new progress in pyrosequencing for automated quantitative analysis of bi- or multi-allelic sequence...

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New progress in Pyrosequencing for genotyping applications 1

New progress in pyrosequencing for fully automated quantitative analysis of bi- or multi-allelic sequence variations

Gerald Schock, Ph.D.Associate Director PyrosequencingQIAGEN GmbH

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2

QIAGEN products shown here are intended for molecular biology

applications. These products are not intended for the diagnosis,

prevention or treatment of a disease.

For up-to-date licensing information and product-specific

disclaimers, see the respective QIAGEN kit handbook or user

manual. QIAGEN kit handbooks and user manuals are available at

www.QIAGEN.com or can be requested from QIAGEN Technical

Services or your local distributor.

Legal disclaimer

New progress in Pyrosequencing for genotyping applications

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Outline

Challenges in quantitative genotyping analysis

Pyrosequencing technology and workflow in genotyping analysis

Introduction into the new PyroMark Q48 Autoprep

MPD strategy for a seamless, automated Pyrosequencing workflow

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Outline





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PCR Real-time PCR• Detection of single sequence

variationso LOD <1%

• Quantification of single sequence variations o LOD typically <1%

Non-quantitative QuantitativeResult requirement

xsingle mutation or SNP

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PCR Real-time PCR• Detection of single sequence

variationso LOD <1%


Complex analysis

Non-quantitative Quantitative

Simple analysis

Seq

uenc

e va

riatio

n

Result requirement


xx x xx xmultiple mutations or SNPs

ABC D E F

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Sanger sequencing

PCR Real-time PCR

• Detection of sequence variationso LOD approximately 20%o medium to long sequences

• Detection of single sequence variationso LOD <1%


Complex analysis


Simple analysis

Seq

uenc

e va

riatio

n

Result requirement



ABC D E F

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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 variationso LOD approximately 20%o medium to long sequences

• Detection of single sequence variationso LOD <1%


Complex analysis


Simple analysis

Seq

uenc

e va

riatio

n

Result requirement



ABC D E F

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Sequence Chromatograph

Sanger sequencing vs. Pyrosequencing

Dye-terminator sequencing• Labeled chain terminator dideoxynucleotides (ddNTPs)• 4 different fluorescent dyes• Electronic DNA sequence trace (chromatogram) determined by capillary electrophoresis

.Dye-terminator sequencing – limitations• Individual incorporation rate of the dye-labeled ddNTPs into the DNA fragment • Unequal peak heights and shapes in the chromatogram• Dye blobs

Unequal peak heights SNP/Mutation

Sanger sequencing chromatograph does not provided quantitative data

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Sequence Chromatograph


Dye-terminator sequencing• Labeled chain terminator dideoxynucleotides (ddNTPs)• 4 different fluorescent dyes• Electronic DNA sequence trace (chromatogram) determined by capillary electrophoresis

Unequal peak heights SNP/Mutation

Nickel, G.C. et al. Characterizing Mutational Heterogeneity in a Glioblastoma Patient with Double Recurrence. PLoS One, 2012

“… The signals from such mutations [low frequency mutations] are often below the noise threshold in Sanger sequence reads, … it is crucial that researchers do not rely solely on capillary-based sequencing for mutation detection and validation...”

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12Wild-type sequence

GGT>GTT

Gly12Val

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

A: 0%G: 100%

EE SS TT AA CC GG AA5

CC TT CC AA GG10

AA TT GG CC GG15

TT AA GG

0

25

50

75

100

125

150

C4: GNTGRCGTAGGC

Sequence Pyrogram

G/T GG C G AT GG

Mutation

Sequence chromatograph

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Outline





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Pyrosequencing – principle and key features

.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) Real-time pyrophosphate detection for DNA sequencing. 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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Step 1 A sequencing primer is hybridized to a single-stranded PCR amplicon that serves as a template for the Pyrosequencing reaction.

.Based on SEQUENCING-by-SYNTHESIS Principle*


* Ronaghi, M., Uhlén, M., Nyrén, P. (1998) Real-time pyrophosphate detection for DNA sequencing. Science 281:363.

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Step 1 A sequencing primer is hybridized to a single-stranded PCR amplicon that serves as a template for the Pyrosequencing reaction.

Step 2 DNA polymerase catalyzes the incorporation of the deoxyribonucleotide triphosphate (dNTP) into the DNA strand, which will be accompanied by release of pyrophosphate (PPi) in a quantity equimolar to the amount of incorporated nucleotides.

Watch the complete animation at: www.qiagen.com/pyrosequencing-reaction-cascade

.Based on SEQUENCING-by-SYNTHESIS Principle


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Step 3 PPi will be converted to ATP, which in turn generates proportional amounts of visible light. The light is detected by a charge-coupled device (CCD) chip and seen as a peak in the raw data output (Pyrogram). The height of each peak (light signal) is proportional to the number of nucleotides incorporated.




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Step 4 Apyrase continuously degrades unincorporated nucleotides and ATP. When degradation is complete, another nucleotide is added.




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Repeat step 2–4




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Step 5 Addition of dNTPs is performed sequentially.The complementary DNA strand is built up, and the nucleotide sequence is determined from the signal peaks in the Pyrogram trace.

Repeat step 2–4




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Sequencing through unknown regions




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

Di-, tri- and tetra allelic mutations / SNP

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





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Insertions / Deletions

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

C: 57%T: 43%

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



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

C: 57%T: 43%




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



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

C: 57%T: 43%

DNA methylation of multiple CpG sites





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



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

C: 57%T: 43%





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



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

C: 57%T: 43%



QIAGEN webinar:

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

View online at www.qiagen.com



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The workflow for genotyping analysis

Sample collection/

stabilization

DNA purification

Assays&Assay Setup

Pre-Amplification

Data analysis &

interpretation

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QIAGEN solutions for genotyping analysis

Sample collection/

stabilization

DNA purification

Assays&Assay Setup

Pre-Amplification

Data analysis &

interpretation

• RT2 Profiler PCR Arrays/ Assays

• RT2 SYBR® Green qPCR Mastermixes

• PAXgene Blood DNA Tube

• QIAamp• Allprep RNA/ DNA• QIAprep• QIAquick• QIAGEN Plasmid

• QuantiNova • QIAGEN Multiplex

PCR, (HotStarTaq for non-multiplex interests), QIAGEN OneStep Ahead (viral GT)

QIA

GEN

sol

utio

nsIn

stru

men

tsK

its

PCR/qPCR

PCRarrays

• GeneRead DNA Library • GeneRead DNAseq

gene panel• PyroMark Assay Design

SW

NGS/Pyrosequencing

• EZ1 Advanced XL• QIAcube/QIAcube HT• QIAsymphony SP/AS• QIAxpert (Quality

control

• QIAgility• QIAsymphony SP/AS

• QIAxcel• Rotor-Gene Q• PyroMark Q48

Autoprep

• IPA GeneGlobe Data Analysis Center

• PyroMark Q48 Advanced Kit

• RT2 First Strand Kit• RT2 PreAMP cDNA

Synthesis Kit

• REPLI-g Single Cell

• REPLI-g Single Cell • REPLI-g DNA Library• PyroMark PCR Kit

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QIAGENs Pyrosequencing solutions for genotyping analysis

Sample collection/

stabilization

DNA purification

Assays&Assay Setup

Pre-Amplification

Data analysis &

interpretation

• PAXgene Blood DNA Tube

• QIAamp• Allprep RNA/ DNA• QIAprep• QIAquick• QIAGEN Plasmid

QIA

GEN

sol

utio

nsIn

stru

men

tsK

its

• PyroMark Assay Design SW

Pyr

oseq

uenc

ing

• EZ1 Advanced XL• QIAcube/QIAcube HT• QIAsymphony SP/AS• QIAxpert (Quality

control• PyroMark Q48

Autoprep

• PyroMark Q48 Advanced Kit

• QIAgility• QIAsymphony SP/AS

• PyroMark PCR Kit

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Pyrosequencing workflow – assay design


Assay design• Two PCR primers (one is biotinylated)

o Biotin-labeled strand is isolated using Vacuum Prep Workstation• Sequencing primer

o Placed in front of region of interesto Annealed to single-stranded DNA before Pyrosequencing reaction

PCR primer

Region of interest PCR primer

Sequencing primer

Samplepreparation

Assaydesign

PCRAmplification

ssDNApreparation Analysis

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Pyrosequencing workflow - PCR


PCR / RT-PCR• Can use any PCR machine • PyroMark PCR Kit / PyroMark OneStep RT-PCR Kit• Amplify relevant region by PCR (70 - 500 bp)• Can use very short PCR products if desired (i.e. degraded DNA)• One primer has to be biotinylated

Samplepreparation

Assaydesign

PCRAmplification


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Pyrosequencing workflow – template preparation


Template preparation• Separates biotinylated PCR strand from unbiotinylated strand and PCR primers• Streptavidin coated Sepharose beads used for binding biotinylated PCR strand• Immobilization and separation by using

o Sepharose beads and vaccum prep workstation (PyroMark Q96 ID, Q24,, Q24 Adv)o Magnetic sepharose beads (PyroMark Q48 Autoprep)

Samplepreparation

Assaydesign

PCRAmplification


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Samplepreparation

Assaydesign

PCRAmplification


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Samplepreparation

Assaydesign

PCRAmplification


Sequencing primer

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Annealing of Sequencing primer• Sequencing primer only binds to biotinylated PCR strand• ssDNA is needed for binding• Binding done

o Manually (PyroMark Q96 ID, Q24, Q24 MDx, Q24 Adv)o Automated (PyroMark Q48 Autoprep)

Samplepreparation

Assaydesign

PCRAmplification


Sequencing primer

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Pyrosequencing workflow – analysis


Pyrosequencing analysis• Each PCR strand is sequenced individually• Variants are called and quantified according to their peak heights during sequencing

Samplepreparation

Assaydesign

PCRAmplification


C A

G A

C A

G A

X

X

X

X

X

X

X

X

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Pyrosequencing for SNP Detection & Quantification

Detection of tri- allelic SNPs

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Quantitative peak heights to measure allele frequenciesAllele frequency (%)

0%

2%

5%

10%

15%

20%

25%

30%

35%

40%

45%

55%

60%

65%

70%

75%

80%

90%

50%

50%

85%85%

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Quantitative peak heights to measure allele frequencies

Quantitative peak heights

R2 = 0.9993

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100

Allele frequency [%]

Rel. p

eak

heig

ht

SNP E1Linear (SNP E1)

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

50%

85%

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Outline





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PyroMark Q48 Autoprep – Workflow

Workflow comparison of available Pyrosequencing platforms

Samplepreparation

Assay design

PCRAmplification


• PyroMark Q24 Vacuum Workstation


• PyroMark Q24• PyroMark Q24 Adv.• PyroMark Q96 ID

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PyroMark Q48 Autoprep – Workflow

Workflow comparison of available Pyrosequencing platforms

Samplepreparation

Assay design

PCRAmplification




• PyroMark Q24• PyroMark Q24 Adv.• PyroMark Q96 ID

• PyroMark Q48AutoprepNEW

ssDNApreparation

andAnalysis

PyroMark Q48 Autoprep: Simplified workflow combined with advanced Pyrosequencing

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PyroMark Q48 Autoprep – Protocol

Automatic template preparation fully integrated in PyroMark Q48 Autoprep workflow

Loadreagents,

nucleotides,buffers

LoadPCR product& magnetic

beads

InsertAbsorber

strip & Disc

LoadRun files

via USB orEthernet

Automatic Template

Preparation

Pyro-sequencing

Analyzedata

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PyroMark Q48 Autoprep – Dimensions & Weight

Small footprint (1/2 x PyroMark Q24) and low weight (1/3 x PyroMark Q24)

390 mm (L, closed)

300

mm

(H)

560 mm (L, open)250 mm (W)

Small footprint and low weight (8,5 kg)

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PyroMark Q48 Autoprep – SW User Interface

Large and easy to use touch screen and intuitive instrument SW

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PyroMark Q48 Autoprep offers highest degree of automation

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Automated protocol steps along the Pyrosequencing workflow

Loadreagents,

nucleotides,buffers

LoadPCR product

& beads

ManualTemplate

Preparationwith VPWS

Anneal Seq-

primer

Pyro-sequencing

Wash Cartridge &

VPWS

Loadreagents,

nucleotides,buffers

LoadPCR product& magnetic

beads

Automatic Template

Preparation

Anneal Seq-

primer

Pyro-sequencing

PyroMark Q24/Q24 Advanced

PyroMark Q48 Autoprep

• Multi-step pipet 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


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Outline





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Multiple Primer Dispensation for highest automation

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Multiple Primer Dispensation (MPD) theory


PyroMark Q48 Primer Cartridge

PyroMark Q48 Autoprep

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48

Conventional Primer Dispensation theory


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Multiple Primer Dispensation (MPD) theory


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MPD workflow for SNP testing using PyroMark Q48 Autoprep


PTPN22 BACH2 GLIS3 RNLS

INS ERBB3 ORMDL3 IL27

IL2RA DR4 DR3 DQ8

MPD1

MPD2

MPD3

3 different MPD Mixes, each containing 4 different assays: Total of 12 different assays

Data kindly provided by Rainer W. Fürst, Institute of Diabetes Research, Helmholtz Center Munich, Germany

Design assays

Check PCR

product

Check Seq-

primer

Check quality

Pyro-sequencing

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Design assays

Check PCR

product

Check Seq-

primer

Check quality

Pyro-sequencing

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Design assays

Check PCR

product

Check Seq-

primer

Check quality

Pyro-sequencing


Color coding of the analysis software provides a convenient overview about the quality of results obtained. Detailed information is available in each Pyrogram.

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Genotyping of 23 patients with 3 different 4plex MPD mixes using 6 runs


MPD 1 MPD 2 MPD 3PTPN22 BACH2 GLIS3 RNLS INS ERBB3 IL27 ORMDL3 IL2RA DR4 DR3 DQ8

Disc 1

H20 n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a.Patient 1 C/C C/G C/C G/G T/T G/T G/G T/T C/C T/T A/G T/TPatient 2 T/T C/G G/G A/A A/A G/T G/G C/C T/T T/T A/A T/TPatient 3 C/C G/G C/G A/A A/T G/G G/G T/T C/T T/T A/G T/T

Disc 2

Patient 4 C/T C/C G/G A/A A/A G/T G/G C/T T/T C/T G/G C/TPatient 5 C/T G/G C/G A/A A/T G/G G/G T/T T/T C/T A/G C/TPatient 6 C/T C/G C/C A/A A/A T/T G/G T/T T/T C/T A/G C/TPatient 7 C/T C/G C/G A/A A/A G/G G/G T/T T/T C/T A/G C/T

Disc 3

Patient 8 C/C C/C C/G G/G A/A G/T G/G C/T T/T T/T G/G T/TPatient 9 C/C G/G C/G A/G T/T G/T A/G C/T T/T T/T G/G T/T

Patient 10 C/T C/G C/G G/G A/T G/G A/G C/T T/T T/T G/G T/TPatient 11 C/C C/G C/G A/A A/A G/T A/A T/T T/T T/T A/G T/T

Disc 4

Patient 12 C/C G/G C/C A/G A/A G/G A/G C/T T/T C/T G/G C/TPatient 13 C/C G/G C/C A/A A/T G/G A/A T/T T/T C/T G/G C/TPatient 14 C/C C/G C/G A/A A/A G/G A/G T/T T/T C/T G/G C/TPatient 15 C/C C/G C/G A/G A/T T/T A/G C/T C/T T/T G/G T/T

Disc 5

Patient 16 C/C C/G G/G A/G A/T G/G G/G C/C T/T T/T A/G T/TPatient 17 C/C C/G C/G A/A A/A G/T A/G C/T T/T C/T G/G T/TPatient 18 T/T G/G C/G A/G A/A G/G A/G C/T T/T T/T G/G T/TPatient 19 T/T C/C C/C A/A A/A G/T A/G C/C T/T T/T A/A T/T

Disc 6

Patient 20 C/C C/G G/G A/A A/A G/T A/G C/T T/T C/T A/G C/TPatient 21 C/C C/G C/G A/A T/T G/T A/G C/C C/T T/T A/A T/TPatient 22 C/C G/G G/G A/A A/T T/T A/G T/T C/C T/T A/G T/TPatient 23 C/C C/G G/G A/G A/T G/G G/G C/T C/C C/T A/G C/T

Multiple Primer Dispensation dramatically decreases hands-on time and thus increases throughput in SNP typing.


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PyroMark Q48 Autoprep – Testimonial

54New progress in Pyrosequencing for genotyping applications

Rainer W. Fürst, PhD, Institute of Diabetes Research, Helmholtz Center Munich, Germany

“Straightforward for first-time users, reduced hands-on time and a doubled sample throughput with the opportunity for multiple primer dispensation are considerable advantages of the new PyroMark Q48 Autoprep System. The PyroMark Q48 Autoprep System is a next-level of Pyrosequencing methodology!”

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Summary

PyroMark Q48 Autoprep for genotyping

• Cost efficient tool for genotyping of single and complex mutations/SNPs.

• Di-, tri, and tetra-allelic SNPs are analyzed using the same assay, in the same run

• Highly sensitive and reliable quantitative sequencing data

• Fast processing: 48 samples in minutes

• Automated Pyrosequencing through integrated template prep

• MPD strategy offers seamless, automated Pyrosequencing workflow

• Low sample input amounts: 1–10 ng

• Highly sensitive LOD:• 1–2% in mutation analysis

PyroMark Q48 Autoprep: Simplified workflow combined with advanced Pyrosequencing

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Q&A session

Thank you for your attention!

For up-to-date licensing information and product-specific disclaimers for QIAGEN products, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.qiagen.com or can be requested from QIAGEN Technical Services or your local distributor.

New progress in Pyrosequencing for fully automated quantitative analysis of bi- or multi-allelic sequence variationsGerald Schock, PhD.Associate Director PyrosequencingQIAGEN GmbH

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PyroMark Q48 Autoprep on the web

57

More information about the platform, accessories and reagents are available onlinehttps://www.qiagen.com/de/shop/automated-solutions/sequencers/pyromark-q48-autoprep/https://www.qiagen.com/de/shop/automated-solutions/sequencers/pyromark-q48-advanced-reagents/

https://www.qiagen.com/de/shop/automated-solutions/sequencers/pyromark-q48-autoprep/

https://www.qiagen.com/de/shop/automated-solutions/sequencers/pyromark-q48-advanced-reagents/

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PyroMark Q48 Autoprep Intro Page

58

https://www.qiagen.com/de/resources/technologies/pyrosequencing-resource-center/



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Backup slides

Backup slides

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PyroMark instrument comparison

Spec/Feature PyroMark Q48 Autoprep PyroMark Q24 Advanced 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 160-180 bp up to 80 bp

Main Applications SNP, mutationCpG and CpN methylation de-novo sequencing

SNP, mutationCpG and CpN methylation de-novo sequencing

SNP, mutationCpG methylation de-novo sequencing

Adressable Markets/ Main Markets

Genetic testingEpigeneticsMicrobiol IDDrug resistance typing



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

Availibility available in Europe and Asia3)

available available

1) Using Magnetic Streptavidin-Beads; 2) Using Streptavidin-Beads; 3) currently not available in USA & Canada, planned for mid 2016

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

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Pyrosequencing application overview

Pyrosequencing addresses various markets

Methylation StudiesQuantify methylation level of multiple CpG

sites in one assay

Resistance TypingDetect and quantify complex mutations

leading to drug resistance

Cancer MutationsDetect and quantify complex mutations

Microbial IDIdentification and sub-

typing of varies microbial organism

SNP ConfirmationDi-, tri & tetra SNPs in up

to 10 x 96 sample throughput format

ForensicsY-STR markers and SNPs, tissue-specific methylation

detection Pyrosequencing

Biomarker verificationValidation & verification of

GWAS & NGS data

new progress in pyrosequencing for automated quantitative analysis of bi- or multi-allelic sequence...

Healthcare