Sample & Assay Technologies PyroMark Q24 Andrea Tesoriero Application Specialist.

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


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


Pyrosequencing Assays

PCR primerSequencing

primer

Biotinylated PCR primer

Region of interest

Amplification of Region of interest (PCR)

Pyrosequencing Analysis


Pyrosequencing Workflow

PCR

Sample Preparation – 10 min

Pyrosequencing Analysis – 10-60 min/plate


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


Pyrosequencing Workflow Sample Preparation

PCR product immobilized on Sepharose beads

PSQ plate with sequencing primer

EtOH

Denaturation Solution(NaOH)

Washing buffer

WaterVacuum tool


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


Pyrosequencing WorkflowEnzyme Cascade

PPi

ATP Time

Light


Pyrosequencing Workflow A Pyrogram™ is generated



VariableRegion

Reference Sequence

T A GG TTT G C


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



ACTGCCT-------- TGACGGA---

A G C T A G


Homozygous A

GCTGCCT-------- CGACGGA---

A G C T A G

GCTGCCT-------- CGACGGA---Homozygous G

A G C T A G


GCTGCCT-------- CGACGGA---

Heterozygous A/G

Pyrosequencing Workflow Unequivocal genotype classification


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


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


PyroMarkTM Q24 System

InstrumentVacuum Prep. Workstation

Application and Assay Design software

Reagents


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


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.


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


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


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


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


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


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


Mutation exampleThe KRAS assay – codon 12 position 2

A: 0%C: 0%G: 84%T: 16%

A: 0%G: 100%


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%


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%


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


Mutation exampleThe KRAS assay – codon 12 position 1

A: 0%C: 0%G: 61%T: 39%

A: 0%G: 100%


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%


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


Mutation exampleThe KRAS assay – codons 13 and 61

A: 0%C: 0%G: 100%T: 0%

A: 19%G: 81%


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


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


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.


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


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)


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


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


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


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


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


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


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)


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


F Software Overview


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

http://www.pyrosequencing.com/techsupport


PyroMark product lineProduct Offering Overview

Instruments

Sample preparation

Software

Reagents

Assay Kits

PyroMark Q24

PyroMark Q96 ID

PyroMark Q96 MD

PyroMark Q24 Workstation


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


F Gracias por su atención

Sample & Assay Technologies PyroMark Q24 Andrea Tesoriero Application Specialist.

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