PyroMark Q48 Autoprep

For automated Pyrosequencing with integrated template preparation for advanced methylation, mutation and SNP quantification

Features

  • 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
S_4246_PyroMQ48_1699_s_new

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PyroMark Q48 Autoprep Instrument

Cat. No. / ID: 9002471

PyroMark Q48 Instrument, multistep pipet, software and documentation
InstrumentAccessoriesSoftwareReagent
PyroMark Q48 Autoprep
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PyroMark Software
Q48 Reagents
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PyroMark Q48 Autoprep Instrument
System
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PriorityPlus
The PyroMark Q48 Autoprep is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Product Details

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.

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).
See figures

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").
See figures

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.

Services

Have your throughput demands increased? Do you want to expand your application range or further streamline workflows? Do you need to manage more complex samples? Do you need improved connectivity?

If the answer to any of these questions is yes, then QIAGEN’s trade-in/trade-up program is just right for you!

QIAGEN makes it easy to keep up to speed with the latest automation and sample processing technologies. Simply exchange an old instrument for a new one. Improve your efficiency by trading in a competitor instrument or trading up with an older QIAGEN instrument.

Learn more about trade-in/trade-up opportunities.

Supporting data and figures

Specifications

FeaturesSpecifications
ApplicationsSimultaneous 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
Instrument dimensionsW 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
ConnectionsOne USB port, Ethernet connection
HumidityRelative humidity of 20–80% (noncondensing)
Kits designed for this instrumentPyroMark Q48 Advanced Reagents, PyroMark Q48 Advanced CpG Reagents
Operating temperature18–30ºC (64–86ºF)
Overvoltage categoryII
Place of operationFor indoor use only in a draft-free location. No exposure to direct sunlight.
Pollution level2
Power100–240V AC, 50/60 Hz. Mains supply voltage fluctuations are not to exceed ±10% of the nominal supply voltages.
Process temperature28°C ± 0.5°C
Process timeDepends on the number of dispensations
Samples per run (throughput)1–48
SoftwarePyroMark Q48 Advanced Software (to be installed on PC)
TechnologyPyrosequencing
Weight8.5 kg (18.7 lb)
AltitudeUp to 2000 m (6500 ft)

Service Plans

Pyro Q48, Full Agreement, no PM

Cat. No. / ID: 9244445

Instrument repair service for the PyroMark Q48 at regional repair center. Shipping, labor and parts costs included during the Full Agreement period. Loaner instrument provided within 2–3 business days. Instrument repair turnaround time of 7–10 business days.

Resources

Brochures & Guides (1)
Automated Pyrosequencing with integrated template preparation for advanced methylation, mutation and SNP quantification
Safety Data Sheets (1)
Download Safety Data Sheets for QIAGEN product components.
Kit Handbooks (2)
For use with PyroMark Q48 Autoprep, PyroMark Q24 Advanced, PyroMark Q24, PyroMark Q96 ID and PyroMark Q96 MD systems
Instrument User Manuals (1)
Operating Software (1)
This is the latest version of the PyroMark Q48 Autoprep instrument software. The software may only be downloaded by registered users with a registered PyroMark Q48 Autoprep instrument. For updating please follow instructions provided in the user manual chapter 5.4 Upgrading the instrument software. The user manual can be downloaded from the PyroMark Q48 Autoprep webpage.
Quick-Start Protocols (1)

FAQ

Which operating system is compatible with PyroMark IdentiFire Software?

PyroMark IdentiFire Software is compatible with Windows 2000, Windows XP, and Windows 7 (32 bit).

 

FAQ ID - 3340
In which format can PyroMark CpG Assays be ordered?
PyroMark CpG Assay can be ordered in tubes or on 96-well plates. PyroMark CpG Assays, 96 wells, require a minimum order of 24 assays per plate.
FAQ ID -2824
Is there a user manual available for the PyroMark Assay design software?
There is no specific PyroMark Assay Design Software user manual available but a so-called Quick Guide can be downloaded from the PyroMark instrument webpage. Furthermore, the software contains a comprehensive online help (accessible via the Help menu or by pressing the “F1” key).
FAQ ID -2851
Where can I order the Streptavidin Sepharose beads for pyrosequencing?
The recommended Streptavidin Sepharose High Performance beads for pyrosequencing can be ordered at GE Healthcare with the cat. no. 17-5113-01.

The PyroMark Q48 Autoprep protocol uses magnetic streptavidin-coated Sepharose® beads (PyroMark Q48 Magnetic Beads), which bind to the biotinylated PCR strand.

PyroMark Q48 Magnetic Beads can be ordered at QIAGEN with the cat. no. 974203.

FAQ ID -2850
3843-How-do-I-prevent-a-drifting-baseline-in-my-pyrosequencing-pyrogram
Verify that the environmental temperature did not change (e.g., air conditioner cycling on or off) and remains under 32°C during the run. Ensure that reagents have been adapted to room temperature prior to the run.
FAQ ID - 3843
How many nucleotides of a homopolymer can be resolved in pyrosequencing?
In the range of 3−5 bases can be resolved depending on the sequence context and base. If it is possible, sequencing of a homopolymer of more than 3−5 nucleotides should be avoided by resetting the sequencing primer.
FAQ ID -2871
Does the PyroMark Assay Design or application software give any cycling conditions for individual assay primers or a PCR setup pipetting scheme?
The PyroMark Assay Design and application software do not support PCR setup with a pipetting scheme or PCR cycling conditions. General recommendations on how to setup and optimization of the PCR reaction are contained in the PyroMark PCR Handbook.
FAQ ID -2862
Which end of the PCR primer for pyrosequencing should be biotinylated?
In pyrosequencing, the 5' end should be biotinylated, regardless of whether the forward or reverse primer is biotinylated.
FAQ ID -2839
What should be the single peak height for the PyroMark Control Oligo on the different PyroMark instruments?

PyroMark Q24: The mean single peak height is 95 +/- 55 RLU.
PyroMark Q48: The mean single peak height is 70 +/- 40 RLU.
PyroMark Q96 ID: The mean single peak height is 35 +/- 10 RLU.
Pyromark Q96 MD: The mean single peak height should be at least 350 RLU.

FAQ ID -2852
What is included in a PyroMark Custom Assay?
The PyroMark Custom Assay includes a 10x PCR Primer Set (mixture of forward and reverse PCR Primer) and 10x Sequencing Primer. Reagents for performing PCR and pyrosequencing reaction are not included.
FAQ ID -2815
What are the features of PyroMark CpG Assays, for example, in terms of design and validation?
PyroMark CpG Assays are genome-wide, pre-designed methylation assays for pyrosequencing analysis. An optimized design algorithm was used for highly specific assay design and advanced CpG methalytion results.
FAQ ID -2821
What is the sensitivity limitation for pyrosequencing?
In general, the standard claim for pyrosequencing sensitivity is approximately 5%, which is also published in many papers. The actual sensitivity limit is assay dependent and has to be determined individually.
FAQ ID -2840
Which kits can be used in combination with the PyroMark CpG Assays and PyroMark Custom Assays?
QIAamp/DNeasy Kits can be used for DNA isolation, EpiTect Bisulfite Kits for DNA conversion, PyroMark PCR Kit for PCR amplification, EpiTect Control DNA Set for PCR controls, and PyroMark Gold Q24 Reagents or PyroMark Gold Q96 Reagents for the sequencing reaction.

Depending on the platform used, the following reagent kits are required for pyrosequencing:

FAQ ID -2822
How do I reduce background peaks in the pyrosequencing pyrogram?
There are several reasons for a high assay background; the template can form secondary structures that are extended or the primers itself form dimmers that serve as template. Perform accurate sequencing controls (e.g., PCR or sequencing primer only) as recommended in the PyroMark User Manual to observe this kind of background. In addition, an unspecific priming of primer to template or unspecific annealing of sequencing primer to template might also be a background cause. Please check your complete primer design and, if needed, perform a redesign. Try to lower the primer concentration as possible to avoid excess of primer.
FAQ ID -2877
What is the sample throughput of pyrosequencing systems?

PyroMark instruments offer a range of throughput scales. The PyroMark Q24 can process 1–24 samples in parallel, the PyroMark Q48 Autoprep, 1–48; the PyroMark Q96 ID, 1–96; and the PyroMark Q96 MD, 1–96; or the automation option enables automated processing of ten 96-well plates. The sample processing speed depends on the number of nucleotide dispensations necessary for the programmed analysis. Twenty dispensations take approximately 24 minutes on all instruments; thus, 96 samples are typically processed in 10–100 minutes.

 

 

FAQ ID -2215
What is the concentration of PyroMark Control Oligo?
PyroMark Control Oligo has a concentration of 20 µM and is delivered in a volume of 50 µl. Two tubes of 10x dilution buffer (2x 1.7 ml) are delivered with the control oligo.
FAQ ID -2846
How are the PyroMark CpG Assays shipped and stored?
PyroMark CpG Assays are shipped lyophilized at ambient temperatures (20−25°C) and should be stored at −20°C either reconstituted or lyophilized. Repeated freeze−thaw cycles should be avoided. When stored under these conditions, the reconstituted product can be kept for at least 18 months from the date of receipt without reduction in performance.
FAQ ID -2816
How are the PyroMark CpG Assays reconstituted?
The PyroMark CpG Assay is reconstituted as a 10x PCR Primer Set in 550 µl TE, pH 8.0 and the 10x Sequencing Primer is reconstituted in 1175 µl Annealing Buffer if using the PyroMark Q24, 880 µl if using the PyroMark Q96 ID, and 1175 µl if using the PyroMark Q96 MD.
FAQ ID -2817
What concentration should be used for the sequencing primer in pyrosequencing?

Usually the sequencing primer is used at 0.3 µM in annealing buffer but some assays might require additional optimization of the sequencing primer concentration.

For PyroMark Q24 and PyroMark Q96 MD, the final concentration of the sequencing primer is 0.3 µM and, for PyroMark Q96 ID, 0.4 µM.

The PyroMark Q48 Autoprep dispenses the sequencing primers for annealing. The final concentration of sequencing primers in a well is 0.8 µM but may be adapted to optimize assays.

 

FAQ ID -2826
Does QIAGEN offer a design of Custom PyroMark CpG Assays?
No, QIAGEN does not design any Custom PyroMark CpG Assay. Customers have the possibility to order pre-designed, genome-wide PyroMark CpG Assays or order a user-designed assay (e.g., with the PyroMark Assay Design Software or assays known from previous projects or from the literature).
FAQ ID -2818
Will the primer sequence for the PyroMark CpG Assay be provided?
Primer sequences for PyroMark CpG Assays are not provided; they are proprietary.
FAQ ID -2823
Which purity grade is recommended for pyrosequencing primers?
Only the biotinylated primer needs to be HPLC purified, whereas the other primers require standard desalting only.
FAQ ID -2832
Will dUTP in a PCR reaction affect pyrosequencing?
In general, dUTP/UNG treatment should work for pyrosequencing to reduce contamination risk with PCR amplicons from previous PCRs.
FAQ ID -2843
What is the purpose of the unmethylated and unconverted control DNA of the EpiTect PCR Control DNA Set?

The unmethylated and unconverted human control DNA of the EpiTect PCR Control DNA Set allows to check that primers designed for the specific detection of unmethylated and converted DNA (U-converted DNA), and for methylated, converted DNA (M-converted DNA) does not bind to untreated genomic DNA.*

In case bisulfite conversion was not complete, leaving certain unmethylated C residues unconverted, false positives would result if the primer specific for M-converted DNA binds to untreated gDNA.

This control DNA can also be used to check conversion efficiency during bisulfite treatment.

 

*Summary of principle: Methylation of DNA occurs on cytosine residues, especially on CpG dinucleotides enriched in small regions of DNA. Incubation of target DNA with sodium bisulfite, using, for example, EpiTect Bisulfite Kits, results in conversion of unmethylated cytosine residues into uracil, leaving methylated cytosines unchanged.

 

FAQ ID -2007
What kind of reading length can I expect when using pyrosequencing technology for sequence analysis?

Typical reading length using pyrosequencing technology is 40−60 bases. However, as with any sequencing technology, the maximum read length will depend on template secondary structure, base content, quality of PCR-product, and other parameters.

Depending on the sequence to be analyzed, highly accurate read lengths of 140 bases or more can be obtained in just a single reaction with the Q48 PyroMark Autoprep.

 

 

FAQ ID -2216
What is the recommended amplicon size for CpG assays?
The amplicon length should be short (<200 bp). This is critical especially for DNA from FFPE tissue that is often degraded by the fixation so that short fragments are easier to amplify. Moreover, the DNA suffers from harsh bisulfite treatment and might receive further double strand breaks. Therefore, the amplicon size should be kept as short as possible.
FAQ ID -2825
How do I set up a PyroMark CpG Assay?
All relevant information regarding PyroMark CpG Assay setup can be found on the GeneGlobe website. For the Q24 and Q96, the "Sequence to Analyze" and dispensation order should not be copied manually to create a new assay. Instead, the assay file should be downloaded from the web and opened in the PyroMark CpG softwarePyroMark Q96  ID v2.5 (or higher) software, and PyroMark Q24 Software to keep important software settings.

When using the PyroMark CpG assays with the PyroMark Q48, use the "Sequence to Analyze" provided in the "product specification" section to create an assay setup file in the PyroMark Q48 software. This is done by selecting New CpG assay and pasting in the Sequence to Analyze (not the "sequence after bisulfite treatment") into the Sequence Before Bisulfite Treatment field and pressing Create Dispensation order.
FAQ ID -2814