68-62ºC touchdown protocol • Annealing temperatures >65ºC (high GC%) • 2-step 57ºC protocol • Annealing temperatures <60ºC (low GC%)

  26. 68-62ºC touchdown protocol • Increase primer stringency • May improve results for assays that behave in a non-specific manner when cycled at lower temperatures

  27. 2-step 57ºC protocol • Reduce initial primer stringency • May improve primer annealing for low GC% primers

  28. Platforms • The range of platforms that KASP can be run on

  29. Running KASP on qPCR instruments Illumina: Eco RT-PCR BIO-RAD: CFX, iCycleriQ, MyiQ & MyiQ 2 ABI: 7000-series, Viia7, Quant Studio-series Agilent: Mx3000 / 3500P & Mx4000P Thermo Scientific: PikoReal Roche: LC480 vI and vII, LC 2.0 & LC96

  30. Manuals available and coming soon Currently available: • ABI 7900 • ABI 7500 • ABI Viia7 • ABI StepOne and StepOne Plus • BIORAD CFX • Agilent Mx-series • Illumina EcoRT • Roche LC480 (vI and vII) • Workaround document for LC96 Coming soon: • Fluidigm (updated)

  31. Troubleshooting KASP data • How to troubleshoot data and improve results

  32. Common causes of unexpected results • Storage – aliquot, store in dark (Master mix only) • Thaw fully before use • Mix at every stage • DNA concentration • DNA quality • Thermal cycle correctly programmed • Accurate pipetting • KASP Master mix – appropriate ROX level • Plates sealed sufficiently • Plate read below 40ºC • Correct excitation and emission values

  33. Common assay optimisations • Recycling to reach reaction completion • Altering the thermal cycling conditions • 68-62ºC touchdown protocol • 2-step 57ºC protocol • Altering the magnesium concentration of the reactions • Target SNPs in low GC% regions may require more magnesium • Increase incrementally ~0.3 mM • Adding DMSO • Target SNPs in high GC% regions may benefit from addition of DMSO • Add 5-10% DMSO to the final volume of the reaction • Use standard magnesium concentration

  34. Insufficient amplification • Insufficient PCR cycling • Perform recycling • Low GC% • Add magnesium • High GC% • Add DMSO • DNA concentration too low • Use appropriate DNA concentration • Perform recycling • DNA concentration too high • Dilute to minimise effect of inhibitors • Presence of EDTA • Add magnesium Clear genotyping clusters do not form after completion of the thermal cycling protocol

  35. Scattered grouping of genotyping calls • Cross contamination between DNA samples • Check extraction procedures • Check storage protocols • Inconsistent DNA quantity and quality across plate • Normalise samples before performing genotyping • Magnesium concentration is too high for the GC% of the assay • Reductive magnesium titration • Non-optimal cycling conditions • Use alternative conditions (use GC% content as a guide) Genotyping clusters are not distinct or tight, and data points are scattered

  36. Example of optimisation – 68-62ºC touchdown protocol A B 61-55ºC cycling conditions 68-62ºC cycling conditions • Assays that show a lack of specificity can often be improved by increasing the annealing/extension temperature of the thermal cycling conditions from the standard 61-55ºC touchdown to a 68-62ºC touchdown protocol. • Image A - a SNP assay with poor clustering when run with the standard cycling conditions. • Image B - how the clustering data improves when the same assay is run with the 68-62ºC touchdown conditions.

  37. Example #2 of optimisation – 68-62ºC touchdown protocol A B 61-55ºC cycling conditions 68-62ºC cycling conditions • Human assay • Changing the cycling conditions – increased specificity • 3 clear genotyping clusters

  38. Little or no separation of the heterozygous and a homozygous group • More efficient amplification of one of the allele-specific primers compared to the other • KOD upgrade • biased assay • assay re-design (design to opposite strand) The heterozygous group migrates towards one of the homozygous groups, making genotype scoring difficult

  39. Heterozygote group migrates to a lower position on the X- or Y-axis than expected • Assay has been aliquoted without sufficient mixing, or thawed without sufficient mixing. • Mix assay aliquots thoroughly after thawing and before use • Allele-specific primers saturate the fluorescent quenching system. • KOD upgrade • assay re-design (design to opposite strand) Genotyping clusters are distinct but the heterozygous group does not appear in the expected position (lower than the homozygous group)

  40. Too many genotyping groups • Presence of polymorphism(s) within the primer binding site • Try 2-Step 57ºC protocol (diploid species only) • Identify the additional polymorphism(s) using resources available for your species • KOD upgrade • degenerate base for neighbouring SNP More than three genotyping clusters are visible on the plot

  41. Fewer genotyping groups than expected • Population is monomorphic • result is genuine • Minor allele frequency • include a positive control on every plate • Primers annealing to homologous region • identify bases and order an anchored design • KOD upgrade • Polymorphism is not real • choose an alternative polymorphism for analysis Monomorphic results on the genotyping plot

  42. Some samples do not amplify • Inconsistent DNA quantity or quality • normalise DNA samples • consistent extraction procedures? • Arraying of DNA was not performed as expected • investigate arraying procedures • Poor dispensing of reaction mix into wells • Investigate the ROX levels across plate • Target sequence / allele is not present in some samples • Re-examine sequence Some samples amplify as expected, others cluster at the origin (pink)

  43. No pattern to the results • Plate reader fault • re-read plate • Arraying of DNA was not performed as expected • Investigate arraying procedures • Poor dispensing of reaction mix into wells • Investigate the ROX levels across plate • Target sequence / allele is not present • Re-examine sequence • Very slow amplification • Recycle • Use more template DNA Scattered datapoints

  44. Clusters draw together on the plot Distinct genotyping groups are apparent but their position on the cluster plot is skewed The genotyping groups are drawing too closely together, or merging, on the plot All of the datapoints cluster in the top right of the plot • Homology – primers cannot bind specifically to the region of interest

  45. Design Solutions - Dealing with homology

  46. Homology • Primers are annealing to identical homologous regions • Allele-specific primers are not able to bind specifically to their target template • experimentally increase the annealing temperature to force improved specificity (annealing stringency) • Add 5% DMSO – may work • KOD upgrade • anchored primers – perform a BLAST, identify unique bases

  47. Assay design solutions • KASP is great for finding solutions for polyploid organisms • Similar solutions can be used for regions of high identity or pseudogenes within genome. • Assays can be designed within highly polymorphic regions by adaption of primers. • Large InDel region can be screened for. • KASP is PCR so can be optimised in similar ways to increase the AT / GC range

  48. Solutions for homology and polyploid organisms. • Solutions to tricky problems can often be found for some assays by anchoring • More solutions come from having the correct sequence giving more options for designing better primers. • Primers with degeneracy can be used to avoid associated problem of highly polymorphic regions • Designing in either orientation is often a very easy solution for genomes that are not well annotated Chicken is a very good example and works well.

  49. Solutions for homology and polyloidy Region of interest is the top G/T (K) SNP. The region is highly repetitive throughout the genome. Using the unique base T the reverse common primer is 3 prime end ‘anchored’ to differentiate and isolate the correct SNP.

  50. Designing in the opposite orientation • In some instances where genomes are not well annotated, designing in the opposite orientation can solve assay problems. This is usually caused by unknown polymorphism surrounding the SNP of interest. Assay 471 – Unknown SNP

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