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


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.


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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Tm Melting Point


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


HRM applications

� SNP analysis

� Mutation detection and scanning

� Pathogen detection

� Methylation analysis

� STR and VNTR analysis

� Quantification of copy number variants and mosaicism

� …


HRM – An emerging technology

HRM citations/year*

*source: google scholar

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2004 2005 2006 2007 2008 2009 2010

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


Overview

.Agenda






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


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


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


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


Overview

.Agenda






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


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


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


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


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


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%


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


Standard HRM data analysis workflowAll Current Software

Normalisation Difference plot

Autocalling genotypes

Raw data

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SubtractionLine-ofbest fit

Rotor-Gene Q Operating Software

Software


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


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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Fluorescence-Normalized HRM Plot

Rotor-GeneOperatingSoftware

unpublished customer field test data


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Difference Plot – 1st possibility




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Difference Plot – 3rd possibility



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ScreenClust HRM Cluster Plot

Rotor-GeneScreenClust HRM Software


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


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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Software


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


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


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


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


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

?


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


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


Effect of template purity on HRM

.Typical Contaminants� NaCl� KOAc� EDTA� ETOH� Isopropanol� Na Citrate� Phenol

Template


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


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


Overview

.Agenda






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


� 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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M6QIAGEN products used� Type-it HRM PCR Kit � Rotor-Gene 5plex HRM� ScreenClust HRM Software

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


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


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


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


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


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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