PyroMark Q96 ID Software 2.5

Enhanced application software for use with PyroMark Q96 ID systems


  • Intuitive file-based format allows easy updates and use of plug-ins
  • Runs stored as xml files allow more formats and easy data transfer
  • New CpG analysis mode eliminates need for additional software
  • Includes AQ and CpG setup modes for easy assay setup
  • Simultaneous analysis of all application modes in parallel

Product Details

PyroMark Q96 ID software version 2.5 provides the most flexible solution for time- and cost-efficient detection and quantification of genetic variants on PyroMark Q96 ID systems. This enhanced application software contains four analysis modes: AQ (allele quantification), SNP (analysis of SNPs and InDels), CpG (methylation analysis), and SQA (sequence identification). These four different types of analyses can be performed on the same plate, in the same run.


The enhanced PyroMark Q96 ID software 2.5 is now more user-friendly and flexible.

Comparison of application software for PyroMark Q96 ID systems
PyroMark Q96 ID Software 2.5 (new) PyroMark Q96 ID Software (old)
File-based Oracle-based
Four analysis modes (AQ, CpG, SNP, and SQA) Three analysis modes (AQ, SNP, and SQA)
Four assay setup modes (AQ, CpG, SNP, and SQA) Two assay setup modes (SNP and SQA)            
Simultaneous analysis of all application modes in parallel Only consecutive analysis of different modes is possible
Runs stored as xml files Runs stored in an Oracle database
Support for plug-in reports No support for plug-in reports 
Windows 7 (32 bit) compatible  No Windows 7 (32 bit) support 
No SNP multiplexing support SNP multiplexing support


Upgrading from old versions of PyroMark Q96 ID Software

Previous versions of PyroMark Q96 ID Software stored Pyrosequencing runs in an Oracle database. Users upgrading to PyroMark Q96 ID software 2.5 from an Oracle-based version of the application software must first use the PyroMark Data Converter Tool to convert the run database into a format that is compatible with the enhanced software. Please refer to the resources section to download the tool and access detailed instructions for converting the existing run database to a file-based format.


Safety Data Sheets (1)
Download Safety Data Sheets for QIAGEN product components.
Instrument User Manuals (2)
Operating Software (1)
PyroMark Q96 ID Software 2.5
PyroMark Q96 ID Software 2.5 version 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.
Scientific Posters (1)


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
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
I need to buy a new computer for my PyroMark Q96. Can I reinstall the PyroMark Q96 software on my new computer or do I need to purchase a new license?

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.

FAQ ID - 3472
What is a PyroMark instrument method or instrument code?

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.

FAQ ID -2941
What is the reason for a high substrate peak in the pyrosequencing pyrogram?
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.
FAQ ID -2879
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 kind of shaker should be used for the pyrosequencing binding step?
Shaking conditions are 1400 rpm at room temperature. Optimal results are obtained with 2 mm orbital diameter.
FAQ ID -2837
How many times can the cartridges for PyroMark Q24 or PyroMark Q96 ID instruments be reused?
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.
FAQ ID -2863
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
Can the PyroMark Q96 CpG LINE assay be used with an ID system?
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.
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
When do I have to change the pulse settings/methods in a pyrosequencing run setup?
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.
FAQ ID -2942
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
How many times can vacuum troughs be reused with the PyroMark Vacuum Preparation Stations?
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).
FAQ ID -2848
Which heating block is recommended for the pyrosequencing annealing step?
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.
FAQ ID -2838
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
Where can I find explanations to the warning given by the PyroMark software after run data analysis?
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.
FAQ ID -2874
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
What is the reason for split peaks appearing in between dispensations on my pyrosequencing pyrogram?
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.
FAQ ID -2881
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
Is the CpG software included in the PyroMark instruments to study methylation status?
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.
FAQ ID -2842
How many CpG sites are analyzed by the PyroMark CpG LINE assay?
The PyroMark Q24 LINE-1 assay covers three CpG sites, and the LINE-1 assay for PyroMark Q96 MD covers four sites.
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 is the reason for signals ceasing in the middle of a pyrosequencing run?
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.
FAQ ID -2875
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
Can unused wells in a pyrosequencing plate be used in the next run?
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.
FAQ ID -2872
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
How do I prevent a drifting baseline in my pyrosequencing pyrogram? If this method cannot be selected automatically in the application software, you can download the method/code file from the instrument webpage.
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.
FAQ ID -2878