PyroMark Q96 Systems

For reagents and support of discontinued instrument PyroMark Q96 ID

PyroMark Gold Q96 Reagents (5 x 96)

Cat no. / ID. 972804

For performing Pyrosequencing reactions on the PyroMark Q96 ID (5 x 96) and PyroMark Q96 MD (15 x 96).

ReagentKitAssayAccessoriesSoftware

Q96 Reagents

PyroMark Kits

Assays and Tests

Buffers

Q96 Sample Prep Thermoplate Low

Consumables

PyroMark Software

Type

Gold Q96 Reagents

Gold Q96 SQA Reagents

Gold Q96 CDT Reagents

Reactions

5 x 96

50 x 96

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Features

Consider upgrading to the PyroMark Q48 Autoprep System for these advantages

Automation of the Pyrosequencing protocol
Longer read lengths with the Advanced chemistry
Compact instrument size
Enabled for connectivity
Touch screen display that guides users through the process

Product Details

The PyroMark Q96 MD has been discontinued and is no longer supported.

The PyroMark Q96 ID was discontinued in 2016 and the instrument is no longer actively sold. Existing instruments will be supported until the end of 2023. Reagents will continue to be available until the end of 2023.

To future-proof your Pyrosequencing assays, consider upgrading your work to using a PyroMark Q48 Autoprep System, which is actively sold and supported by QIAGEN.

Service Plans

Premium Cover Agreement, PyM Q96 ID

Cat. No. / ID. 9241827

1 year coverage of travel, labor, and spare parts. The instrument is repaired at the customer site with an onsite response time of 24 hours. The agreement includes a preventive maintenance/inspection visit.

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Pyro Q96 ID, Full Agreement

Cat. No. / ID. 9241823

On-site PyroMark Q96 ID instrument repair, including travel, labor and parts, for a period of one year. Response time of two business days. Includes one Preventive Maintenance or Inspection Service during the Full Agreement period.

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Pyro Q96 ID, Preventive Subscription

Cat. No. / ID. 9243548A

One on-site Preventive Maintenance or Inspection Service visit for the PyroMark Q96 ID System, including travel, labor and parts. Includes a 10% discount on repair services during the Preventive Subscription period.

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Resources

Scientific poster

Download Safety Data Sheets for QIAGEN product components.

A Pyrosequencing method for the forensic identification of tissue source using QIAGEN’s PyroMark Q24 Advanced system.

For use with PyroMark Q48 Autoprep, PyroMark Q24 Advanced, PyroMark Q24, PyroMark Q96 ID and PyroMark Q96 MD systems

For performing Pyrosequencing reactions on the PyroMark Q96 ID, PyroMark Q96 MD, and PyroMark Q96 MD Automated

PyroMark Assay Design Software version 2.0.2 is compatible with Windows 7 and Windows 10 (64 bit) operating systems. This software may only be downloaded by registered users with a valid PyroMark Assay Design Software license. If you do not have a valid software license, contact your QIAGEN sales representative.

PyroMark Q96 ID Software 2.5 version 2.5.10.7 is the application software for setting up, performing and analyzing runs on PyroMark Q96 ID instruments. The main feature of this release is 64bit compatibility with Windows 7 operating systems.

This software can only be downloaded by users with a registered PyroMark Q96 ID instrument.

FAQ

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.

2007

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.

2215

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.

2216

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 software, PyroMark 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.

2814

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.

2815

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.

2816

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.

2817

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).

2818

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.

2821

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:

PyroMark Q96 ID and MD: PyroMark Gold Q96 Reagents
PyroMark Q24: PyroMark Gold Q24 Reagents
PyroMark Q24 Advanced: PyroMark Q24 Advanced Reagents and PyroMark Q24 Advanced CpG Reagents
PyroMark Q48 Autoprep: PyroMark Q24 Advanced Reagents and PyroMark Q24 Advanced CpG Reagents

2822

Primer sequences for PyroMark CpG Assays are not provided; they are proprietary.

2823

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.

2824

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.

2825

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.

2826

The nucleotides can only be ordered as part of the PyroMark Gold Reagents which also contain enzyme and substrate mix.

2827

Only the biotinylated primer needs to be HPLC purified, whereas the other primers require standard desalting only.

2832

PyroMark Gold Q96 Reagents:

PyroMark Gold Q96 SQA Reagents (1 x 96) should be used for performing SQA analyses on the PyroMark Q96 ID. It can also be used to supplement the PyroMark Gold Q96 Reagents (5 x 96) when long runs lead to a shortage of nucleotides.
PyroMark Gold Q96 Reagents (5 x 96) should be used for SNP and CpG analyses on the PyroMark Q96 ID and MD instruments. On the PyroMark Q96 MD, the kit contains enough for 15x96 plates.
PyroMark Gold Q96 CDT Reagents (6 x 96) should be used only with CDTs on the PyroMark Q96 MD instrument, for performing SNP and CpG anslyses. The nucleotides are pre-diluted for use with CDTs, so this is not to be used on ID instrument or with NDTs on the MD.
PyroMark Gold Q96 Reagents (50 x 96) should be used only with the PyroMark Q96 MDA (automated option) for SNP anslyses. Not for use with the PyroMark Q96 ID or non-automated MD.

See the PyroMark Gold Q96 Reagents Handbook for additional information.

2836

Shaking conditions are 1400 rpm at room temperature. Optimal results are obtained with 2 mm orbital diameter.

2837

A heating block that can heat up to 80−90°C is recommended. A solid block is preferable. For the PyroMark Q24 the surface area must be 50 mm x 60 mm and for PyroMark Q96 ID/MD 120 mm x 80 mm.

2838

In pyrosequencing, the 5' end should be biotinylated, regardless of whether the forward or reverse primer is biotinylated.

2839

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.

2840

The PyroMark Q96 MD uses the NDT and CDT to dispense nucleotides, while the Q24 and Q96 ID use cartridges.

NDT means nucleotide dispensing tip. This is the original dispensing tip which is more sensitive to dust but dispenses lower nucleotide volumes and enables longer read length.

CDT means caplillary dispensing tip and is comparable to Q24 and Q96 ID cartridges. They dispense a higher volume but are easier in handling.

The NDT and CDT use two different tip holders which cannot be interchanged

2841

The PyroMark Q24 software and the new PyroMark Q96 ID software version 2.5 support CpG analysis in the CpG mode. The ORACLE-based PyroMark Q96 ID software version and PyroMark Q96 MD software do not support CpG analysis. In this case, an independent, additional software is needed which is the PyroMark CpG software version 1.0.

2842

In general, dUTP/UNG treatment should work for pyrosequencing to reduce contamination risk with PCR amplicons from previous PCRs.

2843

Reconstiuted enzyme and substrate of PyroMark Gold Reagents, should not be vortexed since this could lead to conformational changes which affect the activity.

2844

The PyroMark Q96 ID software version 1.0 contains a SNP mode for simplex and multiplex entries (and Allele Quantification analyses can be performed for simplex entries) and an SQA mode for de novo sequencing analysis. This software version does not contain a CpG mode for methylation analysis, however the PyroMark CpG software v1.0 can be used.

2845

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.

2846

There is no precise recommendation how many times these troughs on the PyroMark Vacuum Preparation Stations (Q24 and Q96) can be reused. It depends on the individual handling and cleaning (with water).

2848

The new PyroMark Assay Design Software Version 2.0 contains new algorithms to facilitate CpG assay design and enables PCR and sequencing primer design for all pyroseqeuncing applications and PyroMark instruments.

2849

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.

2850

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).

2851

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.

2852

The PyroMark Q24 LINE-1 assay covers three CpG sites, and the LINE-1 assay for PyroMark Q96 MD covers four sites.

2858

The PyroMark CpG LINE-1 assay is specific for human DNA and was not tested on mouse DNA. Mouse LINE-1 elements differ slightly in sequence and may not be detected.

2859

The PyroMark Q96 CpG LINE-1 assay can only be used with a PyroMark Q96 MD system because the PyroMark Q96 ID instrument does not have a camera that is sensitive enough. For the PyroMark Q24, there is a dedicated PyroMark Q24 LINE-1 assay.

2861

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.

2862

When cleaning and storing the cartridges properly, the Q24 cartridge can be used up to 30 times and Q96 ID cartridge up to 20 times. The cartridge product sheet and PyroMark Q24/ID user manual contain guidelines how to clean the cartridges correctly.

2863

The PyroMark Q96 MD HS Capillary Tip and HS Reagent Tip can be used up to 20 times and the PyroMark Q96 HS Nucleotide Tip up to 10 times.

2865

The PyroMark Q96 MD software contains a SNP mode for simplex and multiplex entries (and Allele Quantification analyses can be performed for simplex entries). This software version does not contain a CpG mode for methylation analysis, however the PyroMark CpG software v1.0 can be used. SQA analysis for de novo sequencing is not possible.

See our Product Selection Guide for additional information on CpG supplementary software.

2866

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.

2871

In principle, it is possible to use so far unused pyrosequencing wells for the next run and leave the wells that are already used empty. However, due to contamination risk when cleaning and handling plates, QIAGEN does not recommend this.

2872

The PyroMark Q24 application software a PyroMark Q24 Software user guide with all essential information about warnings and software features, which can be found contains under Help (press F1). The Pyromark ID/MD software also contains a software guide under Help. Moreover, the individual instrument user manuals contain helpful information in the troubleshooting section.

2874

The cartridge needle can be blocked or damaged, causing a dispensation error. Clean the cartridge following the guidelines or repeat the run with a new cartridge. On the other hand, if high amounts of template have been used resulting in very high signals (>100 RLU), the substrate for the sequencing reaction might be depleted. In this case, template conditions should be optimized.

2875

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.

2877

Let the PyroMark instrument warm up (approx. 60 min) to adapt to room temperature before use. Make sure the ambient room temperature is within range 18−28°C.

2878

Usually pyrophosphate or dATP/ATP contamination in the sample or in the buffer can cause a high substrate peak. Large amounts of pyrophosphate are generated in the PCR reaction and might be carried over to the sequencing reaction. Check the PyroMark buffers and reagents and use new ones.

2879

The PyroMark cartridge needle can be blocked or damaged. Clean the cartridge or exchange with a new one. Check for correct reagent cartridge and cartridge method used in the run. Check if the reagent cartridge cover was closed properly. Make sure that the cartridge was dry after cleaning because nucleotide droplets might be caught at the needle tip and might fall down at any time, or exchanged.

2881

An instrument method or instrument code encodes the individual pulse time settings of specific cartridge lot batch. These pulse time settings change when, for example, a new batch of capillaries is used with slight variations in the needle diameter. For larger diameters, the pulse settings are lowered to dispense the correct volume of liquid. In addition, the viscosity of enzyme and substrate mixes can change, which influences dispensing volumes.

The individual instrument method/code number is printed on the cartridge label. The corresponding methods/code settings can be downloaded as a file from the respective instrument webpage and opened in the PyroMark application software.

2941

Always check for the actual method/code number printed on the cartridge label. Make sure that you choose this method/code number when setting up the pyrorun in the application software. If this method cannot be selected automatically in the application software, you can download the method/code file from the instrument webpage.

2942

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

3340

If you need to purchase a new computer for your PyroMark Q96, or if you need to reinstall, upgrade, or replace the operating system (OS), you can reinstall the PyroMark Q96 software without purchasing a new license.

3472

TB Management

Transplant

Infectious Disease

Oncology

Sexual & Reproductive Health

Sample Processing

Solutions for Laboratory-Developed Tests

PyroMark Q96 Systems

For reagents and support of discontinued instrument PyroMark Q96 ID

PyroMark Gold Q96 Reagents (5 x 96)

Features

Product Details

Service Plans

Premium Cover Agreement, PyM Q96 ID

Pyro Q96 ID, Full Agreement

Pyro Q96 ID, Preventive Subscription

Resources

FAQ

TB Management

Transplant

Infectious Disease

Oncology

Sexual & Reproductive Health

Sample Processing

Solutions for Laboratory-Developed Tests

Welcome to QIAGEN!

PyroMark Q96 Systems

For reagents and support of discontinued instrument PyroMark Q96 ID

PyroMark Gold Q96 Reagents (5 x 96)

Features

Product Details

Service Plans

Premium Cover Agreement, PyM Q96 ID

Pyro Q96 ID, Full Agreement

Pyro Q96 ID, Preventive Subscription

Resources

FAQ