Dna Methylation Analysis in a Single Day - Download the Slides
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The document describes the capabilities and features of the new Pyromark Q48 Autoprep for DNA methylation analysis, which allows researchers to obtain insights in a single day. It outlines advancements in pyrosequencing technology, bisulfite conversion efficiency, and methodologies for analyzing both 5-methylcytosine and 5-hydroxymethylcytosine. The document emphasizes the importance of overcoming bottlenecks in methylation analysis to enhance data accuracy and streamline workflows.
Dna Methylation Analysis in a Single Day - Download the Slides
- 1. Sample to Insight DNA methylation analysis in a single day Gerald Schock, Ph.D. Associate Director, Pyrosequencing The new PyroMark Q48 Autoprep DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 1
- 2. Sample to Insight Legal disclaimer 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. DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 2
- 3. Sample to Insight Outline DNA methylation analysis and bisulfite conversion1 5-Hydroxymethylcytosine analysis2 5-mC and 5-hmC analyses by Pyrosequencing – technology and workflow3 Addressing bottlenecks in bisulfite conversion by 'advanced Pyrosequencing'4 Streamlining Pyrosequencing analysis using PyroMark Q48 Autoprep5 DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 3
- 4. Sample to Insight Outline DNA methylation analysis and bisulfite conversion1 5-Hydroxymethylcytosine analysis2 5-mC and 5-hmC analyses by Pyrosequencing – technology and workflow3 Addressing bottlenecks in bisulfite conversion by 'advanced Pyrosequencing'4 Streamlining Pyrosequencing analysis using PyroMark Q48 Autoprep5 DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 4
- 5. Sample to Insight Epigenetics DNA methylation and the use of epigenetic marker analysis C 5mC ~5% DNA regions enriched with CpG dinucleotides are localized in promoters and regulatory elements (5’ upstream regions) – Methylation regulates gene expression . .Epigenetic mechanisms • DNA (CpG) methylation • Histone modification • microRNA CH2OH CH3CH3CH3CH3 * CH3CH3CH3 CpG islandCpG shoresCpG shelfCpG canyon DNA DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 5
- 6. Sample to Insight Common technologies in epigenetic DNA methylation analysis 6 Technologies used at different steps within a project DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep Screening / Discovery Verification / Validation Functional studies • Genome-wide analysis ◦ Next Generation Sequencing (NGS) ◦ Arrays • Targeted analysis ◦ NGS panels ◦ HRM • Single CpG sites ◦ MS-PCR* ◦ Real-time MS-PCR* • Single or multiple sites ◦ Sanger sequencing ◦ Pyrosequencing • Single CpG sites ◦ Real-time MS-PCR* • Single or multiple sites ◦ Pyrosequencing ◦ Mass Array Genome coverage Sample number *MS-PCR: Methylation-Specific PCR
- 7. Sample to Insight Technologies for the verification of DNA methylation results DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 7 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/SNP or CpG site xx x xx x multiple mutations or CpG sites ABC D E F
- 8. Sample to Insight Epigenetics DNA methylation and the use of epigenetic marker analysis C 5mC ~5% DNA methylation and hydroxymethylation cannot be analyzed directly by PCR-based technologies. Unmethylated sites must be selectively converted before analysis. CH2OH CH3CH3CH3CH3 * CH3CH3CH3 CpG islandCpG shoresCpG shelfCpG canyon DNA DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 8 5hmC 0.03-0.7% *
- 9. Sample to Insight Bisulfite treatment for DNA methylation analysis Selective conversion of unmethylated CpG by bisulfite treatment • Bisulfite converts cytosines into uracil ◦ Methylated cytosines in CpG sequences remain unaffected ◦ All unmethylated CpG sequences are converted to UpG • Uracil is replaced by thymine during PCR reaction Principal steps of bisulfite treatment DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 9 G G T C A G T G A C/ m C G G G T U A G T G A U/ m C G G G T T A G T G A T/C G Bisulfite conversion PCR amplification
- 10. Sample to Insight Improving bisulfite conversion efficiency Bottlenecks or challenges Incomplete bisulfite conversion • Unmethylated cytosines remain as cytosines • False positive methylation results • Incorrect data interpretation Issues in Pyrosequencing derived from incomplete bisulfite conversion • Bisulfite conversion of genomic DNA using a kit from supplier Z • Ten nanograms of the resulting DNA including unconverted genomic DNA and bisulfite converted DNA were coamplified and then sequenced by Pyrosequencing DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 10 G G T C A G T G A C/ m C G G G T U/C A G T G A U/C/ m C G G G T T/C A G T G A T/C/C G Incomplete Bisulfite conversion PCR amplification
- 11. Sample to Insight Improving bisulfite conversion efficiency EpiTect Bisulfite Technology A unique buffer – the DNA Protect Buffer • Facilitates formation of single-stranded DNA • Ensures complete cytosine conversion ◦ Internal data: 99.4%–99.8% (bisulfite sequencing) ◦ Published user data: Taylor et al. 2007: 98.8% (454® Sequencing) Meissner et al. 2008: >99% (Solexa® Genome Analyzer) • Prevents DNA fragmentation during bisulfite conversion reaction ◦ Results in high molecular bisulfite converted DNA DNA Protect Buffer Bisulfite reaction setup Mixed bisulfite reaction Analysis of DNA fragment sizes after bisulfite conversion • A comparison of EpiTect Fast DNA Bisulfite Kit with other supplier kits; 1 µg of gDNA was used for bisulfite conversion • EpiTect Fast DNA Bisulfite Kit showed the lowest level of fragmentation – size ranged from several hundred base pairs to over 6 kb • Other supplier kits showed peaks at 500 bp and 1 kb DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 11
- 12. Sample to Insight Outline DNA methylation analysis and bisulfite conversion1 5-Hydroxymethylcytosine analysis2 5-mC and 5-hmC analyses by Pyrosequencing – technology and workflow3 Addressing bottlenecks in bisulfite conversion by 'advanced Pyrosequencing'4 Streamlining Pyrosequencing analysis using PyroMark Q48 Autoprep5 DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 12
- 13. Sample to Insight Analysis of 5-hydroxymethylation The importance of 5-hydroxymethylcytosine (5-hmC) Key findings • Oxidative product of 5-mC – catalyzed by the TET enzymes • Found in embryonic stem cells and neuronal cells • Promotes gene expression during active demethylation Sources: • Ito, S. et al. (2011) Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science 333, 1300–1303. • Branco, M.R., Ficz, G., and Reik, W. (2011) Uncovering the role of 5-hydroxymethylcytosine in the epigenome. Nat. Rev. Genet. 13, 7–13 DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 13 C 5mC ~5% 5hmC 0.03-0.7%
- 14. Sample to Insight Analysis of 5-hydroxymethylation 5-hmC cannot be discriminated from 5-mC by bisulfite conversion Principal steps of bisulfite treatment • Bisulfite converts cytosines into uracil ◦ (Hydroxy) methylated cytosines in CpG sequences remain unaffected ◦ All unmethylated CpG sequences are converted to UpG • Uracil is replaced by thymine during PCR reaction G G T C A G T G A C/ m C G T T A C/ 5hm C G G G T U A G T G A U/ m C G T T A U/ 5hm C G G G T T A G T G A T/C G T T A T/C G Bisulfite conversion PCR amplification Bisulfite conversion PCR amplification DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 14
- 15. Sample to Insight Analysis of 5-hydroxymethylation Oxidative bisulfite sequencing (oxBs-Seq) to analyze 5-hmC Principal steps of oxBs-Seq • Oxidation of 5-hydroxymethylcytosine (5-hmC) to 5-formylcytosine (5-fC) • Bisulfite conversion of genomic DNA and DNA after oxidation • Discriminate 5-hmC from 5-mC by comparing both sequencing runs Source: Booth, J. M. et al. (2013) Oxidative bisulfite sequencing of 5-methylcytosine and 5-hydroxymethylcytosine. Nature Protocols 8, 1841-1851 oxidation DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 15 G G T C A G T G A C/ m C G T T A C/ 5hm C G G G T U A G T G A U/ m C G T T A U/ 5hm C G G G T T A G T G A T/C G T T A T/C G Bisulfite conversion PCR amplification Bisulfite conversion PCR amplification G G T C A G T G A C/ m C G T T A C/ 5f C G G G T U A G T G A U/ m C G T T A U/U G G G T T A G T G A T/C G T T A T/T G Bisulfite conversion PCR amplification Bisulfite conversion PCR amplification
- 16. Sample to Insight Analysis of 5-hydroxymethylation Oxidative bisulfite sequencing (oxBs-Seq) to analyze 5-hmC Principal steps of oxBs-Seq • Oxidation of 5-hydroxymethylcytosine (5-hmC) to 5-formylcytosine (5-fC) • Bisulfite conversion of genomic DNA and DNA after oxidation • Discriminate 5-hmC from 5-mC by comparing both sequencing runs Source: Booth, J. M. et al. (2013) Oxidative bisulfite sequencing of 5-methylcytosine and 5-hydroxymethylcytosine. Nature Protocols 8, 1841-1851 oxidation DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 16 C / m C / 5hm C C / m C / 5f C T / C / C T / C / T 20% / 70% / 10% 20% / 70% / 10% 80% 70% Methylation measured by Pyrosequencing 80% - 70% = 10% 5h-methylation
- 17. Sample to Insight Outline DNA methylation analysis and bisulfite conversion1 5-Hydroxymethylcytosine analysis2 5-mC and 5-hmC analyses by Pyrosequencing – technology and workflow3 Addressing bottlenecks in bisulfite conversion by 'advanced Pyrosequencing'4 Streamlining Pyrosequencing analysis using PyroMark Q48 Autoprep5 DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 17
- 18. Sample to Insight Pyrosequencing – Principle and key features Pyrosequencing is based on the sequencing-by-synthesis principle Principle • Stepwise synthesis of DNA by addition of nucleotides • Enzyme cascade generates a light signal upon incorporation of nucleotides Step 1: Hybridization of a sequencing primer Source: Ronaghi, M., Uhlén, M., and Nyrén, P. (1998) A sequencing method based on real-time pyrophosphate. Science 281, 363–365. DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 18
- 19. Sample to Insight Pyrosequencing – Principle and key features Pyrosequencing is based on the sequencing-by-synthesis principle Principle • Stepwise synthesis of DNA by addition of nucleotides • Enzyme cascade generates a light signal upon incorporation of nucleotides Steps 2–4: Addition of dNTPs, conversion into light signal and degradation of nucleotides DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 19
- 20. Sample to Insight Pyrosequencing – Principle and key features Pyrosequencing is based on the sequencing-by-synthesis principle Principle • Stepwise synthesis of DNA by addition of nucleotides • Enzyme cascade generates a light signal upon incorporation of nucleotides Step 5: Pyrogram generation and data analysis DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 20
- 21. Sample to Insight Pyrosequencing – Principle and key features Pyrosequencing is based on the sequencing-by-synthesis principle Pyrosequencing data in various application areas Sequencing through unknown regions DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 21 Sequencing through unknown regions
- 22. Sample to Insight Pyrosequencing – Principle and key features Pyrosequencing is based on the sequencing-by-synthesis principle Pyrosequencing data in various application areas Di-, tri- and tetra-allelic mutations/SNP A : 44% C : 0% G : 56% T : 0% DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 22 A: 44% C: 0% G: 56% T: 0% Di-, tri- and tetra-allelic mutations/SNP Sequencing through unknown regions
- 23. Sample to Insight Pyrosequencing – Principle and key features Pyrosequencing is based on the sequencing-by-synthesis principle Pyrosequencing data in various application areas QIAGEN webinar: New progress in Pyrosequencing for automated quantitative analysis of bi- or multi-allelic sequence variations Recorded sessions: https://attendee.gotowebinar.com/ recording/1567587296094690306 DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 23 A: 44% C: 0% G: 56% T: 0% Di-, tri- and tetra-allelic mutations/SNP Sequencing through unknown regions
- 24. Sample to Insight Pyrosequencing – Principle and key features Pyrosequencing is based on the sequencing-by-synthesis principle Pyrosequencing data in various application areas - - - - - - - : 56% ATCTGCC: 44% C: 57% T: 43% Insertions and deletions Sequencing through unknown regions A: 44% C: 0% G: 56% T: 0% Di-, tri- and tetra-allelic mutations/SNP DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 24 Insertions and deletions - - - - - - -: 56% ATCTGCC: 44% C: 57% T: 43%
- 25. Sample to Insight Pyrosequencing – Principle and key features Pyrosequencing is based on the sequencing-by-synthesis principle Pyrosequencing data in various application areas Insertions and deletions - - - - - - -: 56% ATCTGCC: 44% C: 57% T: 43% DNA methylation of multiple CpG sites Sequencing through unknown regions A: 44% C: 0% G: 56% T: 0% Di-, tri- and tetra-allelic mutations/SNP DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 25 DNA methylation of multiple CpG sites
- 26. Sample to Insight Pyrosequencing – Principle and key features Pyrosequencing is based on the sequencing-by-synthesis principle Pyrosequencing data in various application areas DNA methylation of multiple CpG sites Insertions and deletions - - - - - - -: 56% ATCTGCC: 44% C: 57% T: 43% DNA methylation of multiple CpG sites Sequencing through unknown regions A: 44% C: 0% G: 56% T: 0% Di-, tri- and tetra-allelic mutations/SNP DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 26
- 27. Sample to Insight Pyrosequencing workflow for DNA methylation analysis • PAXgene Blood DNA Tube • QIAamp Kits • AllPrep RNA/ DNA Kits • EpiTect Fast DNA Kits • EpiTect Fast LyseAll Kits • EpiTect Fast FFPE Kits • PyroMark Assay Design SW • Pre-designed PyroMark Assays • PyroMark PCR Kit • PyroMark Q48 Autoprep • PyroMark Q48 Advanced Reagents PyroMark Assay Design SW PyroMark Q48 Advanced Kit PyroMark PCR Kit PyroMark Q48 AutoprepQIAcube QIAxpert QIAxcel Advanced QC EpiTect Fast DNA Kits 1–3 h 0.5 days EZ1 Pyro- sequencing Pre- amplification Assay design Sample collection &/ stabilization Bisulfite conversion DNA purification DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 27
- 28. Sample to Insight Pyrosequencing workflow – Assay design Assay design • Two PCR primers (one is biotinylated) ◦ Biotin-labeled strand is isolated using Vacuum Prep Workstation • Sequencing primer ◦ Placed in front of region of interest ◦ Annealed to single-stranded DNA before Pyrosequencing reaction PCR primer Region of interest PCR primer Sequencing primer DNA purification Bisulfite conversion Pre- amplification Assay design ssDNA preparation Pyro- sequencing DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 28
- 29. Sample to Insight Pyrosequencing workflow – PCR PCR or RT-PCR • Can use any PCR machine • PyroMark PCR Kit or PyroMark OneStep RT-PCR Kit • Amplify relevant region by PCR (up to 500 bp) • Can use very short PCR products if desired (i.e., degraded DNA) • One primer has to be biotinylated DNA purification Bisulfite conversion Pre- amplification Assay design ssDNA preparation Pyro- sequencing DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 29
- 30. Sample to Insight Pyrosequencing workflow – Template preparation Annealing of sequencing primer • Sequencing primer only binds to biotinylated PCR strand • ssDNA is needed for binding • Binding done ◦ Manually (PyroMark Q96 ID, Q24, Q24 Advanced) ◦ Automated (PyroMark Q48 Autoprep) DNA purification Bisulfite conversion Pre- amplification Assay design ssDNA preparation Pyro- sequencing Sequencing primer DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 30
- 31. Sample to Insight Measuring frequencies in sequence variations Quantitative peak heights to measure allele frequencies Source: Wasson et al. (2002). Assessing allele frequencies of single nucleotide polymorphisms in DNA pools by Pyrosequencing technology. Biotechniques 32,1144–1152. Even as little as 2% of one allele in 98% of the other could be detected. DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 31 G G T C A G T G A C/ m C G G G T U A G T G A U/ m C G G G T T A G T G A T/C G Bisulfite conversion PCR amplification
- 32. Sample to Insight Measuring frequencies in sequence variations Quantitative peak heights to measure allele frequencies DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 32 G G T C A G T G A C/ m C G G G T U A G T G A U/ m C G G G T T A G T G A T/C G Bisulfite conversion PCR amplification Ratio T:C .A .G .T.A .A.T .C .C .T.G .27% .A .G .T .A .A.T/C .T.T G .A .A
- 33. Sample to Insight Measuring frequencies in sequence variations Built-in quality control: successful bisulfite conversion Ratio T:C C=0% T=100% .A .G .T.A .A.T .C .C .T.G .27% .A .G .T .A .A.T/C .T.T G .A .A Pyrosequencing quantifies CpG methylation down to 5% and below and provides an internal bisulfite conversion control. DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 33 G G T C A G T G A C/ m C G G G T U A G T G A U/ m C G G G T T A G T G A T/C G Bisulfite conversion PCR amplification
- 34. Sample to Insight Complete conversion ensures reliable quantification of DNA methylation EpiTect Bisulfite Technology A unique buffer – the DNA Protect Buffer • Ensures complete cytosine conversion ◦ Internal data: 99.4%–99.8% (bisulfite sequencing) ◦ Published user data: Taylor et al. 2007: 98.8% (454 Sequencing) Meissner et al. 2008: >99% (Solexa Genome Analyzer) Superior bisulfite conversion • Bisulfite conversion of genomic DNA – comparison of EpiTect Fast DNA Bisulfite Kit and a kit from Supplier Z • Unconverted genomic DNA and bisulfite converted DNA were coamplified and then sequenced by Pyrosequencing EpiTect Fast Kits enabled superior bisulfite conversion compared to Supplier Z’s kit. DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 34
- 35. Sample to Insight Outline DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 35 DNA methylation analysis and bisulfite conversion1 5-Hydroxymethylcytosine analysis2 5-mC and 5-hmC analyses by Pyrosequencing – technology and workflow3 Addressing bottlenecks in bisulfite conversion by 'advanced Pyrosequencing'4 Streamlining Pyrosequencing analysis using PyroMark Q48 Autoprep5
- 36. Sample to Insight Incomplete bisulfite conversion may result in shorter read lengths Conventional Pyrosequencing analysis of the LOX gene locus PyroMarkQ24 DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 36 Trusted sequence
- 37. Sample to Insight Incomplete bisulfite conversion may result in shorter read lengths Conventional Pyrosequencing analysis of the LOX gene locus PyroMarkQ24 Trusted sequence Uncertain sequence Conventional Pyrosequencing may show uncertain quantification results in long sequencing runs. DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 37
- 38. Sample to Insight Incomplete bisulfite conversion may result in shorter read lengths Conventional Pyrosequencing analysis of the LOX gene locus PyroMarkQ24 Increasing peak deviation from expected signals in late sequence positions. Trusted sequence Uncertain sequence DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 38
- 39. Sample to Insight Advanced Pyrosequencing increases read length and trust Conventional vs. advanced Pyrosequencing analysis of the LOX gene locus PyroMarkQ24Advanced Advanced Pyrosequencing overcomes issues of incomplete bisulfite conversion. Trusted sequence DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 39 PyroMarkQ24 Trusted sequence Uncertain sequence
- 40. Sample to Insight Advanced Pyrosequencing technology Advanced Pyrosequencing addresses main bottlenecks in conventional Pyrosequencing Advanced Pyrosequencing • Longer Pyrosequencing read length ◦ de novo sequencing ◦ DNA methylation analysis • Increased reliability in quantification of CpG sites and other sequence variations • Increased robustness of assays ◦ Weak sequencing primer binding ◦ Low level incomplete bisulfite conversion • Facilitates easier assay development DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 40
- 41. Sample to Insight Advanced Pyrosequencing technology Optimized software, reagents and instrument firmware work hand-in-hand Advanced Pyrosequencing • Longer Pyrosequencing read length ◦ De novo sequencing ◦ DNA methylation analysis • Increased reliability in quantification of CpG sites and other sequence variations • Increased robustness of assays ◦ Weak sequencing primer binding ◦ Low level incomplete bisulfite conversion • Facilitates easier assay development PyroMark Analysis SW • optimized run setup files (e.g., additional dispensations) DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 41
- 42. Sample to Insight Advanced Pyrosequencing technology Optimized software, reagents and instrument firmware work hand-in-hand Advanced Pyrosequencing • Longer Pyrosequencing read length ◦ De novo sequencing ◦ DNA methylation analysis • Increased reliability in quantification of CpG sites and other sequence variations • Increased robustness of assays ◦ Weak sequencing primer binding ◦ Low level incomplete bisulfite conversion • Facilitates easier assay development PyroMark Analysis SW • optimized run setup files (e.g. additional dispensations) PyroMark Advanced Reagents • optimized nucleotide concentration DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 42
- 43. Sample to Insight Advanced Pyrosequencing technology Optimized software, reagents and instrument firmware work hand-in-hand Advanced Pyrosequencing • Longer Pyrosequencing read length ◦ De novo sequencing ◦ DNA methylation analysis • Increased reliability in quantification of CpG sites and other sequence variations • Increased robustness of assays ◦ Weak sequencing primer binding ◦ Low level incomplete bisulfite conversion • Facilitates easier assay development PyroMark Analysis SW • optimized run setup files (e.g. additional dispensations) PyroMark Advanced Reagents • optimized nucleotide concentration Instrument firmware • optimized dispensation (e.g. double shots) DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 43
- 44. Sample to Insight Advanced Pyrosequencing technology Optimized software, reagents and instrument firmware work hand-in-hand Advanced Pyrosequencing • Longer Pyrosequencing read length ◦ De novo sequencing ◦ DNA methylation analysis • Increased reliability in quantification of CpG sites and other sequence variations • Increased robustness of assays ◦ Weak sequencing primer binding ◦ Low level incomplete bisulfite conversion • Facilitates easier assay development PyroMark Analysis SW • optimized run setup files (e.g. additional dispensations) PyroMark Analysis SW • optimized analysis DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 44 PyroMark Advanced Reagents • optimized nucleotide concentration Instrument firmware • optimized dispensation (e.g. double shots)
- 45. Sample to Insight Advanced Pyrosequencing technology PyroMark platforms offering advanced Pyrosequencing Advanced Pyrosequencing is available on two PyroMark platforms PyroMark Q24 Advanced PyroMark Q48 Autoprep DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 45
- 46. Sample to Insight Outline DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 46 DNA methylation analysis and bisulfite conversion1 5-Hydroxymethylcytosine analysis2 5-mC and 5-hmC analyses by Pyrosequencing – technology and workflow3 Addressing bottlenecks in bisulfite conversion by 'advanced Pyrosequencing'4 Streamlining Pyrosequencing analysis using PyroMark Q48 Autoprep5
- 47. Sample to Insight PyroMark Q48 Autoprep – Workflow Workflow comparison of available Pyrosequencing platforms Sample preparation PCR amplification Assay design ssDNA preparation Analysis • PyroMark Q48 AutoprepNEW • PyroMark Q24 Vacuum Workstation • PyroMark Q96 Vacuum Workstation • PyroMark Q24 • PyroMark Q24 Advanced • PyroMark Q96 ID PyroMark Q48 Autoprep: Simplified workflow combined with advanced Pyrosequencing. DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 47
- 48. Sample to Insight PyroMark Q48 Autoprep – Dimensions and weight Smaller footprint (1/2 x PyroMark Q24) and lighter in weight (1/3 x PyroMark Q24) 250 mm (W) 390 mm (L, closed) 300 mm (H) 560 mm (L, open) DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 48
- 49. Sample to Insight PyroMark Q48 Autoprep – Protocol PyroMark Q48 Autoprep workflow – fully integrated automatic template preparation manual automated manual/automated Load & run files via USB or ethernet Load PCR product & magnetic beads Load reagents, nucleotides and buffers Automatic template preparation Anneal sequencing primer Pyro- sequencing Wash cartridge DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 49
- 50. Sample to Insight PyroMark Q48 Autoprep – SW user interface Large and easy-to-use touch screen and intuitive instrument SW DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 50
- 51. Sample to Insight Summary Improvements in the EpiTect and PyroMark portfolio enhances DNA methylation analysis • Improved bisulfite conversion efficiency ◦ DNA Protect Buffer maximizes output and improves reliability • Analysis of 5-hmC by oxidative bisulfite conversion and sequencing • Pyrosequencing quantifies methylation of consecutive CpG sites ◦ At single site resolution and with a sensitivity of 5% and below ◦ With low sample input amounts: 1–10 ng ◦ Direct quality control of bisulfite conversion during the run • ‘Advanced’ Pyrosequencing for improved and longer read length • Pre-designed assays for CpG islands, HumanMethylation 450k and EpicMethylation arrays • PyroMark Q48 Autoprep automates the main Pyrosequencing workflow steps PyroMark Q48 Autoprep: Simplified workflow combined with advanced Pyrosequencing for longer reads and more accurate methylation analysis. DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 51
- 52. Sample to Insight Pyrosequencing resource center at QIAGEN.com www.qiagen.com/de/resources/technologies/pyrosequencing-resource-center/ DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 52
- 53. Sample to Insight Thank you for attending! Questions? Contact QIAGEN Technical Service Call (US) : 1-800-426-8157 Call (EU) : +49 2103-29-12400 Email: techservice-na@QIAGEN.com techservice-eu@QIAGEN.com QIAwebinars@QIAGEN.com All our solutions from Sample to Insight on: QIAGEN.com DNA methylation analysis in a single day – The new PyroMark Q48 Autoprep 53