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

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

Application software
Reagents
Vacuum Prep.
Workstation
Instrument

• 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

The system
PyroMark Q24 and Vacuum prep workstation

• 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

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.

• 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

1. Reproducible sequence data with built-in QC is most appropriate for a clinical decision
2. Pyrosequencing is easier, faster and cheaper than traditional sequencing
3. Pyrosequencing quantifies the proportion of mutant cells among normal cells
4. In contrast, hybridization assays with differential probes:
– require subjective interpretation
– have no inherent QC

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.

• 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

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.

TM
Bisulfite conversion of DNA
PCR
~ 2h
Sample Prep
~ 15 min
Pyrosequencing
~ 10 min

• 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

• 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

• 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

CpG methylation studies with PyroMark TM Q24
 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

• 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)

CpG Methylation Analysis with PyroMark TM Q24
1.
Bisulfite conversion
2.
PCR amplification m C
C m C
U m C
U
C
T
3.
Pyrosequencing
75%
25%
Degree of methylation is analyzed as a ”C/T SNP” using the AQ mode in the software m C C

1. Bisulfite treatment of denatured DNA m C m C
C U
2. PCR amplification m C C
U T
3. Analysis
Original sequence:
GGA m C / C G C TAG m C / C GA C TG C TA
Bisulfite treatment
GGA m C / U G U TAG m C / U GA U TG U TA
PCR
GGA C / T G T TAG C / T GA T TG T TA

• 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)

 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

Pyrosequencing delivers quantitative and reproducible data

Light
PPi
ATP
Time