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Sample to Insight
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
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
Sample to Insight
New progress in Pyrosequencing for genotyping applications 3
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
Sample to Insight
New progress in Pyrosequencing for genotyping applications 4
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
Sample to Insight
New progress in Pyrosequencing for genotyping applications 5
Challenges in quantitative genotyping analysis
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
Sample to Insight
New progress in Pyrosequencing for genotyping applications 6
Challenges in quantitative genotyping analysis
PCR Real-time PCR• Detection of single sequence
variationso LOD <1%
• Quantification of single sequence variations o LOD typically <1%
Complex analysis
Non-quantitative Quantitative
Simple analysis
Seq
uenc
e va
riatio
n
Result requirement
xsingle mutation or SNP
xx x xx xmultiple mutations or SNPs
ABC D E F
Sample to Insight
New progress in Pyrosequencing for genotyping applications 7
Challenges in quantitative genotyping analysis
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%
• Quantification of single sequence variations o LOD typically <1%
Complex analysis
Non-quantitative Quantitative
Simple analysis
Seq
uenc
e va
riatio
n
Result requirement
xsingle mutation or SNP
xx x xx xmultiple mutations or SNPs
ABC D E F
Sample to Insight
New progress in Pyrosequencing for genotyping applications 8
Challenges in quantitative genotyping 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 variationso LOD approximately 20%o medium to long sequences
• Detection of single sequence variationso LOD <1%
• Quantification of single sequence variations o LOD typically <1%
Complex analysis
Non-quantitative Quantitative
Simple analysis
Seq
uenc
e va
riatio
n
Result requirement
xsingle mutation or SNP
xx x xx xmultiple mutations or SNPs
ABC D E F
Sample to Insight
New progress in Pyrosequencing for genotyping applications 9
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
Sample to Insight
New progress in Pyrosequencing for genotyping applications 10
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
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...”
Sample to Insight
New progress in Pyrosequencing for genotyping applications 11
Sanger sequencing vs. Pyrosequencing
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
Sample to Insight
New progress in Pyrosequencing for genotyping applications 12
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
Sample to Insight
New progress in Pyrosequencing for genotyping applications 13
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
Sample to Insight
New progress in Pyrosequencing for genotyping applications 14
Pyrosequencing – principle and key features
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.
Sample to Insight
New progress in Pyrosequencing for genotyping applications 15
Pyrosequencing – principle and key features
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
Sample to Insight
New progress in Pyrosequencing for genotyping applications 16
Pyrosequencing – principle and key features
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
Sample to Insight
New progress in Pyrosequencing for genotyping applications 17
Pyrosequencing – principle and key features
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
Sample to Insight
New progress in Pyrosequencing for genotyping applications 18
Pyrosequencing – principle and key features
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
Sample to Insight
New progress in Pyrosequencing for genotyping applications 19
Pyrosequencing – principle and key features
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
Sample to Insight
New progress in Pyrosequencing for genotyping applications 20
Pyrosequencing – principle and key features
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
New progress in Pyrosequencing for genotyping applications 21
Pyrosequencing – principle and key features
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
New progress in Pyrosequencing for genotyping applications 22
Pyrosequencing – principle and key features
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
New progress in Pyrosequencing for genotyping applications 23
Pyrosequencing – principle and key features
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
Sample to Insight
New progress in Pyrosequencing for genotyping applications 24
Pyrosequencing – principle and key features
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
New progress in Pyrosequencing for genotyping applications 25
Pyrosequencing – principle and key features
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
Sample to Insight
New progress in Pyrosequencing for genotyping applications 26
The workflow for genotyping analysis
Sample collection/
stabilization
DNA purification
Assays&Assay Setup
Pre-Amplification
Data analysis &
interpretation
Sample to Insight
New progress in Pyrosequencing for genotyping applications 27
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
Sample to Insight
New progress in Pyrosequencing for genotyping applications 28
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
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 interesto Annealed to single-stranded DNA before Pyrosequencing reaction
PCR primer
Region of interest PCR primer
Sequencing primer
Samplepreparation
Assaydesign
PCRAmplification
ssDNApreparation Analysis
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
Samplepreparation
Assaydesign
PCRAmplification
ssDNApreparation Analysis
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)
Samplepreparation
Assaydesign
PCRAmplification
ssDNApreparation Analysis
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)
Samplepreparation
Assaydesign
PCRAmplification
ssDNApreparation Analysis
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)
Samplepreparation
Assaydesign
PCRAmplification
ssDNApreparation Analysis
Sequencing primer
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)
Samplepreparation
Assaydesign
PCRAmplification
ssDNApreparation Analysis
Sequencing primer
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
Samplepreparation
Assaydesign
PCRAmplification
ssDNApreparation Analysis
C A
G A
C A
G A
X
X
X
X
X
X
X
X
Sample to Insight
New progress in Pyrosequencing for genotyping applications 36
Pyrosequencing for SNP Detection & Quantification
Detection of tri- allelic SNPs
Sample to Insight
New progress in Pyrosequencing for genotyping applications 37
Pyrosequencing for SNP Detection & Quantification
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%
Sample to Insight
New progress in Pyrosequencing for genotyping applications 38
Pyrosequencing for SNP Detection & Quantification
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%
Sample to Insight
New progress in Pyrosequencing for genotyping applications 39
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
Sample to Insight
New progress in Pyrosequencing for genotyping applications 40
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
Sample to Insight
New progress in Pyrosequencing for genotyping applications 41
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
Sample to Insight
New progress in Pyrosequencing for genotyping applications 42
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
Sample to Insight
New progress in Pyrosequencing for genotyping applications 43
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)
Sample to Insight
New progress in Pyrosequencing for genotyping applications 44
PyroMark Q48 Autoprep – SW User Interface
Large and easy to use touch screen and intuitive instrument SW
Sample to Insight
PyroMark Q48 Autoprep offers highest degree of automation
45
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
New progress in Pyrosequencing for genotyping applications
Sample to Insight
New progress in Pyrosequencing for genotyping applications 46
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
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
Sample to Insight
Multiple Primer Dispensation for highest automation
48
Conventional Primer Dispensation theory
New progress in Pyrosequencing for genotyping applications
Sample to Insight
Multiple Primer Dispensation for highest automation
49
Multiple Primer Dispensation (MPD) theory
New progress in Pyrosequencing for genotyping applications
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 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
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
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.
Sample to Insight
Multiple Primer Dispensation for highest automation
53
Genotyping of 23 patients with 3 different 4plex MPD mixes using 6 runs
New progress in Pyrosequencing for genotyping applications
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.
Data kindly provided by Rainer W. Fürst, Institute of Diabetes Research, Helmholtz Center Munich, Germany
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!”
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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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New progress in Pyrosequencing for genotyping applications
PyroMark Q48 Autoprep on the web
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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/
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New progress in Pyrosequencing for genotyping applications
PyroMark Q48 Autoprep Intro Page
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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
Genetic testingEpigeneticsMicrobiol IDDrug resistance typing
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