Pyrosequencing is a highly flexible technology based on sequencing-by-synthesis for the rapid and quantitative analysis of any type of sequence variation. The real-time output delivers high-resolution sequence information, making pyrosequencing highly suitable for applications ranging from biallelic or multiallelic SNP analysis, DNA methylation quantification to complex mutation analysis of multiple sequence variations in a single run.
In this slideeck, we introduce the new PyroMark Q48 Autoprep system which enables fully automated template preparation integrated in the pyrosequencing workflow. In addition, a new Multiple Primer Dispensation (MPD) strategy is presented which allows fully automated dispensation of sequencing primer, offering a seamless workflow from samples to quantitative genotyping results.
This slidedeck focuses on the following topics
• Pyrosequencing technology and workflow in genotyping analysis
• Introduction into the new PyroMark Q48 Autoprep
• MPD strategy for a seamless automated pyrosequencing workflow
Join us and learn how you can apply the new pyrosequencing system and protocol to your variant analysis or genotyping research
New Progress in Pyrosequencing for Automated Quantitative Analysis of Bi- or Multi-allelic Sequence Variations Webinar
1. Sample to Insight
New progress in pyrosequencing for fully automated
quantitative analysis of bi- or multi-allelic sequence variations
Gerald Schock, Ph.D.
Associate Director Pyrosequencing
QIAGEN GmbH
New progress in Pyrosequencing for genotyping applications 1
2. 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 genotyping applications
3. Sample to Insight
Outline
New progress in Pyrosequencing for genotyping applications 3
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
4. Sample to Insight
Outline
New progress in Pyrosequencing for genotyping applications 4
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
5. Sample to Insight
Challenges in quantitative genotyping analysis
New progress in Pyrosequencing for genotyping applications 5
PCR Real-time PCR
• Detection of single sequence
variations
o LOD <1%
• Quantification of single
sequence variations
o LOD typically <1%
Non-quantitative QuantitativeResult requirement
x
single mutation or SNP
6. Sample to Insight
Challenges in quantitative genotyping analysis
New progress in Pyrosequencing for genotyping applications 6
PCR Real-time PCR
• Detection of single sequence
variations
o LOD <1%
• Quantification of single
sequence variations
o LOD typically <1%
Complex
analysis
Non-quantitative Quantitative
Simple
analysis
Sequencevariation
Result requirement
x
single mutation or SNP
xx x xx x
multiple mutations or SNPs
ABC D E F
7. Sample to Insight
Challenges in quantitative genotyping analysis
New progress in Pyrosequencing for genotyping applications 7
Sanger sequencing
PCR Real-time PCR
• 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
Sequencevariation
Result requirement
x
single mutation or SNP
xx x xx x
multiple mutations or SNPs
ABC D E F
8. Sample to Insight
Challenges in quantitative genotyping analysis
New progress in Pyrosequencing for genotyping applications 8
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
Sequencevariation
Result requirement
x
single mutation or SNP
xx x xx x
multiple mutations or SNPs
ABC D E F
9. Sample to Insight
Sequence Chromatograph
Sanger sequencing vs. Pyrosequencing
New progress in Pyrosequencing for genotyping applications 9
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
10. Sample to Insight
Sequence Chromatograph
Sanger sequencing vs. Pyrosequencing
New progress in Pyrosequencing for genotyping applications 10
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...”
11. Sample to Insight
Sanger sequencing vs. Pyrosequencing
New progress in Pyrosequencing for genotyping applications 11
12Wild-type sequence
GGT>GTT
Gly12Val
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
Sequence chromatograph
12. Sample to Insight
Outline
New progress in Pyrosequencing for genotyping applications 12
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
13. Sample to Insight
Pyrosequencing – principle and key features
New progress in Pyrosequencing for genotyping applications 13
.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
14. Sample to Insight
Pyrosequencing – principle and key features
New progress in Pyrosequencing for genotyping applications 14
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*
• 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.
15. Sample to Insight
Pyrosequencing – principle and key features
New progress in Pyrosequencing for genotyping applications 15
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
• Stepwise synthesis of DNA by addition of nucleotides
• Enzyme cascade generates a light signal upon incorporation of nucleotides
16. Sample to Insight
Pyrosequencing – principle and key features
New progress in Pyrosequencing for genotyping applications 16
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.
Watch the complete animation at: www.qiagen.com/pyrosequencing-reaction-cascade
.Based on SEQUENCING-by-SYNTHESIS Principle
• Stepwise synthesis of DNA by addition of nucleotides
• Enzyme cascade generates a light signal upon incorporation of nucleotides
17. Sample to Insight
Pyrosequencing – principle and key features
New progress in Pyrosequencing for genotyping applications 17
Step 4
Apyrase continuously degrades
unincorporated nucleotides and ATP.
When degradation is complete, another
nucleotide is added.
Watch the complete animation at: www.qiagen.com/pyrosequencing-reaction-cascade
.Based on SEQUENCING-by-SYNTHESIS Principle
• Stepwise synthesis of DNA by addition of nucleotides
• Enzyme cascade generates a light signal upon incorporation of nucleotides
18. Sample to Insight
Pyrosequencing – principle and key features
New progress in Pyrosequencing for genotyping applications 18
Repeat step 2–4
Watch the complete animation at: www.qiagen.com/pyrosequencing-reaction-cascade
.Based on SEQUENCING-by-SYNTHESIS Principle
• Stepwise synthesis of DNA by addition of nucleotides
• Enzyme cascade generates a light signal upon incorporation of nucleotides
19. Sample to Insight
Pyrosequencing – principle and key features
New progress in Pyrosequencing for genotyping applications 19
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
Watch the complete animation at: www.qiagen.com/pyrosequencing-reaction-cascade
.Based on SEQUENCING-by-SYNTHESIS Principle
• Stepwise synthesis of DNA by addition of nucleotides
• Enzyme cascade generates a light signal upon incorporation of nucleotides
20. Sample to Insight
Pyrosequencing – principle and key features
New progress in Pyrosequencing for genotyping applications 20
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
21. Sample to Insight
Pyrosequencing – principle and key features
New progress in Pyrosequencing for genotyping applications 21
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
22. Sample to Insight
Pyrosequencing – principle and key features
New progress in Pyrosequencing for genotyping applications 22
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
23. Sample to Insight
Pyrosequencing – principle and key features
New progress in Pyrosequencing for genotyping applications 23
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
DNA methylation of multiple CpG sites
.Based on SEQUENCING-by-SYNTHESIS Principle
• Stepwise synthesis of DNA by addition of nucleotides
• Enzyme cascade generates a light signal upon incorporation of nucleotides
24. Sample to Insight
Pyrosequencing – principle and key features
New progress in Pyrosequencing for genotyping applications 24
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
25. Sample to Insight
Pyrosequencing – principle and key features
New progress in Pyrosequencing for genotyping applications 25
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
QIAGEN webinar:
Advanced single base resolution DNA methylation
and mutation analysis in long sequence runs using
Pyrosequencing
View online at www.qiagen.com
.Based on SEQUENCING-by-SYNTHESIS Principle
• Stepwise synthesis of DNA by addition of nucleotides
• Enzyme cascade generates a light signal upon incorporation of nucleotides
26. Sample to Insight
The workflow for genotyping analysis
New progress in Pyrosequencing for genotyping applications 26
Sample
collection/
stabilization
DNA
purification
Assays&
Assay Setup
Pre-
Amplification
Data analysis
&
interpretation
27. Sample to Insight
QIAGEN solutions for genotyping analysis
New progress in Pyrosequencing for genotyping applications 27
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)
QIAGENsolutions
InstrumentsKits
PCR/
qPCR
PCR
arrays
• 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
28. Sample to Insight
QIAGENs Pyrosequencing solutions for genotyping analysis
New progress in Pyrosequencing for genotyping applications 28
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
QIAGENsolutions
InstrumentsKits
• PyroMark Assay
Design SW
Pyrosequencing • 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
29. Sample to Insight
Pyrosequencing workflow – assay design
New progress in Pyrosequencing for genotyping applications 29
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 interest
o Annealed to single-stranded DNA before Pyrosequencing reaction
PCR primer
Region of interest
PCR primer
Sequencing primer
Sample
preparation
Assay
design
PCR
Amplification
ssDNA
preparation
Analysis
30. Sample to Insight
Pyrosequencing workflow - PCR
New progress in Pyrosequencing for genotyping applications 30
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
Sample
preparation
Assay
design
PCR
Amplification
ssDNA
preparation
Analysis
31. Sample to Insight
Pyrosequencing workflow – template preparation
New progress in Pyrosequencing for genotyping applications 31
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)
Sample
preparation
Assay
design
PCR
Amplification
ssDNA
preparation
Analysis
32. Sample to Insight
Pyrosequencing workflow – template preparation
New progress in Pyrosequencing for genotyping applications 32
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)
Sample
preparation
Assay
design
PCR
Amplification
ssDNA
preparation
Analysis
33. Sample to Insight
Pyrosequencing workflow – template preparation
New progress in Pyrosequencing for genotyping applications 33
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)
Sample
preparation
Assay
design
PCR
Amplification
ssDNA
preparation
Analysis
Sequencing primer
34. Sample to Insight
Pyrosequencing workflow – template preparation
New progress in Pyrosequencing for genotyping applications 34
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)
Sample
preparation
Assay
design
PCR
Amplification
ssDNA
preparation
Analysis
Sequencing primer
35. Sample to Insight
Pyrosequencing workflow – analysis
New progress in Pyrosequencing for genotyping applications 35
Pyrosequencing analysis
• Each PCR strand is sequenced individually
• Variants are called and quantified according to their peak heights during sequencing
Sample
preparation
Assay
design
PCR
Amplification
ssDNA
preparation
Analysis
C A
G A
C A
G A
X
X
X
X
X
X
X
X
36. Sample to Insight
Pyrosequencing for SNP Detection & Quantification
New progress in Pyrosequencing for genotyping applications 36
Detection of tri- allelic SNPs
37. Sample to Insight
Pyrosequencing for SNP Detection & Quantification
New progress in Pyrosequencing for genotyping applications 37
Quantitative peak heights to measure allele frequencies
Allele frequency (%)
0%
2%
5%
10%
15%
20%
25%
30%
35%
40%
45%
55%
60%
65%
70%
75%
80%
90%
50%
50%
85%
85%
38. Sample to Insight
Pyrosequencing for SNP Detection & Quantification
New progress in Pyrosequencing for genotyping applications 38
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.peakheight
SNP E1
Linear (SNP E1)
Even as little as 2% of one allele
in 98% of the other could be detected
50%
85%
39. Sample to Insight
Outline
New progress in Pyrosequencing for genotyping applications 39
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
40. Sample to Insight
PyroMark Q48 Autoprep – Workflow
New progress in Pyrosequencing for genotyping applications 40
Workflow comparison of available Pyrosequencing platforms
Sample
preparation
Assay
design
PCR
Amplification
ssDNA
preparation
Analysis
• PyroMark Q24
Vacuum Workstation
• PyroMark Q96
Vacuum Workstation
• PyroMark Q24
• PyroMark Q24 Adv.
• PyroMark Q96 ID
41. Sample to Insight
PyroMark Q48 Autoprep – Workflow
New progress in Pyrosequencing for genotyping applications 41
Workflow comparison of available Pyrosequencing platforms
Sample
preparation
Assay
design
PCR
Amplification
ssDNA
preparation
Analysis
• 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
42. Sample to Insight
PyroMark Q48 Autoprep – Protocol
New progress in Pyrosequencing for genotyping applications 42
Automatic template preparation fully integrated in PyroMark Q48 Autoprep workflow
Load
reagents,
nucleotides,
buffers
Load
PCR product
& magnetic
beads
Insert
Absorber
strip & Disc
Load
Run files
via USB or
Ethernet
Automatic
Template
Preparation
Pyro-
sequencing
Analyze
data
43. Sample to Insight
PyroMark Q48 Autoprep – Dimensions & Weight
43
Small footprint (1/2 x PyroMark Q24) and low weight (1/3 x PyroMark Q24)
390 mm (L, closed)
300mm(H)
560 mm (L, open)
250 mm (W)
Small footprint and low weight (8,5 kg)
New progress in Pyrosequencing for genotyping applications
44. Sample to Insight
PyroMark Q48 Autoprep – SW User Interface
New progress in Pyrosequencing for genotyping applications 44
Large and easy to use touch screen and intuitive instrument SW
45. Sample to Insight
PyroMark Q48 Autoprep offers highest degree of automation
45
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 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
New progress in Pyrosequencing for genotyping applications
46. Sample to Insight
Outline
New progress in Pyrosequencing for genotyping applications 46
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
47. Sample to Insight
Multiple Primer Dispensation for highest automation
47
Multiple Primer Dispensation (MPD) theory
New progress in Pyrosequencing for genotyping applications
PyroMark Q48 Primer Cartridge
PyroMark Q48 Autoprep
48. Sample to Insight
Multiple Primer Dispensation for highest automation
48
Conventional Primer Dispensation theory
New progress in Pyrosequencing for genotyping applications
49. Sample to Insight
Multiple Primer Dispensation for highest automation
49
Multiple Primer Dispensation (MPD) theory
New progress in Pyrosequencing for genotyping applications
50. Sample to Insight
Multiple Primer Dispensation for highest automation
50
MPD workflow for SNP testing using PyroMark Q48 Autoprep
New progress in Pyrosequencing for genotyping applications
PTPN22 BACH2 GLIS3 RNLS
INS ERBB3 ORMDL3IL27
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
51. Sample to Insight
Multiple Primer Dispensation for highest automation
51
MPD workflow for SNP testing using PyroMark Q48 Autoprep
New progress in Pyrosequencing for genotyping applications
Design
assays
Check
PCR
product
Check
Seq-
primer
Check
quality
Pyro-
sequencing
52. Sample to Insight
Multiple Primer Dispensation for highest automation
52
MPD workflow for SNP testing using PyroMark Q48 Autoprep
New progress in Pyrosequencing for genotyping applications
Design
assays
Check
PCR
product
Check
Seq-
primer
Check
quality
Pyro-
sequencing
Data kindly provided by Rainer W. Fürst, Institute of Diabetes Research, Helmholtz Center Munich, Germany
Color coding of the analysis software provides a convenient overview about the
quality of results obtained. Detailed information is available in each Pyrogram.
54. Sample to Insight
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!”
55. Sample to Insight
Summary
New progress in Pyrosequencing for genotyping applications 55
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
56. Sample to Insight
Q&A session
New progress in Pyrosequencing for genotyping applications 56
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 variations
Gerald Schock, PhD.
Associate Director Pyrosequencing
QIAGEN GmbH
57. Sample to Insight
PyroMark Q48 Autoprep on the web
57
More information about the platform, accessories and reagents are available online
New progress in Pyrosequencing for genotyping applications
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/
58. Sample to Insight
PyroMark Q48 Autoprep Intro Page
58New progress in Pyrosequencing for genotyping applications
https://www.qiagen.com/de/resources/technologies/pyrosequencing-resource-center/
59. Sample to Insight
Backup slides
New progress in Pyrosequencing for genotyping applications 59
Backup slides
60. Sample to Insight
PyroMark instrument comparison
60
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,
mutation
CpG and CpN methylation
de-novo sequencing
SNP,
mutation
CpG and CpN methylation
de-novo sequencing
SNP,
mutation
CpG methylation
de-novo sequencing
Adressable Markets/
Main Markets
Genetic testing
Epigenetics
Microbiol ID
Drug resistance typing
Genetic testing
Epigenetics
Microbiol ID
Drug resistance typing
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
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
New progress in Pyrosequencing for genotyping applications
61. Sample to Insight
Pyrosequencing application overview
New progress in Pyrosequencing for genotyping applications 61
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 ID
Identification 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
Editor's Notes
For screening and discovery technologies are used which provide a large genome coverage whereas for V and V technologies are used which can handle large sample numbers at a reasonable price. Because you agree it doesn’t make sense to use NGS for hundreds of samples. There are different technologies which can be used…… but Pyro has some striking advantages when to comes and price/speed and sensitivity . In the past sanger has been used quite often for the verification of sequencing data.
For screening and discovery technologies are used which provide a large genome coverage whereas for V and V technologies are used which can handle large sample numbers at a reasonable price. Because you agree it doesn’t make sense to use NGS for hundreds of samples. There are different technologies which can be used…… but Pyro has some striking advantages when to comes and price/speed and sensitivity . In the past sanger has been used quite often for the verification of sequencing data.
For screening and discovery technologies are used which provide a large genome coverage whereas for V and V technologies are used which can handle large sample numbers at a reasonable price. Because you agree it doesn’t make sense to use NGS for hundreds of samples. There are different technologies which can be used…… but Pyro has some striking advantages when to comes and price/speed and sensitivity . In the past sanger has been used quite often for the verification of sequencing data.
For screening and discovery technologies are used which provide a large genome coverage whereas for V and V technologies are used which can handle large sample numbers at a reasonable price. Because you agree it doesn’t make sense to use NGS for hundreds of samples. There are different technologies which can be used…… but Pyro has some striking advantages when to comes and price/speed and sensitivity . In the past sanger has been used quite often for the verification of sequencing data.