Rotor-Gene Q — Pure Detection - University of Hong...
Transcript of Rotor-Gene Q — Pure Detection - University of Hong...
Sample & Assay Technologies
Rotor-Gene Q — Pure Detection
High Resolution Melting
From Assay to Analysis
Nan Fang, Ph.D.
Senior ScientistR&D
QIAGEN
Sample & Assay Technologies
Overview
.Agenda
� Introduction into HRM� The principle� Advantages� Applications overview
� HRM in detail� Analysis� Dye
� Critical factors� Reaction chemistry� Instrument and software� Cycling conditions� Assay design� Template
� Application data and summary
The applications presented here are for research purposes. Not for diagnostic use.
Sample & Assay Technologies
The HRM principle
� HRM characterizes double-stranded PCR products based on the melting behavior� Different DNA sequences melt at different specific temperatures
� This is measured in real time using special intercalating dyes� The fluorescence decreases during DNA dissociation
� The HRM software plots fluorescence decrease vs. temperature
Temperature
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Sample & Assay Technologies
Advantages of HRM
� Enables highly specific and accurate detection of genetic variations
� Allows further downstream analysis of amplicon (e.g. by sequencing)
� Less expensive than alternative methods
� Easy to use
� Fast
� Flexible
� Versatile
Sample & Assay Technologies
HRM applications
� SNP analysis
� Mutation detection and scanning
� Pathogen detection
� Methylation analysis
� STR and VNTR analysis
� Quantification of copy number variants and mosaicism
� …
Sample & Assay Technologies
HRM – An emerging technology
HRM citations/year*
*source: google scholar
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Ririe, Rasmussen and Wittwer Product Differentiation by Analysis of DNA MeltingCurves during the Polymerase Chain ReactionAnal. Biochem., 1997, vol. 245
HRM is expected to become a standard technology for genotyping applicationsHRM is expected to become a standard technology for genotyping applications
Sample & Assay Technologies
Overview
.Agenda
� Introduction into HRM� The principle� Advantages� Applications overview
� HRM in detail� Analysis� Dye
� Critical factors� Reaction chemistry� Instrument and software� Cycling conditions� Assay design� Template
� Application data and summary
Sample & Assay Technologies
HRM Analysis - Five steps from PCR to result
Step 1: AmplificationWas the amplification successful?
Step 2: Melt curve analysisCheck to verify amplification specificity
Step 3: NormalisationSelect suitable samples and analysis range
Step 4: Difference plotSelect the reference genotype
Step 5: Autocalling genotypesunknowns will be either related to known genotypes or will be marked as variation
Sample & Assay Technologies
HRM and SNP genotyping
SNP Class* Base Change Typical TM Shift Abundance (in humans)
I C/T and G/A Large>0.5°C
Very Small <0.2°C
64%
II C/A and G/T 20%
III C/G 9%
IV A/T 7%
.SNP (= Single Nucleotide Polymorphism)
� Single nucleotide variation
� Can have major impact on how individuals respond to
� Disease
� Environmental factors (e.g. bacteria, viruses, chemicals)
� Drugs and other therapies
� SNP maps help to identify multiple genes associated with complex diseases (e.g. cancer, diabetes, vascular disease)
Source: Wikipedia
Reliable SNP genotyping requires clear resolution of ∆TM < 0.2°CReliable SNP genotyping requires clear resolution of ∆TM < 0.2°C
* Classification according to Venter et. al., 2002
Sample & Assay Technologies
Traditional dye technology for melting analysis
SYBR® Green I
Some dye molecules relocate as melting begins and do not contribute to fluorescence decrease
. SYBR™ Green I is toxic to PCR,so low concentration is needed
. Unsaturated binding may allow dye relocation during melts, making it less suitable for HRM
Non saturating intercalating dyes,e.g. SYBR Green
Unoccupied positions
Sample & Assay Technologies
Novel dye technology for melting analysis
Saturating intercalating dyes,e.g. EvaGreen
Dye saturation leaves no room for relocation events during melting
. “Saturation” dyes are much less toxic, so concentration used can be higher
. This reduces dye relocation events and improve melting resolution
All positions occupied
Saturating dyes like EvaGreen are required for high resolution melt analysisSaturating dyes like EvaGreen are required for high resolution melt analysis
Sample & Assay Technologies
Overview
.Agenda
� Introduction into HRM� The principle� Advantages� Applications overview
� HRM in detail� Analysis� Dye
� Critical factors� Reaction chemistry� Instrument and software� Cycling conditions� Assay design� Template
� Application data and summary
Sample & Assay Technologies
The amplification step in HRM analysis
� Specific PCR products are vital for optimal results in HRM
� Classical melt analysis is recommended in HRM method development
to monitor specificity of PCR amplification
High quality PCR is imperative for good HRM resultsHigh quality PCR is imperative for good HRM results
specific product unspecific products
Reactionchemistry
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Components of the Type-Itand Epitect HRM kits
.Enzyme: HotStarTaq Plus DNA Polymerase� Activation within 5 minutes� Unmatched specificity and sensitivity
.Buffer:� Unique combination of K+ and NH4
+ ions� High specificity� No optimization of PCR parameters necessary
Dye: EvaGreen� Saturating dye� Distinct melting curves
.Q-Solution� Improves amplification of difficult loci
.
Reactionchemistry
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Successful genotyping of class IV SNP Reactionchemistry
Only optimized chemistry allows clear resolution of minute TM differencesOnly optimized chemistry allows clear resolution of minute TM differences
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Instrumentation prerequisites for HRM analysis
� Temperature
� Thermal variability from sample-to-sample must be minimal
� RGQ has an optimal temperature uniformity of 0.01°C
� Fluorescence
� Low “noise” from detector system
� High stability of excitation light and uniform illumination
� High intensity excitation tuned to the optimal dye wavelength
� RGQ: low-noise photomultiplier & and high power HRM LED
� Combined prerequisites
� High data density i.e. high number of fluorescence data points
per degree thermal transition
� RGQ with up to 50 points/°C (0.02°C resolution)
Instrument precision is imperative for accurate HRM resultsInstrument precision is imperative for accurate HRM results
Instrumentation
Sample & Assay Technologies
Instrument benchmarking by SNP differentiationRotor-Gene with best performance of all cyclers Instrumentation
Two genotypesdistinguished
Comparison of various block cyclers and the Rotor-Gene for HRM performanceFrom Herrmann et al; Clinical Chemistry 53, 2007; 1544-1548
Block cycler with temp. uniformity: ± 0.4 °CHigh fluorescence noise
� all traces intercalating� no genotypes resolved
Block cycler with temp. uniformity: ± 0.5 °C
� several traces intercalating� only one genotype resolved
Block cycler with temp. uniformity: ± 0.2 °C
� homozygous tracesintercalating
� only two genotypesresolved
Rotor-Gene HRMwith temp. uniformity: ± 0.01 °C
� all traces separated� all 4 genotypes unambiguously
resolved� only real-time cycler resolving
class IV SNP homozygous samples
Sample & Assay Technologies
Combined mode of cycler and chemistry
A: EpiTect HRM Kit and Rotor-Gene Q B: HRM Kit and Instrument from Supplier AII
Instrumentation &chemistry
Only the combination of optimal chemistry and instrumentation ensures reliable resultsOnly the combination of optimal chemistry and instrumentation ensures reliable results
Confidence threshold:95%
Sample & Assay Technologies
Maximum flexibility for analysisRGQ software methods fro HRM analysis
� All melt modules can be used with HRM datasets� Melt analysis
� Normalisation & Differentiation� Preferably used during method development
� Standard HRM analysis� Normalisation & Subtraction� Genotyping via autocalling
� ScreenClust HRM analysis� Normalisation plus statistical analysis� Large & difficult datasets and screening applications
� Check and validate your data with more state-of-the art algorithms on the RGQ� Find new HRM results with the unique analysis methods such as ScreenClust
Concentration Analysis
End-Point (+/-) Analysis
Scatter Graph
Allelic Discrimination
REST
Comparative Quantitation
��CT Relative Quantitation
2 Standard Curves Relative Quantitation
Standard Curve Absolute Quantitation
ScreenClust HRM Analysis
Standard HRM Analysis
Melt Analysis
qPCR modules Melt modules
Software
Sample & Assay Technologies
Standard HRM data analysis workflowAll Current Software
Normalisation Difference plot
Autocalling genotypes
Raw data
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Rotor-Gene Q Operating Software
Software
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Difference plot Auto calling Results
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� Genotypes automatically called by comparing samples and controls in difference plot
� Confidence value (%) is calculated as integrity check for auto called results
� Samples below user-set confidence threshold will be marked as a variation (here: 95%)
Control (100%)
Variation (< 95%)
Rotor-Gene Q software conveniently identifies known genotypes or variations Rotor-Gene Q software conveniently identifies known genotypes or variations
Data analysisAutocalling & confidence percentage
Software
Sample & Assay Technologies
Why is the standard HRM workflow often unsatisfactoryUnmatched needs for HRM analysis
Standard HRM software packages:
� plot and analyse only melt curve shape and position� interpretation based on operator experience� no standardisation� manual intervention limits screening applications � autocalling often fails for minute difference samples
� lack rigorous statistical interpretation of data sets� limited confidence and comparability of results
� always need control samples for genotyping� limits the number of unknowns in a run� new polymorphisms often stay undetected
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Real-life performance testHow many genotypes do you see?
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Sample & Assay Technologies
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Difference Plot – 1st possibility
Rotor-GeneOperatingSoftware
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Sample & Assay Technologies
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Real-life performance testHow many genotypes do you see?
Difference Plot – 2nd possibility
Rotor-GeneOperatingSoftware
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Sample & Assay Technologies
Real-life performance testHow many genotypes do you see?
Difference Plot – 3rd possibility
Rotor-GeneOperatingSoftware
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Real-life performance testHow many genotypes do you see?
ScreenClust HRM Cluster Plot
Rotor-GeneScreenClust HRM Software
unpublished customer field test data
Only ScreenClust HRM Software accurately separates all genotypesStandardized process to retrieve the correct result
Only ScreenClust HRM Software accurately separates all genotypesStandardized process to retrieve the correct result
Sample & Assay Technologies
Rotor-Gene ScreenClust HRM SoftwareHow does it work
.Uses innovative statistical methods� Analyzes the differences between all samples within one HRM run � Groups all samples according to the alleles into clusters � Displays the clusters graphically
.Advantages:� Superior auto calling of genotypes� Automatic detection of new mutations� Provides statistical values for maximum confidence in the results� Minimized efforts and highly standardized processes� Orthogonal approach e.g. for validation purposes
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Advanced Statistical Data Analysis WorkflowRotor-Gene ScreenClust HRM Software ONLY
Normalisation Residual plotRaw data
Line-ofbest fit
DifferentiationAveragingSubtraction
Autocalling genotypes Principal component analysisClustering
supervised
unsupervised
Software
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ScreenClust HRM WorkflowClustering
Supervised: � groups are known and
controls are available for each group
� sample is assigned to wild type or mutant or heterozygous group ...
� allows autocalling based on several control samples
� method of choice for genotyping
Unsupervised: � groups are unknown or
not all of the controls are available
� sample is assigned to cluster 1, or cluster 2 or...
� allows hypothesis free analysis� method of choice to detect new
genetic polymorphism
� The human eyes can easily see “clusters” but the software must decide onfacts using algorithms to define which sample belongs to which “cluster”
� Two modes: unsupervised and supervised clustering
Software
Sample & Assay Technologies
Current paper for ScreenClust HRM qPCR special edition in METHODS
� METHODS Volume 50, Issue 4, Pages S10-S14 (April 2010)� allows to get a deeper insight in the methods and workflows� incldues supplemental on-line material with formulas and description of the algorithms� gives some application examples
Software
Sample & Assay Technologies
Software: a critical success factor ScreenClust example for a difficult SNP genotyping data set
.A/T Class IV SNP� minute differences between homozygote alleles (< 0.1°C) � impossible to resolve genotypes in a normalized melt plot with Rotor-Gene Q operating Software
.ScreenClust HRM� provides unambiguous detection� clear automated clustering of all genotypes� exploits the full resolving power of RGQ instrument and type-it HRM chemistry
QIAGEN products used� Type-it HRM PCR Kit � Rotor-Gene 5plex HRM� ScreenClust HRM Software
wild type
heterozygote
mutant
Software
Sample & Assay Technologies
HRM analysis – melting of amplicon at � 72°C
72 °C
� melting of amplicons at � 72°C results in partial dye release already during the extension step
amplification plots display lower plateaus and therefore seem to indicate lower product yields
UB = 100% unmethylated, bisulfite converted human DNA
MB = 100% methylated, bisulfite converted human DNA
Target: CpG island of the MLH1 gene
Cycling
?
Sample & Assay Technologies
Modified HRM cycling protocol
3-step cycling withextension at 72°C
Cycling
3-step cycling withextension at 68°C
Ensure that cycling conditions allow amplification of all genotypesEnsure that cycling conditions allow amplification of all genotypes
Sample & Assay Technologies
Guidelines for assay design, cycling and analysis
.Assay design� Design assays with PCR product lengths of 70–350 bp� For SNP analysis, use of PCR products of 70–150 bp is recommended� The melting temperature of primers used for PCR with subsequent HRM analysis
should be at least 56°C� Design assays with a single melting domain
(http://www.bioinformatics.org/meltsim/wiki/Main/HomePage)
.Run � Follow cycling guidelines as recommended in the Type-It and EpiTect HRM manual,
respectively.� Initially determine the melting point for each new HRM PCR product. Run HRM
analysis to span a temperature range from 65°–95°C. For time savings, perform subsequent HRM analyses from 5°C below the lowest TM of all expected PCR products to 5°C above the highest TM of all PCR products.
.Analysis� Check that the PCR result contains only specific product� Exclude samples showing primer dimers or nonspecific products from analysis
Sample & Assay Technologies
Effect of template purity on HRM
.Typical Contaminants� NaCl� KOAc� EDTA� ETOH� Isopropanol� Na Citrate� Phenol
Template
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Template purity – effect of contaminantsNaCl
Template
Effect of NaCl on TM
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Concentration [mM]
TM [°C]
Salts increase the TM of PCR ampliconsSalts increase the TM of PCR amplicons
Sample & Assay Technologies
Conclusions and recommendations
.Conclusions
� Differences in final reaction compositions result in different melting behaviour
and lead to wrong genotype classification
.Recommendations
� Use identical method for DNA isolation for all samples
� Use identical batch of reagents for DNA isolation, including resuspension / elution
buffer for all samples
� Be careful to avoid contamínant carryover in your sample
Template
Sample & Assay Technologies
Overview
.Agenda
� Introduction into HRM� The principle� Advantages� Applications overview
� HRM in detail� Analysis� Dye
� Critical factors� Reaction chemistry� Instrument and software� Cycling conditions� Assay design� Template
� Application data and summary
Sample & Assay Technologies
Application example: SNP genotyping Detection of point mutations in the human KRAS gene
Analysis of point mutations in the human KRAS geneAnalysis of point mutations in the human KRAS gene
QIAGEN products used� Type-it HRM PCR Kit � Rotor-Gene 5plex HRM� Standard HRM analysis module
Assay source:Do et al.: High resolution melting analysis for rapid and sensitive EGFR and KRAS mutation detection in formalin fixed paraffin embedded BiopsiesBMC Cancer 2008, 8:142
Sample & Assay Technologies
� Supervised analysis of various gene mutations� normalized melt plot with almost identical curve shape making genotyping very challenging� 7 genotypes (6 mutations and wild type) called successfully by ScreenClust HRM � 3 cluster plots here as the first 3 principal components were required for discrimination
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Assay source:Do et al.: High resolution melting analysis for rapid and sensitive EGFR and KRAS mutation detection in formalin fixed paraffin embedded BiopsiesBMC Cancer 2008, 8:142
Application example: Mutation scanningSuccessful scanning of insertions/deletions in EGFR exon 19
Sample & Assay Technologies
Application example: Methylation analysisVarious ratios of methylated and unmethylated DNA-APC
QIAGEN products used� EpiTect Bisulfite Kit� EpiTect HRM PCR Kit � Rotor-Gene 5plex HRM� Standard HRM analysis
module
� Analysis of a CpG island from the promoter region of the APC gene(adenomatosis polyposis coli)
� Mixtures of methylated and unmethylated DNA as template� Clear resolution of similar methylation degrees down to 5% sensitivity
Sample & Assay Technologies
Application Example: Allelic ratio analysis
.Four allelic ratios of the factor V Leiden (G1691A) polymorphism� difficult to resolve allelic ratios down to 2.5%� minimal melt curve shape differences for 2.5%, 5% and 10% mutations
.Rotor-Gene Q plus ScreenClust HRM analysis� provides unambiguous resolution of all allelic ratios also in unsupervised mode� Sensitivity < 2.5% allelic ratios often needed for somatic mutation research
QIAGEN products used� RGQ 5plex HRM� ScreenClust HRM
Sample & Assay Technologies
Application Example: Pathogen typing with HRM Bacteria species typing for veterinary diagnostics
from N. Jeffrey et al Microbiology 2007 153: 2679-2688
� Mycoplasma synoviae is an economically important pathogen of poultry worldwide� Causing respiratory infection and synovitis� HRM analyses allows detection and identification of all M. synoviae strains � Rapid and effective analysis for outbreaks situations (single test in less than 2 h)
Classification of Mycoplasma Synoviae strains with HRM
QIAGEN products used:RLT lysis buffer Qiaex II matrixRW1 Buffer QIAquick PCRRotor-Gene Q 5plex HRM
Sample & Assay Technologies
Summary
� HRM is a powerful emerging technique to investigate genetic differences offering
� Throughput & speed
� Cost effectiveness & convenience
� Broad application range
� Outstanding performance in HRM analysis requires:
� Reliable template purity
� Highly specific HRM PCR kits with saturating dye
� qPCR and HRM instrument with superior temperature uniformity
� Powerful software packages for any kind of data analysis
Sample & Assay Technologies
The complete solution for HRM analysis
gDNA preparation from any sample type� Manual or automated for any throughput
Highly specific HRM PCR kits � Mandatory for good HRM results
Most precise real-time cycler on the market� Prerequisite for accurate HRM analysis
Powerful analysis software packages� Easy and reliable data acquisition and interpretation� A variety of different algorithms for full flexibility
Reliable results by dedicated solutions for an entire HRM workflow Reliable results by dedicated solutions for an entire HRM workflow
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Questions and Answers