Transcript PyroMark™Q24 - CMGM Stanford

PyroMark™Q24
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
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
PyroMark Q24 System
Reagents
Application
software
Instrument
Vacuum Prep.
Workstation
The PyroMark Q24 System
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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
PyroMark™Q24
The system
PyroMark Q24 and Vacuum prep workstation
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
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.
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
Pyrosequencing for the clinic
• Pyrosequencing offers a Gold
Standard for genetic testing
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
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.
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
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.
PyroMarkTM Q24
CpG Methylation
Bisulfite
conversion
of DNA
PCR
~ 2h
Sample Prep
~ 15 min
Pyrosequencing
~ 10 min
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
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
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
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
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)
CpG Methylation Analysis with PyroMarkTMQ24
1. Bisulfite conversion
2. PCR amplification
mC
mC
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mC
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3. Pyrosequencing
75%
25%
Degree of methylation is
analyzed as a ”C/T SNP”
using the AQ mode in
the software
mC
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CpG Methylation Analysis
1. Bisulfite treatment of
denatured DNA
mC
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mC
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2. PCR amplification
mC
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3. Analysis
Original sequence:
GGAmC/CGCTAGmC/CGACTGCTA
Bisulfite
treatment
GGAmC/UGUTAGmC/UGAUTGUTA
PCR
GGAC/TGTTAGC/TGATTGTTA
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)
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
Pyrosequencing delivers
quantitative and reproducible data
Pyrosequencing Technology
Light
PPi
ATP
Time
PyroMark Q24