Значение исследований ...€¦ · sample to insight Значение...

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Значение исследований метилирования генома на примере использования PyroMark Q48. Демонстрация прибора

Pyrosequencing technology for mutation and methylation analysis 1

Alexey A. Apchelimov, Ph.D.Director, QIAGEN Russia

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Mol Cell Biol. 2010 Oct; 30(20): 4758–4766.

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Outline


Curr Genomics. 2011 Nov; 12(7): 486–505.

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4

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Environ Health Perspect. 2006 Mar; 114(3): A160–A167.

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• More than 464,000 (230.000 in Europe) new cases diagnosed in 2012

• Over 131.000 deaths in Europe in 2012

Breast Cancer Incidence in Europe

PROM-10435-001 11/17

Breast Cancer (BC) is the most common malignancy in women

29%

13%

7%6%4%

4%

4%

3%

3%

27%

Cancer Incidence in European Women

Breast

Colorectal

Lung

Corpus uteri

Ovary

Cervix uteri

Stomach

Melanoma

Pancreas

Other / unspec.

Cancer incidence in women: 1,606,252 cases per year in Europe

Ferlay, J. et al. (2014) GLOBOCAN 2012 v1.1, Cancer Incidence and Mortality Worldwide: IARC

CancerBase No. 11 Lyon, France: International Agency for Research on Cancer. Available from:

http://globocan.iarc.fr

7

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PITX2 Methylation in Breast Cancer

PROM-10435-001 11/17

PITX2 gene promotor methylation

• PITX2-gene is regulated by methylation within the promotor region

• PITX2 has been reported as prognostic and predictive marker in breast cancer (1–3)

• PITX2 methylation can be reliably assessed by real-time PCR technology in formalin-fixed, paraffin-embedded (FFPE) tissue (2)

• Several retrospective studies have shown a strong correlation between PITX2 promoter methylation and outcome measures such as progression-free survival (PFS), metastasis-free survival (MFS), disease-free survival (DFS) and overall survival (OS) (1–4)

• PITX2 has been shown to be predictive for therapy response to anti-estrogens and anthracycline-based chemotherapy (ANT) (2–4)

1. Nimmrich, I., et al. (2008) Breast Cancer Res Treat 111:429.

2. Harbeck, N., et al. (2008). J Clin Oncol 26: 5036.

3. Absmaier, M., et al. (2017). Int. J. Oncology (in press).

4. Hartmann, O., et al., (2009). Clin Cancer Res 15: 315.

PITX2 methylation: a novel biomarker for breast cancer therapy selection

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Sample to Insight

PROM-10435-001 11/17

Risk Categories and Response Prediction for Operated BC

Low risk~10%

Intermediate risk~65%

High risk~25%

Operated Breast Cancer

Clinico-pathological risk, e.g. St. Gallen classification (1, 2)

Prognosis by gene expression (3)

e.g. Endopredict®

No CTx

~50%Low risk

~60%High risk

~40%

CTx

~50%

AC:

ANT:

CMF:

CTx:

D:

EC:

P:

Doxorubicin / Cyclophosphamide

Anthracycline-based CTx

Cyclophosphamide / Methotrexate / Fluorouracil

Chemotherapy

Docetaxel

Epirubicin / Cyclophosphamide

Paclitaxel

Response prediction by PITX2

methylation (4)

High

ANTe.g. EC or AC

followed by P or D*

ANT-response probability

Alternative CTxe.g. DC or P

or CMF*

Low1. Goldhirsch, A., et al. (2007) Annals of Oncology 18:1133.

2. Bauer, K., et al.(2010) BMC Cancer 10:228.

3. Kiechle, M. (2014) Frauenarzt 55:232.

4. Aubele, M., et al. (2017) Disease Markers,

https://doi.org/10.1155/2017/4934608.

* According to current guidelines, e.g. AGO-guidelines 2017

9

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DNA methylation changes during aging


Sci Adv. 2016 Jul; 2(7): e1600584.

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Genome Biol. 2017; 18: 238.

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Epigenetic Reprogramming


Waddington, 1957

Development. 2009 Feb 15; 136(4): 509–523.

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Pyrosequencing – principle and key features

14

.Based on SEQUENCING-by-SYNTHESIS Principle

European Inventor Award 2013: Pål Nyrén for inventing pyrosequencing

Picture courtesy of: http://www.epo.org/learning-events/european-inventor/finalists/2013/nyren.html/

Ronaghi, M., Uhlén, M., Nyrén, P. (1998)

Real-time pyrophosphate detection for

DNA sequencing. Science 281:363.

Advanced single base resolution DNA methylation and mutation analysis in long sequence runs using Pyrosequencing

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Pyrosequencing – principle and key features


Technology – based on SEQUENCING-by-SYNTHESIS*

.Principle

• Stepwise synthesis of DNA by addition of nucleotides

• Enzyme cascade generates a light signal upon incorporation of nucleotides

* Ronaghi, M., Uhlén, M., Nyrén, P. (1998) A sequencing method based on real-time pyrophosphate. Science. 281:363.

Step 1 Hybridization of a sequencing primer

Step 2-4 Addition of dNTP, conversion into light

signal, degradation of nucleotides

Step 5 Pyrogram generation and data analysis

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Pyrosequencing application overview

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Pyrosequencing addresses various markets

Methylation Studies

Quantify methylation level of multiple CpG

sites in one assay

Resistance Typing

Detect and quantify

complex mutations leading to drug resistance

Cancer Mutations

Detect and quantify

complex mutations

Microbial IDIdentification and sub-

typing of varies microbial

organism

SNP Confirmation

Di-, tri & tetra SNPs in up to 10 x 96 sample throughput format

Forensics

Y-STR markers and SNPs, tissue-specific methylation

detection

Pyrosequencing

Biomarker Verification

Validation & verification of GWAS & NGS data


../../../SuckauL/Eigene Dateien/My Documents/Lokale Daten/Epigenetics/ACTUAL/Biotage/Presentations/PyroID & Methylation/Frida/TechSupport/Presentationer/Pooling.ppt#1. Benefits of analyzing sample pools

../../../SuckauL/Eigene Dateien/My Documents/Lokale Daten/Epigenetics/ACTUAL/Biotage/Presentations/PyroID & Methylation/Frida/TechSupport/Presentationer/CpG methylation.ppt#1. PowerPoint Presentation

../../../SuckauL/Eigene Dateien/My Documents/Lokale Daten/Epigenetics/ACTUAL/Biotage/Presentations/PyroID & Methylation/Frida/TechSupport/Presentationer/Chimerism.ppt#1. Hematopoetic Chimerism

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Pyrosequencing applications


Results provided by Pyrosequencing for various applications

Pyrosequencing provids all information needed for epigentic, genetic, and microbial

marker analysis

Sequencing through unknown regions


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marker analysis

A: 44%C: 0%G: 56%T: 0%

Di-, tri- and tetra-allelic mutations / SNP

Insertions / Deletions

- - - - - - - : 56%ATCTGCC: 44%

C: 57%T: 43%



- - - - - - - : 56%ATCTGCC: 44%

C: 57%T: 43%

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marker analysis

DNA methylation of multiple CpG sites

A: 44%C: 0%G: 56%T: 0%



- - - - - - - : 56%ATCTGCC: 44%

C: 57%T: 43%



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marker analysis


A: 44%C: 0%G: 56%T: 0%



- - - - - - - : 56%ATCTGCC: 44%

C: 57%T: 43%



Sample to Insight

Measuring frequencies in sequence variations

Advanced single base resolution DNA methylation and mutation analysis in long sequence runs using

Pyrosequencing21

Example: DNA methylation analysis

.A G T T A C G A C A .Sequence to be analyzed:

.After bisulfite conversion:

.A G T T A C G A C A .A G T T A Cm

G A C A .and

.A G T T A T G A T A .A G T T A Cm

G A T A .and

.Analyzed sequence:

X

.A .G .T .A .A.T/C .T.T G .A

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Pyrosequencing22


Ratio

T:C

.A G T T A C G A C A


G A C A .and


G A T A .and

.A .G .T .A .A.T .C .C .T.G

.27%

.Nucleotides added:

X

.A .G .T .A .A.T/C .T.T G .A

.A

.Sequence to be analyzed:


.Analyzed sequence:

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Pyrosequencing23


Ratio

T:C



G A C A .and


G A T A .and

.A .G .T .A .A.T .C .C .T.G

.27%

X

.A .G .T .A .A.T/C .T.T G .A

.A.Nucleotides added:


.Analyzed sequence:


Sample to Insight



Pyrosequencing24


Ratio

T:C

C=0%

T=100%



G A C A .and


G A T A .and

.A .G .T .A .A.T .C .C .T.G

.27%

.Built-in quality control: successful bisulfite conversion

X

.A .G .T .A .A.T/C .T.T G .A

.A.Nucleotides added:


.Analyzed sequence:


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25

Quantitative peak heights to measure allele frequencies

Wasson et al. 2002. Assessing allele frequencies of single nucleotide polymorphisms in dna pools by Pyrosequencing

technology. BioTechniques 32:1144–1152.

Even as little as 2% of one allele in 98% of the other could be detected


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Common technologies in epigenetic and genetic projects

26

Technologies used at various steps reflect need for coverage and costs for sample processing

Screening /

Discovery

Verification /

Validation

Functional

studies

• Genomewide analysis

◦ Next Generation

Sequencing (NGS)

◦ Arrays

• Targeted analysis

◦ NGS panels

◦ HRM

• Single site analysis

◦ PCR or MS-PCR*

◦ qPCR or qMS-PCR*

◦ Sanger sequencing

◦ Pyrosequencing

• Multiple site analysis

◦ Sanger sequencing

◦ Pyrosequencing

• Single site analysis

◦ qPCR or MS-PCR*

◦ Pyrosequencing

• Single or multiple site

analysis

◦ Pyrosequencing

◦ Mass Array

*MS-PCR: Methylation-Specific PCR

Pyrosequencing technology for mutation and methylation analysis

Genome coverage

Sample number

Sample to Insight


27


Screening /

Discovery

Verification /

Validation

Functional

studies



Genome coverage

Sample number

Genomewide analysis

Targeted analysis

Somatic mutation frequencies

Analysis of single/multiple CpGsLife Technologies

ABI - SOLiD

Illumina

HiSeq/MiSeq

Life Technologies

IonTorrent

Illumina

Microarrays

Roche 454

Roche 454 Junior

QIAGEN

GeneReader NGS

Sanger

sequencing

PCR / MSP*

GeneRead DNAseq

Gene Panel

EpiTect Methyl

qPCR

High-Resolution

Melting (HRM)

Pyrosequencing

Real-time PCR /

MethyLight

Sample to Insight


28


Screening /

Discovery

Verification /

Validation

Functional

studies



Genomewide analysis

Targeted analysis

Somatic mutation frequencies

Analysis of single/multiple CpGsLife Technologies

ABI - SOLiD

Illumina

HiSeq/MiSeq

Life Technologies

IonTorrent

Illumina

Microarrays

Roche 454

Roche 454 Junior

QIAGEN

GeneReader NGS

Sanger

sequencing

PCR / MSP*

GeneRead DNAseq

Gene Panel

EpiTect Methyl

qPCR

High-Resolution

Melting (HRM)

Pyrosequencing

Real-time PCR /

MethyLight

Genome coverage

Sample number

Sample to Insight

Challenges in quantitative DNA analysis


Sanger sequencing Pyrosequencing

PCR Real-time PCR

• Quantification of sequence

variations

o LOD down to 1–2%

o short to medium sequences

• Detection of sequence

variations

o LOD approximately 20%

o medium to long sequences

• Detection of single sequence

variations

o LOD <1%

• Quantification of single

sequence variations

o LOD typically <1%

Complex

analysis

Non-quantitative Quantitative

Simple

analysis

Sequ

ence v

ariation

Result requirement

xsingle sequence variation

xx x xx xmultiple sequence variations

ABC D E F

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Sanger Sequencing vs Pyrosequencing


Sanger Sequencing for genetic analysis

Sequence Chromatograph

12Wild Type Sequence

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

Sequence Pyrogram

G/T GG C G AT GG

Mutation

GGT>GTT

Gly12Val

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Pyrosequencing at a glance


Traditional Sanger Sequencing vs Pyrosequencing for genetic analysis

Sanger Sequencing PyroMark Pyrosequencing

Application Sequencing of long sequences for

SNP / Mutation Analysis

Fragment Analysis

Detection of short/medium sequences for

SNP / Mutation Analysis

Microbial Identification

Frequency

analysis

hardly quantifies variant frequencies

below 15-20%

safely quantifies variant frequencies as low as

1-5%

Time 6-8 h,

(including PCR & cycle sequencing)

2-3 h

(including PCR)

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Pyrosequencing at a glance


Traditional Sanger Sequencing vs Pyrosequencing for epigenetic analysis

Bisulfite Sanger Sequencing PyroMark Pyrosequencing

Application Cloned Sanger sequencing for

Methylation Analysis

Detection of short/medium sequences for

Methylation Analysis

Frequency

analysis

Depending on number of clones. safely quantifies variant frequencies as low as

1-5%

Time weeks 2-3 h

(including PCR)

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Outline


Pyrosequencing technology

Positioning

Applications

Workflows

PyroMark Q48 Autoprep

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Epigenetics


DNA Methylation and the use of epigentic marker analysis

.Epigenetics mechanisms

• DNA (CpG) Methylation

• Histone Modification

• micro RNA

C 5mC~5%

Epigenetic marker analysis requires sensitive quantification of multiple, consecutive

CpGs or CpNs

CpG island

CH3CH3CH3CH3CH2OH CH3CH3

*

CH3

CpG shoresCpG shelfCpG canyon

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Epigenetics






• micro RNA

C 5mC~5%

CpG island

CH3

CH3CH3CH3CH3 CH3

Gene expression

No gene expressionX


CpGs or CpNsAlja Videtic Paska, Petra Hudler. Aberrant methylation patterns in cancer: a clinical view.

Biochemia Medica 2015;25(2):161-76.

http://www.biochemia-medica.com/taxonomy/term/1303


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Epigenetics






• micro RNA

C 5mC~5%

CpG island

CH3

CH3CH3CH3CH3 CH3

Gene expression

No gene expressionX






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Epigenetics






• micro RNA

C 5mC~5%

CpG island

CH3

CH3CH3CH3CH3 CH3

Gene expression

No gene expressionX


CpGs or CpNs

therascreen MGMT Pyro Kit

For sensitive detection of MGMT methylations

• Intended to provide clinicians with information to aid the selection of glioblastoma

multiforme patients more likely to benefit from alkylating agents (temozolomide).

• The kit enables the quantitative measurement of methylation in four CpG sites in

exon 1 of the human MGMT gene

• Bisulfite converted genomic DNA is amplified by PCR and sequenced

• One methylation assay per sample

2% 2% 3% 4%

EE SS GG TT CC GG TT5AA TT CC AA GG

10TT CC GG TT CC

15AA TT GG TT TT

20CC GG

0

100

200

300

400

MGMT Unmethylated

69% 69% 73% 73%

EE SS GG TT CC GGTT5AA TT CC AA GG

10TT CC GGTT CC

15AA TT GGTT TT

20CC GG

0

100

200

300

400

500

MGMT Methylated

*CE-IVD: requires PyroMark Q24 MDx

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Epigenetics






• micro RNA

C 5mC~5%

CpG island

CH3

CH3CH3CH3CH3 CH3

Gene expression

No gene expressionX


CpGs or CpNsShen-Gunther et al, 2016. Molecular Pap smear: HPV genotype and DNA methylation of ADCY8, CDH8, and ZNF582 as an integrated biomarker for high-grade

cervical cytology. Clin Epigenetics. 2016; 8(1): 96.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5022163/


http://scialert.net/abstract/?doi=tmr.2010.1.15

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Epigenetics






• micro RNA

C 5mC~5%

CpG island

CH3

CH3CH3CH3CH3 CH3

Gene expression

No gene expressionX


CpGs or CpNsShen-Gunther et al, 2016. Molecular Pap smear: HPV genotype and DNA methylation of ADCY8, CDH8, and ZNF582 as an integrated biomarker for high-grade

cervical cytology. Clin Epigenetics. 2016; 8(1): 96.


Figure 1. Protocol schema

and representative images of

cervical cytology and cervical

carcinoma cell lines used in

the study. a Sample

collection, DNA extraction,

HPV genotyping by Sanger

sequencing, and CpG profiling

of gene-specific promoters by

pyrosequencing


http://scialert.net/abstract/?doi=tmr.2010.1.15

Sample to Insight

Outline



Positioning

Applications

Workflows

Life Science/Research

Molecular Diagnostics


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Pyrosequencing solutions for genetic and epigenetic analysis

42

• PAXgene Blood

DNA Tube

• QIAamp Kits

• AllPrep RNA/

DNA Kits

• EpiTect Fast

DNA Kits

• EpiTect Fast

LyseAll Kits

• EpiTect Fast

FFPE Kits

• PyroMark Assay

Design SW

• Pre-designed

PyroMark

Assays

• PyroMark PCR

Kit

• PyroMark Q48

Autoprep

• PyroMark Q48

Advanced

Reagents

Sample

collection &/

stabilization

DNA

purification

Assay

design

Bisulfite

conversion

Pre-

amplification

Pyro-

sequencing

PyroMark Assay

Design SW

PyroMark Q48

Advanced Kit

PyroMark PCR Kit

PyroMark Q48

Autoprep


QIAcube QIAxpert QIAxcel Adv.QC

EpiTect Fast DNA

1-3 hrs up to 0,5 days

EZ1

For epigentics only

depending on sequencing length

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Outline



Positioning

Applications

Workflows

Life Science/Research

Molecular Diagnostics


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Pyrosequencing solutions for MDx applications

44

• QIAamp Kits

e.g.

QIAamp FFPE

DNA tissue Kit

• EpiTect Fast

DNA Kits

• EpiTect Fast

LyseAll Kits

• EpiTect Fast

FFPE Kits

• PyroMark PCR Kit

• Therascreen Pyro

kits:

◦ EGFR

◦ KRAS

◦ NRAS

◦ RAS Extension

V2

◦ BRAF

◦ GIST Kit

◦ UGT1A1

◦ MGMT

• PyroMark Q24

Vacuum Prep

Worksation MDx

• PyroMark Q24 MDx

• Therascreen Pyro

kits:

◦ EGFR

◦ KRAS

◦ NRAS

◦ RAS Extension

V2

◦ BRAF

◦ GIST Kit

◦ UGT1A1

◦ MGMT

• PyroMark Q24 MDx

Software

• Therascreen

plugins:

◦ EGFR

◦ KRAS

◦ NRAS

◦ RAS Extension

V2

◦ BRAF

◦ GIST Kit

DNA

purification

Sample

Prep

Bisulfite

conversion

Pyro-

sequencingAnalysis

PyroMark Q24 MDx


EpiTect Fast DNA

2-3 hrs 1-2 hrs

For MGMT only

PyroMark Q24 VPWS

Pre-

amplification

PyroMark Q24 MDx

SW & Plugins

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therascreen portfolio to address cancer biomarkers

Disease Areas Brain

MGMT

IDH1 & IDH2

(BRAF)

Colorectal

KRAS

NRAS

BRAF

UGT1A1

Melanoma

BRAF

NRAS

NSCLC

EGFR

ALK

KRAS

(NRAS)

(BRAF)

GIST

KIT & PDGFRA

(BRAF)

www.mycancergenome.org

Leukemia (ipsogen)

JAK2 V617F

BCR-ABL1

PML-RARA

WT1

NPM1

….

Industry-leading

portfolio of solid

tumor

companion

diagnostics


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Facilitating Personalized Healthcare


• Global leader in partnering with the pharmaceutical industry for companion diagnostic development and global commercialization.

• Being at the forefront in developing companion diagnostics using genomic insights to guide the selection of medicines.

• Kits covering about 30 biomarkers have been marketed, for a variety of automation platforms and biological sample types.

• Expand the pipeline of Sample to Insight technologies for PHC and submit more tests for regulatory clearance or approval.

• Numerous co-development partnershipsare underway with leading pharmaceutical and biotech companies.

QIAGEN’s commitment to Pharma Companion Diagnostics

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Outline



Positioning

Applications

Workflows


Sample to Insight



PyroMark Q48 Autoprep –

simplified Pyrosequencing with

automated template preparation

Including

• Fully quantitative CpG, CpN, mutation,

and SNP analysis

• Single base resolution

• Sensitive analysis down to LOD of 1-5%

• Minimal input amounts of 1-10 ng

• Highly suitable for FFPE samples

• Fast sample to result analysis in a few hours

• Automated template preparation

• Immediate and straight forward data analysis

Sample to Insight

PyroMark Q48 Autoprep – Workflow


Workflow comparison of available Pyrosequencing platforms

Sample

preparation

Assay

design

PCR

amplification

ssDNA

preparationAnalysis

• PyroMark Q24

Vacuum Workstation

• PyroMark Q96

Vacuum Workstation

• PyroMark Q24

• PyroMark Q24 Adv.

• PyroMark Q96 ID

• PyroMark Q48

AutoprepNEW

ssDNA

preparation

and

analysis

PyroMark Q48 Autoprep:

Simplified workflow combined with advanced Pyrosequencing

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PyroMark Q48 Autoprep – Components


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PyroMark Q48 Discs

Polypropylene discs for 48 samples

Divided into 4 segments, each for 12 samples

Easy to handle, requiring no clamps

to hold in place

Reusable up to 4 times1)

1) For unused wells only

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Injector cartridges

Integrated part of the instrument

Includes filters to prevent blocking of needles

Internal calibration data stored on cartridge

Features a real time drop-sensor

Automatic dispensation of up to 3 (4)1) different

Sequencing primers

Automatic dispensation of 3 (4)1) MPD2) Mixes

1) Requires manual pipetting of binding buffer 2) MPD: Multiple Primer Dispensation

Sample to Insight



Large and easy to use touch screen

Guides user through every protocol step

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Network access

Enables remote access to run files via a TCP/IP network

through the Ethernet port

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PyroMark Q48 Autoprep – Protocol

Automatic template preparation fully integrated in PyroMark Q48 Autoprep workflow


Load

Run files

via USB or

Ethernet

Load

reagents,

nucleotides,

buffers

Load

PCR product

& magnetic

beads

Automatic

Template

Preparation

Anneal

Sequencing

primer

Pyro-

sequencing

Wash

Cartridge

55

manual automated manual/automated

Sample to Insight

PyroMark Q48 Autoprep offers highest degree of automation

56

Automated protocol steps along the Pyrosequencing workflow

Load

reagents,

nucleotides,

buffers

Load

PCR product

& beads

Manual

template

preparation

with VPWS

Anneal

Seq-

primer

Pyro-

sequencing

Wash

Cartridge &

VPWS

Load

reagents,

nucleotides,

buffers

Load

PCR product

& magnetic

beads

Automatic

template

preparation

Anneal

Seq-

primer

Pyro-

sequencing

PyroMark Q96 MD, Q96 ID, Q24, and Q24 Advanced


• Multi-step pipette

can be used for

pipetting beads

• Automatic

pipetting system

can be used

manual automated manual/automated

• Automatic dispensation

of up to 3 Seq primers

• Manual dispensation

for 4 or more Seq

primers

• Automatic dispensation

of 3 MPD* Mixes

*MPD: Multiple Primer Dispensation

Wash

Cartridge


Sample to Insight

PyroMark instrument comparison

57

Spec/Feature PyroMark Q48 Autoprep PyroMark Q24 MDx PyroMark Q96 ID

# of preps, format 48, disc format 24, plate format 96, plate format,

Footprint small medium large

Template Prep Integrated 1) separate vacuum prep

workstation 2)

separate vacuum prep

workstation 2)

Sequencing length up to 160-180 bp up to 80 bp up to 80 bp

Main Applications SNP,

mutation

CpG and CpN methylation

de-novo sequencing

therascreen Pyro kits

(mutation and methylation)

SNP,

mutation

CpG methylation

de-novo sequencing

Adressable Markets/

Main Markets

Genetic testing

Epigenetics

Microbiol ID

Drug resistance typing

Genetic testing/MDx Genetic testing

Epigenetics

Microbiol ID

Drug resistance typing

Throughput 4-6 runs, 48 samples each 4-6 runs, 24 samples each 4-6 runs, 96 samples each

CE-IVD mark Not available available Not available

1) Using Magnetic Streptavidin-Beads; 2) Using Streptavidin-Beads

Comparison of PyroMark Q48 Autoprep, PyroMark Q24 Advanced, and PyroMark Q96 ID


Sample to Insight



PyroMark Q48 Autoprep –

simplified Pyrosequencing with

automated template preparation

Including

• Fully quantitative CpG, CpN, mutation,

and SNP analysis

• Single base resolution

• Sensitive analysis down to LOD of 1-5%

• Minimal input amounts of 1-10 ng

• Highly suitable for FFPE samples

• Fast sample to result analysis in a few hours

• Automated template preparation

• Immediate and straight forward data analysis