Sample to Insight 1 Overcome challenges in epigenetics research - DNA methylation techniques, pyrosequencing and applications Part 1: DNA Methylation – an Essential Element in Epigenetics Facts 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 Pyrosequencing QIAGEN GmbH
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New Progress in Pyrosequencing for DNA Methylation
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Sample to Insight
1
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
Sample to Insight
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 epigentic applications
Sample to Insight
Common technologies in epigenetic DNA methylation analysis
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Technologies used at different steps within a project
New progress in Pyrosequencing for epigentic applications
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
Sample to Insight
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
Sample to Insight
Challenges in Pyrosequencing DNA methylation analysis
5
Current challenges observed during Pyrosequencing analysis
� Insufficient read length
� Issues sequencing through homopolymer T sequences
New progress in Pyrosequencing for epigentic applications
Sample to Insight
Outline
New progress in Pyrosequencing for epigentic applications 6
Challenges in Pyrosequencing DNA methylation analysis
Pyrosequencing technology and workflow
"Advanced Pyrosequencing" technology
Introduction into the new PyroMark Q48 Autoprep
Sample to Insight
Pyrosequencing – principle and key features
New progress in Pyrosequencing for epigentic applications 7
.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
Sample to Insight
Pyrosequencing – principle and key features
New progress in Pyrosequencing for epigentic applications 8
Sequencing through unknown regions
Sequencing through unknown regions
.Based on SEQUENCING-by-SYNTHESIS Principle
• Stepwise synthesis of DNA by addition of nucleotides• Enzyme cascade generates a light signal upon incorporation of nucleotides
Sample to Insight
Pyrosequencing – principle and key features
New progress in Pyrosequencing for epigentic applications 9
A: 44%C: 0%G: 56%T: 0%
Di-, tri- and tetra-allelic mutations / SNP
A: 44%C: 0%G: 56%T: 0%
Di-, tri- and tetra-allelic mutations / SNP
Sequencing through unknown regions
.Based on SEQUENCING-by-SYNTHESIS Principle
• Stepwise synthesis of DNA by addition of nucleotides• Enzyme cascade generates a light signal upon incorporation of nucleotides
Sample to Insight
Pyrosequencing – principle and key features
New progress in Pyrosequencing for epigentic applications 10
A: 44%C: 0%G: 56%T: 0%
Di-, tri- and tetra-allelic mutations / SNP
Sequencing through unknown regions
.Based on SEQUENCING-by-SYNTHESIS Principle
• Stepwise synthesis of DNA by addition of nucleotides• Enzyme cascade generates a light signal upon incorporation of nucleotides
QIAGEN webinar:
New progress in Pyrosequencing forautomated quantitative analysis of bi- or multi-allelic sequence variations
Recorded sessions:View online at www.qiagen.com
Sample to Insight
Pyrosequencing – principle and key features
New progress in Pyrosequencing for epigentic applications 11
Insertions / Deletions
- - - - - - - : 56%ATCTGCC: 44%
C: 57%T: 43%
A: 44%C: 0%G: 56%T: 0%
Di-, tri- and tetra-allelic mutations / SNP
Insertions / Deletions
- - - - - - - : 56%ATCTGCC: 44%
C: 57%T: 43%
Sequencing through unknown regions
.Based on SEQUENCING-by-SYNTHESIS Principle
• Stepwise synthesis of DNA by addition of nucleotides• Enzyme cascade generates a light signal upon incorporation of nucleotides
Sample to Insight
Pyrosequencing – principle and key features
New progress in Pyrosequencing for epigentic applications 12
DNA methylation of multiple CpG sites
A: 44%C: 0%G: 56%T: 0%
Di-, tri- and tetra-allelic mutations / SNP
Insertions / Deletions
- - - - - - - : 56%ATCTGCC: 44%
C: 57%T: 43%
DNA methylation of multiple CpG sites
Sequencing through unknown regions
.Based on SEQUENCING-by-SYNTHESIS Principle
• Stepwise synthesis of DNA by addition of nucleotides• Enzyme cascade generates a light signal upon incorporation of nucleotides
Sample to Insight
QIAGEN’s Pyrosequencing solutions for DNA methylation analysis
13
• 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
New progress in Pyrosequencing for epigentic applications
QIAcube QIAxpert QIAxcel Adv.QC
0,5 days 0,5 days
PyroMark Assay Design SW
PyroMark Q48 Advanced Kit
PyroMark PCR KitEpiTect Fast DNA
Sample to Insight
Pyrosequencing workflow – Assay design
New progress in Pyrosequencing for epigentic applications 14
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
Sample to Insight
Pyrosequencing workflow – Assay design
New progress in Pyrosequencing for epigentic applications 15
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
Sample to Insight
Pyrosequencing workflow – PCR
New progress in Pyrosequencing for epigentic applications 16
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
Sample to Insight
Pyrosequencing workflow – Template preparation
New progress in Pyrosequencing for epigentic applications 17
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
Sample to Insight
Pyrosequencing workflow – Template preparation
New progress in Pyrosequencing for epigentic applications 18
Annealing of Sequencing primer• Sequencing primer only binds to biotinylated PCR strand• ssDNA is needed for binding• Binding done
New progress in Pyrosequencing for epigentic applications 19
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
Sample to Insight
Measuring frequencies in sequence variations
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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.
New progress in Pyrosequencing for epigentic applications
Even as little as 2% of one allele in 98% of the other could be detected
Sample to Insight
Measuring frequencies in sequence variations
New progress in Pyrosequencing for epigentic applications 21
New progress in Pyrosequencing for epigentic applications 22
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
Sample to Insight
Improving bisulfite conversion efficiency
New progress in Pyrosequencing for epigentic applications 23
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
Sample to Insight
Outline
New progress in Pyrosequencing for epigentic applications 24
Challenges in Pyrosequencing DNA methylation analysis
Pyrosequencing technology and workflow
"Advanced Pyrosequencing" technology
Introduction into the new PyroMark Q48 Autoprep
Sample to Insight
Advanced Pyrosequencing Technology
25
Advanced Pyrosequencing addresses main bottlenecks in conventional Pyrosequencing
New progress in Pyrosequencing for epigentic applications
� Insufficient read length
� Issues sequencing through homopolymer T sequences
Analysis of DNA methylation - Principle of bisulfite conversion
New progress in Pyrosequencing for epigentic applications 26
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
Sample to Insight
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 bis. reality ~0.1%
after bis. reality ~0.1%
after bis. reality ~0.1%
New progress in Pyrosequencing for epigentic applications
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
Sample to Insight
Effects of low level incomplete bisulfite conversion
28
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
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 bis. reality ~0.1%
after bis. reality ~0.1%
after bis. reality ~0.1%
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
New progress in Pyrosequencing for epigentic applications
Sample to Insight
Multiple dispensations act as “catch up” in Advanced Pyrosequencing
29
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
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
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
after bis. reality ~99.5%
after bis. reality ~0.1%
after bis. reality ~0.1%
after bis. reality ~0.1%
correct calculation
New progress in Pyrosequencing for epigentic applications
Sample to Insight
Incomplete bisulfite conversion may result in short read length
New progress in Pyrosequencing for epigentic applications 30
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
Sample to Insight
Advanced Pyrosequencing increases read length and trust
New progress in Pyrosequencing for epigentic applications 31
Conventional vs. Advanced Pyrosequencing analysis of the LOX gene locusP
yroM
ark
Q24
Pyr
oMar
k Q
24 A
dvan
ced
trusted sequence uncertain sequence
trusted sequence
Advanced Pyrosequencing overcomes issues of low level incomplete bisulfite conversion
Sample to Insight
Issues with quantification of CpG methylation in homopolymers
New progress in Pyrosequencing for epigentic applications 51
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