sample & assay technologies pyromark q24 andrea tesoriero application specialist
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Sample & Assay Technologies
PyroMark™ Q24
Andrea TesorieroApplication Specialist
Sample & Assay Technologies- 2 -
Agenda
Introduction
Pyrosequencing technology
Pyrosequecing workflow
PyroMark Q24
Instrument
System
Software
AQ workflow
– Mutation example – KRAS
CpG Workflow
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What is Pyrosequencing?
Sequencing by Synthesis: Ronaghi M., Uhlén M., Nyrén P. (1998)
Real-Time Pyrophosphate Detection for DNA Sequencing.
Science 281:363-365
Sequence based technology in real time
Simple and robust
No separation, gel, label or probes
In built controls
Flexible
in throughput
in assay design
in type of applications
quantitation
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Pyrosequencing Assays
PCR primerSequencing
primer
Biotinylated PCR primer
Region of interest
Amplification of Region of interest (PCR)
Pyrosequencing Analysis
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Pyrosequencing Workflow
PCR
Sample Preparation – 10 min
Pyrosequencing Analysis – 10-60 min/plate
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Pyrosequencing Workflow
PCR and Sample preparation
1. PCR with one of the
primers biotinylated
2. Immobilize biotinylated
PCR products onto
streptavidin coated beads
3. Separate strands by
denaturation in NaOH
4. Wash /neutralize the
immobilized strand
5. Anneal sequencing primer
Run Pyrosequencing 10-100 min
1-2 h
10-15 min
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Pyrosequencing Workflow Sample Preparation
PCR product immobilized on Sepharose beads
PSQ plate with sequencing primer
EtOH
Denaturation Solution(NaOH)
Washing buffer
WaterVacuum tool
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Pyrosequencing Workflow Sample preparation
1-24 post-PCR samples prepared in parallel in less than 15 minutes
Minimal pipetting
Actual hands-on time, less than 1 minute
Plastic waste reduced to a minimum
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Pyrosequencing WorkflowEnzyme Cascade
PPi
ATP Time
Light
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Pyrosequencing Workflow A Pyrogram™ is generated
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Pyrosequencing Workflow A Pyrogram™ is generated
VariableRegion
Reference Sequence
T A GG TTT G C
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Sequence to Analyse = a/gCTGCCT
Genotype = A/G Heterozygote
C T C GT TCA G
Negative controls Reference Peaks
Single height ref peaksDouble height ref peak2 half height peaks
Pyrosequencing Workflow A Pyrogram™ is generated
Sample & Assay Technologies- 13 -
ACTGCCT-------- TGACGGA---
A G C T A G
ACTGCCT-------- TGACGGA---
Homozygous A
GCTGCCT-------- CGACGGA---
A G C T A G
GCTGCCT-------- CGACGGA---Homozygous G
A G C T A G
ACTGCCT-------- TGACGGA---
GCTGCCT-------- CGACGGA---
Heterozygous A/G
Pyrosequencing Workflow Unequivocal genotype classification
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PyroMarkTM Q24
A complete solution for Mutation and Methylation Analysis
CpG methylation Allele quantification Mutation analysis
A Small Smart Affordable Pyrosequencing System with a 24 well plate format
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Reagent cartridge
24 well plate
24 individual CCD Chips
X-Y drive
Mixer and thermostat
Detection
PositioningTA C G
PyroMark™ Q24 Instrument Design
Ink jet delivery
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PyroMarkTM Q24 System
InstrumentVacuum Prep. Workstation
Application and Assay Design software
Reagents
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PyroMark™ Q24Benefits
F An extremely easy to use system - Up to 24 samples in parallel
Small Footprint Takes very little bench space Measuring only H420xW390xD525mm
Robust Can be run at ambient temperatures between 15°C
and 32°C. Runs can be set up and analyzed on any PC anywhere,
running PyroMark™Q24 Software. Information is transferred between the instrument and
computer via a USB memory stick. Conforms to EU IVDD so suitable for clinical use in
Europe
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PyroMark™ Q24 Software
The software has two analysis modes:
F AQ - A variety of quantification studies and SNP analysis.
F CpG - Methylation analysis of multiple consecutive CpG sites. AQ assays and CpG assays can be performed on the same
PyroMark™ Q24 Plate.
5 multi licenses
You can toggle between the analysis modes in the analysis view of the software, by selecting AQ or CpG in the toolbar.
Both modes offer detailed report information that can be exported to html, Excel, text and PDF formats.
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PyroMark™ Q24 software AQ mode
F Frequency calculations of variable positions in sequence context including;
Single variable position AG/TC Multiple variable positions AG/TCAG/TCAG/T/AC Di- Tri- or Tetra-allelic mutations GA/C/G/TA
High resolution of individual sites Quality assessment of individual sites and sequence context SNP analysis (Multiple positions, Di- Tri- or Tetra-allelic variants) Analysis of SNPs in the presence of CpG sites
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Mutation exampleKRAS
Mutations in the KRAS gene results in a constitutively active KRAS protein which leads to abnormal cell growth, proliferation and differentiation.
KRAS is frequently mutated in Colorectal cancer Lung cancer
The most common KRAS mutations are found in codons 12,13 and 61
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Codon 61
FP
FPB
Seq
RPB
RPSeq
GGT GGC GTAGG
TCCA GTT CTC
Codon 12 & 13
Codon 12+13
G G T G G C
7477
Base
pair
sub
stit
uti
on
Codon 61
C A A
70
Base
pair
sub
stit
uti
on
Wt seq
Flexible assay design facilitates analysis of contiguous, multivariable mutations
G
T
C
A
= Guanine
= Cytosine= Thymine
= Adenine
Mutation exampleKRAS – mutation frequency
Sample & Assay Technologies- 22 -
Mutation exampleThe KRAS 2.0 assay
12 13 61
C A AG CG G T G
A A AA CA A T A
C C CC CC C T G
G CT T T G
Codons
Wt seq
Multi-variable
mutations
BND
FP Seq
RPB
NNt RVcCodon 12 & 13
Codon 61FPB
Seq RP
G G A
C T T
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Mutation exampleThe KRAS 2.0 assay
Efficient detection and quantification of mutations in codons 12, 13 and 61 with built in quality control
1 well/sample for all Codon 12 & 13 mutations (9 reported mutations) 1 well/sample for all Codon 61 mutations (7 reported mutations)
Provides high quality data of DNA from fresh, frozen and paraffin-embedded tumor samples
Sequence context provides built-in control and eliminates false positives/negatives
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Mutation exampleThe KRAS assay – Wild Type
A: 0%C: 0%G: 100%T: 0%
A: 0%G: 100%
EE SS TT AA CC GG AA
5
CC TT CC AA GG
10
AA TT GG CC GG
15
TT AA GG
0
25
50
75
100
125
A1: GNTGRCGTAGGC
A: 0%C: 0%G: 0%T: 100%
EE SS TT CC GG TT CC
5
AA GG TT GG AA
10
CC
0
50
100
150
200
B1: CTCNTGACCT
Sample & Assay Technologies- 25 -
Mutation exampleThe KRAS assay – codon 12 position 2
A: 0%C: 0%G: 84%T: 16%
A: 0%G: 100%
EE SS TT AA CC GG AA
5
CC TT CC AA GG
10
AA TT GG CC GG
15
TT AA GG
0
25
50
75
100
125
150
C4: GNTGRCGTAGGC
A: 0%C: 8%G: 92%T: 0%
A: 0%G: 100%
EE SS TT AA CC GG AA
5
CC TT CC AA GG
10
AA TT GG CC GG
15
TT AA GG
0
10
20
30
40
50
60
C6: GNTGRCGTAGGC
A: 16%C: 0%G: 84%T: 0%
A: 0%G: 100%
EE SS TT AA CC GG AA
5
CC TT CC AA GG
10
AA TT GG CC GG
15
TT AA GG
0
25
50
75
100
125
A2: GNTGRCGTAGGC
GGT>GCT
Gly12Ala
GGT>GAT
Gly12Asp
GGT>GTT
Gly12Val
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Mutation exampleThe KRAS assay – codon 12 position 1
A: 0%C: 0%G: 61%T: 39%
A: 0%G: 100%
EE SS TT AA CC GG AA
5
CC TT CC AA GG
10
AA TT GG CC GG
15
TT AA GG
0
25
50
75
100
125
A6: NGTGRCGTAGGC
A: 58%C: 1%G: 41%T: 0%
A: 0%G: 100%
EE SS TT AA CC GG AA
5
CC TT CC AA GG
10
AA TT GG CC GG
15
TT AA GG
0
50
100
150
C6: NGTGRCGTAGGC
GGT>TGT
Gly12Cys
GGT>AGT
Gly12Ser
Sample & Assay Technologies- 27 -
Mutation exampleThe KRAS assay – codons 13 and 61
A: 0%C: 0%G: 100%T: 0%
A: 19%G: 81%
EE SS TT AA CC GG AA
5
CC TT CC AA GG
10
AA TT GG CC GG
15
TT AA GG
0
25
50
75
100
125
150
C4: GNTGRCGTAGGC
A: 0%C: 1%G: 28%T: 71%
EE SS TT CC GG TT CC
5
AA GG TT GG AA
10
CC
0
50
100
150
200
B1: CTCNTGACCT
GGC>GAC
Gly13Asp
CAA>CAC
Gln61His
TTG>GTG
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Mutation exampleThe KRAS assay – Conclusions
1 PCR to cover ALL mutations in codons 12 & 13 Minimises amount of gDNA needed (10ng)
Optional second PCR to cover all mutations in codon 61 Provides results for rarer but important mutations not covered by
other methods
Fast results PCR product to quantitative result in ~ 30 minutes for 24
samples
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CpG methylation analysis
1. Bisulfite conversion
2. PCR amplification
3. Pyrosequencing
mC C
C U
T
CC
U
mC C
25%75%
Degree of methylation is automatically analyzed by the software.
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CpG methylation analysisAssay design
PCR primer
CpG sites
CpG island
All primers are located in non-variable regions, in between CpG sites
Enables analysis of several adjacent CpG sites with one sequencing primer
Freedom in positioning of the sequencing primer distance from CpG site orientation of assay
PCR primer
Sequencing primer
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CpG methylation analysisA range of analysis possibilities
Any single CpG site
Multiple consecutive CpG sites
One gene at a time Several genes in the same analysis (analyze
up to 24 different assays in one run)
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Benefits of Pyrosequencing for CpG methylation analysis
Quantitative analysis of multiple consecutive sites
Flexible assay design Forward – reverse/ Upper – lower
Flexible primer positioning
Built-in Bisulfite treatment control
Excellent Performance Accuracy
Precision
Reproducibility over time
Confidently discern even small changes in methylation
Fast results
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Benefits of Pyrosequencing for CpG methylation analysis Built-in quality control of bisulfite treatment
Before bisulfite treatment
Analyzed sequence
Any C not followed by a G gives bisulfite QC
TYGATATGGTTYGGTTGGGTTYGTGTTTYGTTGGTTTTGGGYGTTAGTAAGYGYGGGTYGGGYGGGGT
CCGACATGGCCCGGTTGGGCCCGTGCTTCGCTGGCTTTGGGCGCTAGCAAGCGCGGGCCGGGCGGGGC
RASSF1A gene
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Benefits of Pyrosequencing for CpG methylation analysis Accuracy - Linear response in measured methylation
Linear response of methylation measured by Pyrosequencing in PCR-amplified products generated from controlled dilutions of in-vitro methylated (IVM) genomic DNA with unmethylated DNA (IVM DNA is 80% methylated).
Sequence to analyze:
GGGTGGGGYGGATYGYGTGYGT
Normal DNA
Colon cancer DNA
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Benefits of Pyrosequencing for CpG methylation analysis Accuracy – using different sequencing primers
Methylation levels are consistent even when using different primers
Each methylation value is the mean of 3 replicates
MLH1 gene
Seq. Primer 1
Seq. Primer 2
Seq. Primer 3
73 bases
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Benefits of Pyrosequencing for CpG methylation analysis Consistent precision among CpG sites
Confidently measure the individual degree of methylation in adjacent CpG sites, even at long distances from the sequencing primer
Pos. 1 Pos. 2 Pos. 3 Pos. 4 Pos. 5 Pos. 6 Pos. 7 Pos. 8 Pos. 9 Pos. 10
MGMT gene
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Benefits of Pyrosequencing for CpG methylation analysis Quantification of individual CpG sites
Methylation pattern in RASSF1A in neighboring CpG sites in 4 tumor samples (duplicate runs)
Methylation levels may vary from site to site
Pyrosequencing detects site variation reproducibly
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Imprinting
PWS: lack of paternal contributionpaternal deletion (70%)maternal uniparental disomy (25%)
AS: lack of a maternal contribution maternal deletion (70%)paternal uniparental disomy (5%)
Gene expression dependent on the parent of origin
Prader-Willi Syndrome (PWS)Angelman Syndrome (AS)
Neuro developmental disorders caused by a deficiency with 15q parental contributions
methylated on the maternal chromosomeremains unmethylated on paternal chromosome
(UPD-receive two copies of a chromosome from one parent)
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Benefits of Pyrosequencing for CpG methylation analysis Flexible assay design
Analysis in either direction – Prader Willi/Angelman
Forward assay: C/T
Reverse assay: G/A
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F Software Overview
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PyroMark™ product lineCancer mutations, methylation, clinical genetics
PyroMark RUO Optimized PCR and Pyrosequencing protocols, built-in quality control
Cancer Mutations: KRAS, BRAF CpG Methylation: p16, MLH1, LINE-1, MGMT Clinical Genetics: APOE, HFE, MTHFR, Prader-Willi/Angelman
Genetic tests that show real sequence information.
PyroMark Assay Database over 1,000 optimized & wet-tested assay designs Continuous updates Online access: www.pyrosequencing.com/techsupport
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PyroMark product lineProduct Offering Overview
Instruments
Sample preparation
Software
Reagents
Assay Kits
PyroMark Q24
PyroMark Q96 ID
PyroMark Q96 MD
PyroMark Q24 Workstation
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F Genetic VariationF SNP & mutation analysisF Simplex & multiplex SNP genotyping F Tri/tetra-allelic mutations F
QuantificationF CpG methylation analysis
Analysis of polyploid genomesAllele-specific gene expressionSNP pooling studiesViral/bacterial loadLoss of heterozygosityGene copy number
F
Short DNA sequencing Microbial identificationSpecies discrimination Sub-typing Resistance detectionSequence signaturesDNA bar-codingSequence variationSequence verificationForensicsClone checking
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F Gracias por su atención