1. PyroMark™Q24

2. A Small Smart Affordable Pyrosequencing System
PyroMark Q24
A Small Smart Affordable Pyrosequencing System
with a 24 well format
A Complete solution for Mutation and Methylation Analysis
CpG methylation
Allele quantification
Mutation analysis

3. Pyrosequencing PyroMark™Q24
Pyrosequencing technology offers an established genetic analysis method based on the principles of sequencing by synthesis.
PyroMark Q24, a system running up to 24 samples in parallel.
Pyrosequencing assays are simple, fast and robust with inbuilt controls
The results are shown in real time on the instrument display

4. PyroMark Q24 System Reagents Application software Instrument
Vacuum Prep. Workstation

5. The PyroMark Q24 System PyroMark Q24 Instrument
PyroMark Q24 Analysis Software
PyroMark Q24 Vacuum Prep Workstation
Reagents, Consumables and Accessories
PyroMark Q24 Validation Oligo
PyroMark Control Oligo
PyroMark Q24 RUO
Assay DB updated with 69 new CpG methylation assays

6. PyroMark™Q24
The system
PyroMark Q24 and Vacuum prep workstation

7. Benefits of PyroMark™Q24
An extremely easy to use system
Up to 24 samples in parallel
Control oligo delivered with the system ensures proper installation
Validation oligo is available to check the performance of the system
Small Footprint
Takes very little bench space
measuring only H420xW390xD525mm
Robust
Can be run at ambient temperatures between 15°C and 32°C.
Accurate

8. PyroMark™Q24
Runs can be set up and analyzed on any computer anywhere, running PyroMark™Q24 Software.
Information is transferred between the instrument and computer via a USB memory stick.

9. Pyrosequencing Technology
A non destructive technology that enables further template analysis
Ideal for paraffin embedded samples
An extensive database of assays is available via the Biotagebio.com site

10. Pyrosequencing for the clinic
Pyrosequencing offers a Gold Standard for genetic testing
Reproducible sequence data with built-in QC is most appropriate for a clinical decision
Pyrosequencing is easier, faster and cheaper than traditional sequencing
Pyrosequencing quantifies the proportion of mutant cells among normal cells
In contrast, hybridization assays with differential probes:
require subjective interpretation
have no inherent QC

11. Vacuum Prep Workstation
The PCR products have to be processed to yield single-stranded DNA to which a sequencing primer can be annealed.
Samples to be analyzed using PyroMark™Q24 should be prepared, using PyroMark™Q24 Vacuum Prep Workstation.

12. Q24 Vacuum Prep Workstation
1-24 post-PCR samples prepared in parallel in less than 15 minutes
Minimal pipetting
Actual hands-on time, less than 1 minute
Plastic waste reduced to a minimum

13. PyroMark Q24 Software
The software has two analysis modes:
AQ A variety of quantification studies and SNP analysis.
CpG Methylation analysis in multiple consecutive CpG sites.
AQ assays and CpG assays can be performed on the same PyroMark™Q24 Plate.
You can toggle between the analysis modes in the analysis view of the software, by selecting AQ or CpG in the toolbar.
Both modes offer detailed report information that can be exported to html, excel, text and pdf formats.

14. PyroMarkTM Q24 CpG Methylation Bisulfite conversion of DNA PCR
Sample Prep
Pyrosequencing
~ 2h
~ 15 min
~ 10 min

15. CpG Methylation Analysis
Quantitative analysis of multiple consecutive sites
Flexible assay design
Forward – reverse/ Upper – lower
Flexible primer positioning
Built-in Bisulfite treatment control
Excellent Performance
- Accuracy
- Precision
- Reproducibility over time
Confidently discern even small changes in methylation
Fast results

16. CpG mode Analyze methylation in the presence of SNP:s
Suitable for analysis of fresh-frozen, fixed and paraffin-embedded specimens
Present deviation from predefined methylation ranges
Quality assessment of individual sites and sequence context
Mean methylation for the whole assay shown
for quick overview of results
Many assays have been validated using Pyrosequencing technology
24 samples analyzed in parallel in 1 hour after PCR

17. A Robust, Accurate System
Accuracy - Linear response in measured methylation
Methylation levels are consistent even when using different primers
Confidently measure the individual degree of methylation in adjacent CpG sites, even at long distances from the sequencing primer
Easily handles varying methylation levels from site to site
Detects site variation reproducibly

18. CpG methylation studies with PyroMarkTMQ24
Any single CpG site
Multiple consecutive CpG sites
One gene at a time
Several genes in the same analysis (analyze up to 24 different assays in one run)
Global methylation level
Estimate the global methylation levels using repetitive elements

19. Quantification of individual CpG sites
Methylation levels may vary from site to site
Pyro Q-CpG detects site variation reproducibly
Methylation pattern in RASSF1A in neighboring CpG sites in 4 tumor samples (duplicate runs)

20. CpG Methylation Analysis with PyroMarkTMQ24mC mC
1. Bisulfite conversion
2. PCR amplification
3. Pyrosequencing C U mC C U T
25%
75%
Degree of methylation is analyzed as a ”C/T SNP” using the AQ mode in the software mC C

21. CpG Methylation Analysis
1. Bisulfite treatment of
denatured DNA
Original sequence:
GGAmC/CGCTAGmC/CGACTGCTA mC mC
Bisulfite treatment C U
2. PCR amplification
GGAmC/UGUTAGmC/UGAUTGUTA mC C
PCR U T
GGAC/TGTTAGC/TGATTGTTA
3. Analysis

22. AQ mode
Frequency calculations of variable positions in sequence context including;
Single variable position AG/TC
Multiple variable positions AG/TCAG/TCAG/T/AC
Di- Tri- or Tetra-allelic mutations GA/C/G/TA
High resolution of individual sites
Quality assessment of individual sites and sequence context
SNP analysis (Multiple positions, Di- Tri- or Tetra-allelic variants)

23. AQ mode Quantification of di-, tri-, or tetra-allelic mutations
Analysis of SNPs in the presence of methylation
Fast results – typically 15 minutes after PCR to run a typical SNP plate
multiple plates can be run each day

24. Pyrosequencing delivers quantitative and reproducible data
A unique property of Pyrosequencing detection is that peak height is directly proportional to the relative quantity of a given allele. This makes possible the quantification of the relative amount of mutant alleles to normal alleles, the importance of which is illustrated in the genotyping of the JAK2 mutation.

25. Pyrosequencing Technology
Time
Light
PPi
ATP

26. PyroMark Q24