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PyroMark Supplementary Software

For expanded assay setup and analysis with PyroMark systems


  • User-friendly interface increases the range of analyses available
  • Driven by drop-down menus to ensure correct selection of parameters
  • Enable user-designed assays with algorithms for optimization of primers

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PyroMark Assay Design SW 2.0 (5)

Cat. No. / ID: 9019079

Five additional software licenses. Requires PyroMark Assay Design SW 2.0
Expanded SW
Analysis SW
PyroMark Assay Design SW 2.0
PyroMark IdentiFire SW 1.0
The PyroMark Supplementary Software 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 supplementary software expands assay flexibility and analysis breadth of Pyrosequencing analysis. Intuitive and user-friendly, these software products give the user access to optimized assay design for genotyping, allele quantification, methylation analysis, and de novo sequencing, as well as a framework for sequence comparison with entries in a local sequence database.


PyroMark Assay Design SW 2.0
The flexible PyroMark Assay Design Software 2.0 optimizes Pyrosequencing analyses. Featuring algorithms for full quality control and smooth import of assays into any PyroMark instrument, this software was constructed based on years of expertise in Pyrosequencing and PCR optimization. With a few clicks, the software designs PCR and sequencing primers for genotyping, allele quantification, sequence analysis, and methylation analysis. For methylation analysis, the software anticipates the bisulfite converted sequence and highlights CpG sites and non-CpG cytosines that can serve as controls.
PyroMark CpG SW 1.0
When analyzing multiple samples, each potentially with several methylation sites, it is important to have the aid of software that enables visualization of sequence-wide patterns, as well as details of specific variable positions. CpG analysis with PyroMark CpG SW 1.0 for the PyroMark Q96 ID and PyroMark Q96 MD provide that functionality. Standard calculations include methylation frequency, quality assessment, mean methylation values per well and replicates, statistical methylation patterns of multiple consecutive sites, and deviation from expected methylation patterns. Results are displayed in simple-to-understand graphical and numerical representations.
PyroMark IdentiFire SW 1.0
PyroMark IdentiFire SW 1.0 facilitates the compilation of a local sequence database, against which Pyrosequencing output are rapidly matched. The raw data, matched hits, and percent concordance of each hit are presented in detailed identification reports. This software is designed to interface directly with the PyroMark Q96 ID, but it also accepts run files imported from the PyroMark Q24.


Safety Data Sheets (1)
Download Safety Data Sheets for QIAGEN product components.
Scientific Posters (1)
Operating Software (1)
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.


3340 - Which operating system is compatible with PyroMark IdentiFire Software?

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


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 which 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 catalog 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 catalog 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
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 how to setup and optimization of the PCR reaction are contained in the PyroMark PCR handbook.
FAQ ID -2862
Which analyses can be performed with the PyroMark Q96 ID software version 1.0?
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.
FAQ ID -2845
Which analyses can be performed with the PyroMark Q96 MD software?

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.

FAQ ID -2866
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 which are extended or the primers itself form dimmers which 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
How many times can vacuum troughs be re-used 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 re-used. It depends on the individual handling and cleaning (with water).
FAQ ID -2848
What concentration should be used for the sequencing primer in pyrosequencing?

Usually the sequencing primer is used at 0.3µM 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 0.3µM and for PyroMark Q96 ID 0.4µM.

The PyroMark Q48 Autoprep dispenses the sequencing primers for annealing. The final concentration of sequencing primers in a well is 0.8µM, 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
What are the changes of PyroMark Assay Design Software Version 2.0 compared to 1.0?
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.
FAQ ID -2849
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.  Pyrosequencing primers can be ordered here.
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 fall down at any time. or exchanged.
FAQ ID -2881
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
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 is the recommended amplicon size for CpG assays?
The amplicon length should be short (<200bp). This is critical especially for DNA from FFPE tissue which 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’s possible to use so far unused pyrosequencing wells for the next run and leave the already used wells empty. However, due to contamination risk when cleaning and handling plates QIAGEN does not recommend this.
FAQ ID -2872
How do I prevent a drifting baseline in my pyrosequencing pyrogram?
Let the PyroMark instrument warm up (about 60 minutes) to adapt to room temperature before use. Make sure the ambient room temperature is within range 18-28°C.
FAQ ID -2878