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PyroMark Q48 Autoprep

For automated Pyrosequencing with integrated template preparation for advanced methylation, mutation and SNP quantification
  • Fully automated protocol requires less manual interaction
  • Convenient control with intuitive instrument and analysis software
  • Higher throughput allows more samples per run, more runs per day
  • Best Pyrosequencing performance for long and reliable sequence runs
  • Even higher automated throughput with multiple primer dispensation
  • More insight from quantitative DNA methylation analysis
PyroMark Q48 Autoprep integrates fully automated template preparation into the Pyrosequencing protocol. The new design and software double the throughput of PyroMark Q24 Advanced, while keeping the improved performance, making it ideally suited for functional studies, as well as for verification and validation of large numbers of samples from NGS and array experiments.
Cat No./ID: 9002470
PyroMark Q48 Autoprep System
PyroMark Q48 Instrument, multistep pipet, software, documentation and installation
Cat No./ID: 9002471
PyroMark Q48 Autoprep Instrument
PyroMark Q48 Instrument, multistep pipet, software and documentation
Cat No./ID: 9002472
PyroMark Q48 Autoprep Priority
PyroMark Q48 Instrument, multistep pipet, software, documentation, installation and 2-year warranty
Cat No./ID: 9002473
PyroMark Q48 Autoprep PrioPLUS
PyroMark Q48 Instrument, multistep pipet, software, documentation, installation and 3-year warranty
Cat No./ID: 9024321
PyroMark Q48 Cartridge Set
Set of all three replacement cartridges for PyroMark Q48 Autoprep instrument; includes Nucleotide Cartridge, Reagent Cartridge and Primer Cartridge
Cat No./ID: 9024322
PyroMark Q48 Nucleotide Cartridge
Replacement PyroMark Q48 Nucleotide Cartridge for PyroMark Q48 Autoprep instrument
Cat No./ID: 9024323
PyroMark Q48 Reagent Cartridge
Replacement PyroMark Q48 Reagent Cartridge for PyroMark Q48 Autoprep instrument
Cat No./ID: 9024324
PyroMark Q48 Primer Cartridge
Replacement PyroMark Q48 Primer Cartridge for PyroMark Q48 Autoprep instrument
Cat No./ID: 9024325
PyroMark Q48 Software License (1)
Coming Soon
One additional license for PyroMark Q48 Software. Only valid together with PyroMark Q48 Autoprep
Cat No./ID: 9024326
PyroMark Q48 Software License (5)
Coming Soon
Five additional licenses for PyroMark Q48 Software. Only valid together with PyroMark Q48 Autoprep
Cat No./ID: 974230
PyroMark Q48 AutoPrep Starter Kit
€1,129.60
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PyroMark Q48 Magnetic Beads (300), PyroMark Q48 Advanced CpG Reagents (4 x 48), PyroMark Control Oligo, PyroMark Q48 Discs (50) and PyroMark Q48 Absorber Strips (100)
Cat No./ID: 974901
PyroMark Q48 Discs (50)
€122.00
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50 discs for automated template preparation and Pyrosequencing reactions using a PyroMark Q48 Autoprep
Cat No./ID: 974912
PyroMark Q48 Absorber Strips (100)
€99.60
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100 absorber strips used for liquid collection during automated template preparation with PyroMark Q48 Autoprep
The PyroMark Q48 Autoprep is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
0
Excluding well-to-well and disc-to-disc cross contamination.
In run A, PCR products and non-template controls (NTC) were loaded into a Q48 disc in alternating order. The Pyrosequencing results show no cross-contamination between disc wells. Run B was performed with NTC samples only and without replacing the absorber strip from the first run. The results exclude any cross-contamination from one run to another. Results shown in relative light units.
1
The principle of multiple primer dispension (MPD).
A sequencing primer mixture is loaded into 1 reservoir of the primer cartridge and dispensed to 4 different wells. Each primer only anneals to its specific target sequence and will be elongated during the Pyrosequencing reaction. Primers in each MPD mix should be designed and checked to avoid formation of primer–dimers or binding to another PCR template.
2
Compatibility among PyroMark platforms for mutation analysis.
The NRAS Pyro assay was used to measure mutation frequencies in defined mixtures of wildtype and mutated NRAS sequences. Nine different mixtures representing frequencies of 0, 5, 10, 25, 50, 75, 90, 95, and 100% were analyzed using three different PyroMark platforms. The mutation frequencies measured were plotted against the expected frequencies. The data reveals that all three platforms gave the same results, showing that previously designed assays can be transferred among various PyroMark platforms.
3
Compatibility among PyroMark platforms for methylation analysis.
ER-alpha methylation was measured in defined mixtures of methylated and unmethylated control DNA, representing methylation degrees of 0, 50 and 100%, using four different PyroMark platforms. The methylation measured for one CpG site was plotted against the expected percentage of methylated DNA. All four platforms gave the same results, showing that previously designed assays can be transferred among various PyroMark platforms.
4
Principle of Pyrosequencing – step 1.
A DNA segment is amplified, and the strand to serve as the Pyrosequencing template is biotinylated. After denaturation, the biotinylated single-stranded PCR amplicon is isolated and allowed to hybridize with a sequencing primer. The hybridized primer and single-stranded template are incubated with the enzymes DNA polymerase, ATP sulfurylase, luciferase and apyrase, as well as the substrates adenosine 5' phosphosulfate (APS) and luciferin.
5
Principle of Pyrosequencing – step 2.
The first deoxribonucleotide triphosphate (dNTP) is added to the reaction. DNA polymerase catalyzes the addition of the dNTP to the squencing primer, if it is complementary to the base in the template strand. Each incorporation event is accompanied by release of pyrophosphate (PPi), in a quantity equimolar to the amount of incorporated nucleotide.
6
Principle of Pyrosequencing – step 3.
ATP sulfurylase converts PPi to ATP in the presence of adenosine 5' phosphosulfate (APS). This ATP drives the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP. The light produced in the luciferase-catalyzed reaction is detected by CCD sensors 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.
7
Principle of Pyrosequencing – step 4.
Apyrase, a nucleotide-degrading enzyme, continuously degrades unincorporated nucleotides and ATP. When degradation is complete, another nucleotide is added.
8
Principle of Pyrosequencing – step 5.
Addition of dNTPs is performed sequentially. It should be noted that deoxyadenosine alpha-thio triphosphate (dATPαS) is used as a substitute for the natural deoxyadenosine triphosphate (dATP), since it is efficiently used by the DNA polymerase, but not recognized by the luciferase. As the process continues, the complementary DNA strand is elongated, and the nucleotide sequence is determined from the signal peaks in the Pyrogram trace.
Performance
Advanced technology, software and chemistry for long and reliable sequence runs
PyroMark Q48 Autoprep features improved chemistry and instrument operation algorithms that significantly increase assay read length and accuracy in base calling, mutation analysis and methylation quantification compared to PyroMark Q96 and PyroMark Q24 systems. Assay read length in previous systems was limited by background peaks and reduced light signals in the sequencing reaction. The updated PyroMark “Advanced” chemistry and algorithms reduce this background, thereby increasing read length and reliability. Depending on the sequence to be analyzed, highly accurate read lengths of 140 or more bases can be obtained in just a single reaction.

Increased throughput for more SNP and mutation assays per run
Multiple Primer Dispensation (MPD) is a strategy to increase the capacity of automated sequencing primer dispensation in case more assays are needed than cartridge reservoirs are available. The PyroMark Q48 Autoprep Primer Cartridge has 3 reservoirs, but if more assays are needed per disc, the primers can be mixed and filled into the same reservoir of the primer cartridge. After template preparation, primer mixes are dispensed into the wells automatically. Since only one PCR template is present in each well, only the corresponding sequencing primer will bind to the template. All other primers will stay in solution but will not bind (see The principle of multiple primer dispensation). Primers in each MPD mix should be designed and checked to avoid formation of primer–dimers or binding to another PCR template.

Compatibility of assays among different PyroMark platforms
Previously designed Pyrosequencing assays are readily compatible with the new PyroMark Q48 Autoprep instrument and the “Advanced” chemistry. Data indicate that the same mutation frequencies and methylation quantification results are obtained when an assay is run on the various PyroMark platforms (see Compatibility among PyroMark platforms for methylation analysis and Compatibility among PyroMark platforms for mutation analysis). The cross-platform compatibility is also independent of the distance of the analyzed site away from the sequencing primer. This is particularly important when analyzing multiple sequence variations in a single run, which is typical for complex mutation assays or methylation analysis of consecutive CpG sites.

New disc design enables magnetic template preparation without cross contamination
PyroMark Q48 Discs are specially designed to automate template preparation and Pyrosequencing in the same instrument without manual user interaction. All buffers used for template preparation are efficiently removed from the sample and the disc during the run without the risk of any cross-contamination from well-to-well of one run or from disc-to-disc between subsequent runs (see Excluding well-to-well and disc-to-disc cross-contamination).
Principle
The PyroMark Q48 Autoprep uses proven real-time sequence-based Pyrosequencing technology for detection and quantification in genetic analyses and epigenetic methylation studies. The system can analyze up to 48 samples simultaneously. An easy-to-use, automated protocol prepares single-stranded DNA samples without manual interaction from the user. This protocol uses magnetic streptavidin-coated Sepharose beads (PyroMark Q48 Magnetic Beads), which bind to the biotinylated PCR strand. Annealing of sequencing primers can be automated for up to four different sequencing primers. If more sequencing primers are used, the primers can be manually added to the single-stranded DNA samples.

Steps of the Pyrosequencing reaction:
Step 1: A DNA segment is amplified, and the strand to serve as the Pyrosequencing template is biotinylated. After denaturation, the biotinylated single-stranded PCR amplicon is isolated and allowed to hybridize with a sequencing primer. The hybridized primer and single-stranded template are incubated with the enzymes DNA polymerase, ATP sulfurylase, luciferase and apyrase, as well as the substrates adenosine 5' phosphosulfate (APS) and luciferin (see figure "Principle of Pyrosequencing – step 1").

Step 2:
The first deoxribonucleotide triphosphate (dNTP) is added to the reaction. DNA polymerase catalyzes the addition of the dNTP to the squencing primer, if it is complementary to the base in the template strand. Each incorporation event is accompanied by release of pyrophosphate (PPi), in a quantity equimolar to the amount of incorporated nucleotide (see figure "Principle of Pyrosequencing – step 2").
 
Step 3: ATP sulfurylase converts PPi to ATP in the presence of adenosine 5' phosphosulfate (APS). This ATP drives the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP. The light produced in the luciferase-catalyzed reaction is detected by CCD sensors 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 (see figure "Principle of Pyrosequencing – step 3").

Step 4:
Apyrase, a nucleotide-degrading enzyme, continuously degrades unincorporated nucleotides and ATP. When degradation is complete, another nucleotide is added (see figure "Principle of Pyrosequencing – step 4").

Step 5: Addition of dNTPs is performed sequentially. It should be noted that deoxyadenosine alpha-thio triphosphate (dATPαS) is used as a substitute for the natural deoxyadenosine triphosphate (dATP), since it is efficiently used by the DNA polymerase, but not recognized by the luciferase. As the process continues, the complementary DNA strand is elongated, and the nucleotide sequence is determined from the signal peaks in the Pyrogram trace (see figure "Principle of Pyrosequencing – step 5").
Procedure
A run file is created using the PyroMark Q48 Advanced software, and is transferred to the instrument via a USB drive or network connection. Reagents, nucleotides and buffers are loaded into the PyroMark Q48 Autoprep cartridges, according to the volumes indicated on the touchscreen. The biotinylated PCR product and magnetic streptavidin-coated Sepharose beads are loaded into the wells of the Q48 Disc, and the disc and an absorber strip are placed into the instrument. Preparation of the single-stranded template, along with sequencing primer annealing are now completely automated, and require no additional user interaction. Up to 3 separate sequencing primers or Multiple Primer Dispensation (MPD) mixes can be dispensed automatically. Optional, manual primer addition further increases the number of different assays per run.
Applications
Pyrosequencing is increasingly important for research applications in a variety of disciplines. Whether examining drug-resistance development in pathogens, the role of epigenetic DNA methylation in gene expression regulation, genetic markers for specific phenotypes in livestock or polymorphisms in forensic samples of mitochondrial DNA, PyroMark platforms enable powerful and versatile analysis of genetic and epigenetic variation. In addition, because Pyrosequencing integrates sequence detection and quantification, the enhanced analysis resolution can lead to new discoveries.
Software
PyroMark Q48 Autoprep Software, installed on a PC, enables comprehensive analysis of your results. The software contains 4 analysis modes – AQ, SNP, CpG and SEQ. The AQ mode can be used for a variety of quantification studies of mutations such as SNPs and InDels. It is suitable for analyzing single and multivariable positions, as well as di-, tri- and tetra- allelic mutations. The SNP mode provides genotype analysis of SNPs and InDels. The CpG mode enables methylation analysis of single or multiple CpG or CpN sites and provides a built-in control for the bisulfite treatment. The SEQ mode is used for base calling of unknown sequences.

The PyroMark Q48 Autoprep Software is user-friendly and intuitive and provides convenient and improved tools for run analysis. If a problem occurs during the run, or if the system detects an inconsistency with an assay, the software provides specific warning information for each individual well. A "Warning Info" button gives access to additional information about the warning along with recommendations for troubleshooting and preventing its occurrence in subsequent assays.
Features
Specifications
Altitude Up to 2000 m (6500 ft)
Applications Simultaneous running of multiple assays and assay types including SNP, AQ, CpG and SQA to achieve methylation analysis, de novo sequencing, mutation characterization including In/Dels, speciation, quantitative allele sequencing and SNP genotyping
Connections One USB port, Ethernet connection
Humidity Relative humidity of 20–80% (noncondensing)
Instrument dimensions W X D X H: 250 mm (9.8 in.) X 300 mm (11.8 in.) X 300 mm (11.8 in.) with chamber lid and injector cover closed; 250 mm (9.8 in.) X 560 mm (22 in.) X 390 mm (15.4 in.) with chamber lid and injector cover open
Kits designed for this instrument PyroMark Q48 Advanced Reagents, PyroMark Q48 Advanced CpG Reagents
Operating temperature 18–30ºC (64–86ºF)
Overvoltage category II
Place of operation For indoor use only in a draft-free location. No exposure to direct sunlight.
Pollution level 2
Power 100–240V AC, 50/60 Hz. Mains supply voltage fluctuations are not to exceed ±10% of the nominal supply voltages.
Process temperature 28°C ± 0.5°C
Process time Depends on the number of dispensations
Samples per run; throughput 1–48
Software PyroMark Q48 Advanced Software (to be installed on PC)
Technology Pyrosequencing
Weight 8.5 kg (18.7 lb)
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