New Progress in Pyrosequencing for DNA Methylation

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Overcome challenges in epigenetics research - DNA methylation techniques, pyrosequencing and applications

Part 1: DNA Methylation – an Essential Element in EpigeneticsFacts and Technologies

Part 2: Accurate DNA methylation analysis with successful conversion

Part 3: Downstream application of Pyrosequencing

“New progress in Pyrosequencing for automated single base resolution DNA methylation analysis for epigenetic research”

Welcome to a 3-part series on epigenetic research

New progress in Pyrosequencing for epigentic applications

Gerald Schock, Ph.D.Associate Director PyrosequencingQIAGEN GmbH

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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


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Common technologies in epigenetic DNA methylation analysis

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Technologies used at different steps within a project


Screening / Discovery

Verification /Validation

Functionalstudies

• Genome-wide analysis

◦ Next Generation Sequencing (NGS)

◦ Arrays

• Targeted analysis

◦ NGS panels

◦ HRM

• Single CpG sites

◦ MS-PCR*

◦ Real-time MS-PCR*

• Single or multiple sites

◦ Sanger sequencing

◦ Pyrosequencing

• Single CpG sites

◦ Real-time MS-PCR*

• Single or multiple sites

◦ Pyrosequencing

◦ Mass Array

Genome coverage

Sample number

*MS-PCR: Methylation-Specific PCR

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Outline

New progress in Pyrosequencing for epigentic applications 4

Challenges in Pyrosequencing DNA methylation analysis

Pyrosequencing technology and workflow

"Advanced Pyrosequencing" technology

Introduction into the new PyroMark Q48 Autoprep

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Current challenges observed during Pyrosequencing analysis

� Insufficient read length

� Issues sequencing through homopolymer T sequences

� Challenging assay optimization

� Incomplete bisulfite conversion

� Tedious ssDNA preparation

Genomic sequenceTTCGCGATTGAATTCGAAAGACTCTCTTCGGCGGATGAAAGTCGTTATCTCTTGGTTGGTTGAGTTATAGTCTT


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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) 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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Sequencing through unknown regions


.Based on SEQUENCING-by-SYNTHESIS Principle


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





QIAGEN webinar:

New progress in Pyrosequencing forautomated quantitative analysis of bi- or multi-allelic sequence variations

Recorded sessions:View online at www.qiagen.com

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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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DNA methylation of multiple CpG sites

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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QIAGEN’s Pyrosequencing solutions for DNA methylation analysis

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• 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

• PyroMark CpGAssays

◦ CpG islands

◦ Methylation Arrays

• PyroMark PCR Kit

• PyroMark Q48 Autoprep

• PyroMark Q48 Advanced Reagents

Samplecollection &/ stabilization

DNApurification

Assay design

Bisulfiteconversion

Pre-amplification

Pyro-sequencing

PyroMark Q48 Autoprep


QIAcube QIAxpert QIAxcel Adv.QC

0,5 days 0,5 days

PyroMark Assay Design SW

PyroMark Q48 Advanced Kit

PyroMark PCR KitEpiTect Fast DNA

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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 interestPCR primer

Sequencing primer

DNApurification

Bisulfiteconversion

Pre-amplification

Assay design

ssDNA preparation

Pyro-sequencing

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Pyrosequencing workflow – Assay design


Pre-designed Assays• CpG island

o > 30,000 human, > 30,000 mouse, and > 24,000 rat assays• Human methylation array validation assays

o > 600,000 assayso 84.2% coverage and success rate for HM450 BeadChip Array

PCR primer

Region of interestPCR primer

Sequencing primer

DNApurification

Bisulfiteconversion

Pre-amplification

Assay design

ssDNA preparation

Pyro-sequencing

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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 (up to 500 bp)• Can use very short PCR products if desired (i.e. degraded DNA)• One primer has to be biotinylated

DNApurification

Bisulfiteconversion

Pre-amplification

Assay design

ssDNA preparation

Pyro-sequencing

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

DNApurification

Bisulfiteconversion

Pre-amplification

Assay design

ssDNA preparation

Pyro-sequencing

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Pyrosequencing workflow – Template preparation


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 Adv)o Automated (PyroMark Q48 Autoprep)

DNApurification

Bisulfiteconversion

Pre-amplification

Assay design

ssDNA preparation

Pyro-sequencing

Sequencing primer

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Measuring frequencies in sequence variations


Example: DNA methylation analysis

.A G T T A C G A C A

.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

.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:

.After bisulfite conversion:

.Analyzed sequence:

Ratio T:C

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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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Example: DNA methylation analysis

Ratio T:CC=0%

T=100%

.A G T T A C G A C A

.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

.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:

.After bisulfite conversion:

.Analyzed sequence:

.Sequence to be analyzed:

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Improving bisulfite conversion efficiency


Superior bisulfite conversion. Following bisulfite conversion of genomic DNA using the EpiTect Fast DNA Bisulfite Kit or a kit from Supplier Z, unconverted genomic DNA and bisulfite converted DNA were coamplified and then sequenced by Pyrosequencing. EpiTect Fast Kits showed better results when a limited amount of DNA was analyzed (10 ng).

DNApurification

Bisulfiteconversion

Pre-amplification

Assay design

ssDNA preparation

Pyro-sequencing

EpiTect Fast Bisulfite conversion� Complete conversion ensures reliable quantification of DNA methylation

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Improving bisulfite conversion efficiency


EpiTect Fast Bisulfite conversion� Complete conversion ensures reliable quantification of DNA methylation

QIAGEN webinar:

“Accurate DNA methylation analysis by successful bi sulfite conversion”Dr. Wei Cao, Senior Global Market Manager

Recorded sessions:View online at www.qiagen.com

DNApurification

Bisulfiteconversion

Pre-amplification

Assay design

ssDNA preparation

Pyro-sequencing

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Outline






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Advanced Pyrosequencing Technology

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Advanced Pyrosequencing addresses main bottlenecks in conventional Pyrosequencing


� Insufficient read length

� Issues sequencing through homopolymer T sequences

� Challenging assay optimization

� Incomplete bisulfite conversion

� Tedious ssDNA preparation

Genomic sequenceTTCGCGATTGAATTCGAAAGACTCTCTTCGGCGGATGAAAGTCGTTATCTCTTGGTTGGTTGAGTTATAGTCTT

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Analysis of DNA methylation - Principle of bisulfite conversion


Bisulfite conversion – the key step in DNA methylation analysis

• Crucial step in methylation analysis workflow• Efficient base conversion required for accurate assessment of methylation status

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Effects of low level incomplete bisulfite conversion

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Sample: unmethylated DNA

C T C T C AgDNA G A C G A TT T T T T A G A T G A Tafter bis. ideally

T T T T T A G A T G A T

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

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

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

after bis. reality ~99.5%





After bisulfite conversion, some C nucleotides in somemolecules remain unconverted.Conversion efficiency of EpiTect Kits is around 99.4–99.6%. Other kits show conversion efficiencies of 95–98%.

Incomplete bisulfite conversion may affect Pyrosequencing results

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Effects of low level incomplete bisulfite conversion

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Sample: unmethylated DNA (conventional Pyrosequencing)

C T C T C A

T T T T T A

gDNA G A C G A T

G A T G A T








T T T T T A G A T G A Tafter bis. ideally

x%

T A G A T C G A T

single peak

5x peak

wrong calculation

peak deviations


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Multiple dispensations act as “catch up” in Advanced Pyrosequencing

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Sample: unmethylated DNA (Advanced Pyrosequencing)

0%

T A G A T C G

T T T T T A G A T G A T




A T

single peak

5x peak

+C+T

less peak deviations→ longer reads

C T C T C AgDNA G A C G A TT T T T T A G A T G A Tafter bis. ideally





correct calculation


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Incomplete bisulfite conversion may result in short read length


Conventional Pyrosequencing analysis of the LOX gene locusP

yroM

ark

Q24

trusted sequence uncertain sequence

Increasing peak deviation from expected signals in late sequence positions

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Advanced Pyrosequencing increases read length and trust


Conventional vs. Advanced Pyrosequencing analysis of the LOX gene locusP

yroM

ark

Q24

Pyr

oMar

k Q

24 A

dvan

ced


trusted sequence

Advanced Pyrosequencing overcomes issues of low level incomplete bisulfite conversion

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Issues with quantification of CpG methylation in homopolymers

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Genomic sequence

TTCGCGATTGAATTCGAAAGACTCTCTTCGGCGGATGAAAGTCGTTATCTCTTGGTTGGTTGAGTTATAGTCTT

After bisulfite conversion

TTYGYGATTGAATTYGAAAGATTTTTTTYGGYGGATGAAAGTYGTTATTTTTTGGTTGGTTGAGTTATAGTTTT

8xT if C is not methylated

7xT if C is methylated6xT 4xT

His

togr

am


Bisulfite conversion in DNA methylation analysis leads to occurrence of poly T stretches

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Issues with quantification of CpG methylation in homopolymers

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Pyr

oMar

k Q

24



PyroMark Q24 might not distinguish between light signals from 7 or 8 T nucleotides, resulting in uncertain quantification results behind homopolymers

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Improved quantification of CpG methylation in homopolymers

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Pyr

oMar

k Q

24P

yroM

ark

Q24

Adv

ance

d


trusted sequence


PyroMark Q24 Advanced enables reliable quantification of CpG methylation behindand even within a stretch of 8 T nucleotides

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Advanced Pyrosequencing addresses main bottlenecks in conventional Pyrosequencing

Advanced Pyrosequencing

• Longer Pyrosequencing read length

◦ de novo sequencing

◦ DNA methylation analysis

• Increased reliability in quantification of CpGsites and other sequence variations

• Increased robustness of assays

◦ Weak sequencing primer binding

◦ Low level incomplete bisulfite conversion

• Facilitates easier assay development


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Optimized software, reagents and instrument firmware working hand-in-hand

Advanced Pyrosequencing

• Longer Pyrosequencing read length

◦ De novo sequencing

◦ DNA methylation analysis

• Increased reliability in quantification of CpGsites and other sequence variations

• Increased robustness of assays

◦ Weak sequencing primer binding

◦ Low level incomplete bisulfite conversion

• Facilitates easier assay development

PyroMarkAnalysis SW

� optimizedrun setup files (e.g. additional dispensations)

PyroMark Advanced Reagents

� optimized nucleotide concentration

Instrument firmware

� optimized dispensation (e.g. double shots)

PyroMarkAnalysis SW

� optimizedanalysis


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PyroMark Platforms offering Advanced Pyrosequencing

Advanced Pyrosequencing is available on two PyroMark Platforms

PyroMark Q24 Advanced PyroMark Q48 Autoprep


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Outline






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PyroMark Q48 Autoprep – Workflow


Workflow comparison of available Pyrosequencing platforms

Samplepreparation

Assay design

PCRamplification

ssDNApreparation

Analysis

• PyroMark Q24 Vacuum Workstation

• PyroMark Q96 Vacuum Workstation

• 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

Automatic Template

Preparation

Anneal Sequencing

primer

Pyro-sequencing

Wash Cartridge


LoadRun files

via USB orEthernet

Loadreagents,

nucleotides,buffers

LoadPCR product& magnetic

beads

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Automatic Template

Preparation

Anneal Sequencing

primer

Pyro-sequencing

Wash Cartridge


LoadRun files

via USB orEthernet

Loadreagents,

nucleotides,buffers


beads

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Automatic Template

Preparation

Anneal Sequencing

primer

Pyro-sequencing

Wash Cartridge


LoadRun files

via USB orEthernet

Loadreagents,

nucleotides,buffers


beads

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LoadRun files

via USB orEthernet

Loadreagents,

nucleotides,buffers


beads

Automatic Template

Preparation

Anneal Sequencing

primer

Pyro-sequencing

Wash Cartridge

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PyroMark Q48 Autoprep – Dimensions & Weight

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390 mm (L, closed)

300

mm

(H

)

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

Small footprint and low weight (8.5 kg)


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

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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


beads

Automatic template

preparation

Anneal Seq-

primer

Pyro-sequencing

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


• Multi-step pipettecan be used for pipetting beads

• Automaticpipetting systemcan be used

manual automated manual/automated

• Automatic dispensationof up to 3 Seq primers

• Manual dispensationfor 4 or more Seqprimers

• Automatic dispensationof 3 MPD* Mixes

*MPD: Multiple Primer Dispensation

Wash Cartridge


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Summary


PyroMark Q48 Autoprep for “Advanced” Pyrosequencing in DNA methylation analysis

• Cost-efficient tool for analyzing DNA methylation

• Consecutive CpG and CpN sites analyzed independently in a single run

• Improved quantification at any sequence position, even at homopolymer T sequences

• Direct quality control of bisulfite conversion during the run

• Fast processing: 48 samples in minutes

• Automated Pyrosequencing through integrated template prep

• Low sample input amounts: 1–10 ng

• Highly accurate quantification:

• LOD: 5% in methylation analysis

PyroMark Q48 Autoprep: Simplified workflow combined with advanced Pyrosequencing for longer reads and more accurate methylation analysis

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

Request a demo

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Pyrosequencing resource center


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

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PyroMark CpG Assays for methylation array validation


https://www.qiagen.com/shop/genes-and-pathways/custom-products/custom-assay-products/pyromark-cpg-assays-hsmethylation/

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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 automated single base resolution DNA methylation analysis for epigenetic research

Gerald Schock, Ph.D.Associate Director PyrosequencingQIAGEN GmbH

Health & Medicine

New Progress in Pyrosequencing for DNA Methylation