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New Progress in Pyrosequencing for Automated Quantitative Analysis of Bi- or Multi-allelic Sequence Variations Webinar

The document discusses advancements in pyrosequencing technology for automated quantitative analysis of bi- or multi-allelic sequence variations, emphasizing its application in genotyping. It outlines challenges in quantitative genotyping analysis and presents the Pyromark Q48 Autoprep MPD strategy to enhance workflow efficiency. Additionally, it provides an overview of product offerings, assay design, and the sequencing process itself.

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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
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 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
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
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
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
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
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
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
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...”
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
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
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
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.
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
Sample to Insight Pyrosequencing for SNP Detection & Quantification New progress in Pyrosequencing for genotyping applications 36 Detection of tri- allelic SNPs
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%
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%
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
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
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
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
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
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
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
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
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 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
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 3 PTPN22 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/T Patient 2 T/T C/G G/G A/A A/A G/T G/G C/C T/T T/T A/A T/T Patient 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/T Patient 5 C/T G/G C/G A/A A/T G/G G/G T/T T/T C/T A/G C/T Patient 6 C/T C/G C/C A/A A/A T/T G/G T/T T/T C/T A/G C/T Patient 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/T Patient 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/T Patient 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/T Patient 13 C/C G/G C/C A/A A/T G/G A/A T/T T/T C/T G/G C/T Patient 14 C/C C/G C/G A/A A/A G/G A/G T/T T/T C/T G/G C/T Patient 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/T Patient 17 C/C C/G C/G A/A A/A G/T A/G C/T T/T C/T G/G T/T Patient 18 T/T G/G C/G A/G A/A G/G A/G C/T T/T T/T G/G T/T Patient 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/T Patient 21 C/C C/G C/G A/A T/T G/T A/G C/C C/T T/T A/A T/T Patient 22 C/C G/G G/G A/A A/T T/T A/G T/T C/C T/T A/G T/T Patient 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!”
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
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
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/
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/
Sample to Insight Backup slides New progress in Pyrosequencing for genotyping applications 59 Backup slides
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
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

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New Progress in Pyrosequencing for Automated Quantitative Analysis of Bi- or Multi-allelic Sequence Variations Webinar

  • 1.
    Sample to Insight Newprogress 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 QIAGENproducts 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 Newprogress 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 Newprogress 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 Challengesin 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 Challengesin 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 Challengesin 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 Challengesin 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 SequenceChromatograph 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 SequenceChromatograph 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 Sangersequencing 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 Newprogress 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 Theworkflow 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 QIAGENsolutions 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 QIAGENsPyrosequencing 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 Pyrosequencingworkflow – 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 Pyrosequencingworkflow - 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 Pyrosequencingworkflow – 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 Pyrosequencingworkflow – 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 Pyrosequencingworkflow – 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 Pyrosequencingworkflow – 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 Pyrosequencingworkflow – 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 Pyrosequencingfor SNP Detection & Quantification New progress in Pyrosequencing for genotyping applications 36 Detection of tri- allelic SNPs
  • 37.
    Sample to Insight Pyrosequencingfor 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 Pyrosequencingfor 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 Newprogress 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 PyroMarkQ48 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 PyroMarkQ48 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 PyroMarkQ48 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 PyroMarkQ48 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 PyroMarkQ48 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 PyroMarkQ48 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 Newprogress 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 MultiplePrimer 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 MultiplePrimer Dispensation for highest automation 48 Conventional Primer Dispensation theory New progress in Pyrosequencing for genotyping applications
  • 49.
    Sample to Insight MultiplePrimer Dispensation for highest automation 49 Multiple Primer Dispensation (MPD) theory New progress in Pyrosequencing for genotyping applications
  • 50.
    Sample to Insight MultiplePrimer 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 MultiplePrimer 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 MultiplePrimer 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.
  • 53.
    Sample to Insight MultiplePrimer 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 3 PTPN22 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/T Patient 2 T/T C/G G/G A/A A/A G/T G/G C/C T/T T/T A/A T/T Patient 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/T Patient 5 C/T G/G C/G A/A A/T G/G G/G T/T T/T C/T A/G C/T Patient 6 C/T C/G C/C A/A A/A T/T G/G T/T T/T C/T A/G C/T Patient 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/T Patient 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/T Patient 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/T Patient 13 C/C G/G C/C A/A A/T G/G A/A T/T T/T C/T G/G C/T Patient 14 C/C C/G C/G A/A A/A G/G A/G T/T T/T C/T G/G C/T Patient 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/T Patient 17 C/C C/G C/G A/A A/A G/T A/G C/T T/T C/T G/G T/T Patient 18 T/T G/G C/G A/G A/A G/G A/G C/T T/T T/T G/G T/T Patient 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/T Patient 21 C/C C/G C/G A/A T/T G/T A/G C/C C/T T/T A/A T/T Patient 22 C/C G/G G/G A/A A/T T/T A/G T/T C/C T/T A/G T/T Patient 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
  • 54.
    Sample to Insight PyroMarkQ48 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 Newprogress 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&Asession 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 PyroMarkQ48 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 PyroMarkQ48 Autoprep Intro Page 58New progress in Pyrosequencing for genotyping applications https://www.qiagen.com/de/resources/technologies/pyrosequencing-resource-center/
  • 59.
    Sample to Insight Backupslides New progress in Pyrosequencing for genotyping applications 59 Backup slides
  • 60.
    Sample to Insight PyroMarkinstrument 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 Pyrosequencingapplication 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

  • #6 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.
  • #7 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.
  • #8 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.
  • #9 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.
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