PyroMark Q24 Advanced Software

Analysis software for upgrading PyroMark Q24 systems to PyroMark Q24 Advanced


  • Advanced technology, software, and chemistry for long sequence runs
  • Quantitative methylation analysis at consecutive CpG or CpN sites
  • Improved quantification of sequence variations at any sequence position
  • Easy and improved base calling functionality
  • Assay versatility on the same instrument and in the same run

Product Details

PyroMark Q24 Advanced features improved Pyrosequencing technology to provide even better real-time sequence-based detection and quantification than before. PyroMark Q24 Advanced Software enables users to upgrade their existing PyroMark Q24 systems and take advantage of these new benefits and features. The software now has 4 analysis modes: AQ (allele quantification), SNP (genotyping), CpG (methylation at CpG or CpN sites), and SEQ (base-calling of unknown sequences).


Quantification of CpN methylation
The new CpN mode of PyroMark Q24 Advanced now enables methylation analysis of cytosine residues that are not part of CpG sites. Analysis of CpN and CpG sites can be performed together in a single Pyrosequencing reaction. Each of these positions can be selected individually in the software during the run setup (see figure  Quantification of CpN methylation).

Convenient and improved tools for run analysis
The PyroMark Q24 Advanced analysis software is user-friendly and intuitive, and now 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 (see figure  Additional warning information). A "Warning Info" button now gives access to additional information about the warning along with recommendations for troubleshooting and preventing its occurrence in subsequent assays (see figure  Troubleshooting recommendations).

Longer Pyrosequencing runs and improved accuracy
PyroMark Q24 Advanced features improved chemistry and instrument operation algorithms that significantly increase the assay read length and accuracy of the base calling functionality, enabling easy analysis of long de novo sequencing runs. Assay read length was previously limited by background peaks and reduced light signals in the sequencing reaction. The updated chemistry and algorithms of PyroMark Q24 Advanced reduce these background peaks, 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 PyroMark Q24 Advanced reaction (see figure  Long de novo sequencing runs).

Improved methylation analysis at any position
PyroMark Q24 Advanced enables improved methylation quantification in long sequence runs at any sequence position. Previously, analysis of methylation sites further away from the sequencing primer could be uncertain, but now with longer read lengths and higher accuracy, methylation quantification is highly reliable throughout the entire sequencing run (see figure  Analysis of 16 CpG sites in a long sequence run).

Improved sequencing accuracy in homopolymers
Bisulfite conversion in DNA methylation analysis leads to frequent poly T stretches in the nucleotide sequence, and analysis of CpG sites directly after such T homopolymers has previously been challenging due to uncertain quantification of the light signal at these sites. The increased accuracy of PyroMark Q24 Advanced enables reliable quantification of CpG methylation behind and even within a stretch of 8 T nucleotides (see figure  Improved methylation quantification in homopolymers).

Analysis of mutations over long sequences
PyroMark Q24 Advanced also provides reliable quantification of multiple polymorphisms in a single assay. Since single nucleotide polymorphisms (SNPs) and other mutations are often not located close to one another, traditional Pyrosequencing chemistry usually requires separate assays for each mutation site to be analyzed. The new chemistry of PyroMark Q24 Advanced allows much longer runs, enabling reliable analysis of more than one mutation or SNP in the same run (see figure  Quantitative mutation analysis in long sequence runs).
See figures


PyroMark Q24 Advanced Software enables users to upgrade their existing PyroMark Q24 systems and take advantage of the new benefits and features of PyroMark Q24 Advanced. The software is compatible with PyroMark Q24 instruments running firmware 3.0 or higher. Please refer to the Upgrade to PyroMark Q24 Advanced  resource in the "Resources" tab for upgrade instructions and details concerning compatibility.


Pyrosequencing is becoming 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, the PyroMark Q24 Advanced enables 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.

Product comparison
  PyroMark Q24 Advanced PyroMark Q24
Throughput 1–24 samples 1–24 samples
Running volume 25 µl 25 µl 
PCR requirements 5–10 µl
(~0.5–3 pmol of product)
5–10 µl
(~0.5–3 pmol of product)            
Read length (assay- and
10–140 bp or more 10–80 bp
Application software PyroMark Q24 Advanced SW
(requires firmware 1.5.6903 or higher)
PyroMark Q24 SW 2.0
Software functionality SEQ (de novo sequencing)
CpG/CpN methylation
SQA (de novo sequencing)
CpG methylation
Main applications Complex mutation analysis
Epigenetics (CpG and CpN analysis)
Resistance typing and microbial ID  
Mutation analysis
Resistance typing 
Compatible reagents PyroMark Q24 Advanced Reagents
PyroMark Q24 Advanced CpG Reagents  
PyroMark Q24 Gold Reagents 
Sensitivity 2% mutation
98% wt  
2% mutation
98% wt  
Important: PyroMark Q24 Software cannot be used with PyroMark Q24 Advanced Reagents or PyroMark Q24 Advanced CpG Reagents. PyroMark Q24 Advanced Software cannot be used with PyroMark Gold Q24 Reagents. Using the incorrect reagent and software combination will result in incorrect reagent dispensations and lead to inaccurate results.

Supporting data and figures


Safety Data Sheets (1)


Which operating system is compatible with PyroMark Q24 Advanced application software?

The new PyroMark Q24 Advanced Software (version 3.0.1) is compatible with Windows XP, Vista (32 and 64 bit), Windows 7 (32 and 64 bit), and Windows 10 (64 bit).

FAQ ID -3164
Can I reinstall the PyroMark Q24 software on my new computer or following an operating system upgrade, or do I need to purchase a new license?

If you need to install the PyroMark Q24 software on a new computer replacing your old one, or after you reinstall or upgrade the operating system, you can reinstall the PyroMark Q24 software without purchasing a new license.

FAQ ID - 3473

What data are required for trouble-shooting?

For PyroMark Q24, PyroMark Q24 Advanced, PyroMark Q96 ID v2.5, and PyroMark CpG software, data are file-based. The original run files can be attached to email and sent to QIAGEN Technical Service.


For PyroMark Q96 ID 1.0, PyroMark Q96 MD/MDA 1.0, PSQ MA, and PSQ HS/HSA software, data are stored in an Oracle database and need exporting (See Instruction for run file export). Exported data can be attached to email and sent to QIAGEN Technical Service.


The log files from the PyroMark Q96 ID, PyroMark Q96 MD/MDA, PSQ MA, and PSQ HS/HSA instruments provide valuable information. See Instruction for log file retrieval) for log file retrieval instruction


Please send the log file along with the original run file to QIAGEN Technical Service.

FAQ ID -9051
How do I retrieve a backup copy if the data file on the USB stick cannot be opened from PyroMark Q24 or PyroMark Q24 Advanced instruments?

Do not remove the USB stick while data are being copied. Corrupted data files are usually due to the interrupted copying process. Follow these steps to retrieve recently saved runs.


1. When the instrument is not processing, insert a USB stick into the USB port on the instrument.


2. Use the Up and Down buttons to select “Administration” in the menu and press OK.


3. Select “Copy Recently Saved Runs” and press OK.


4. Use the Up and Down buttons to select the run file(s) for retrieval and press Select.


5. When the instrument confirms that the run file(s) has been saved to the USB stick, press Close and remove the USB stick.


In addition, repeat the process with a different USB stick. If the issue persists, please contact QIAGEN Technical Service for further assistance.

FAQ ID -9053
What are the operating system and hardware requirements for PyroMark software?
Why must I update the instrument method when lot numbers of the cartridge/tips change?

PyroMark instruments dispense the correct volume of enzyme mix, substrate mix, and nucleotides using variable pulse times and dispensing pressures. The settings for pulse times and dispensing pressure are specific for each lot of PyroMark dispensing tips and cartridges and need to be updated in the PyroMark software being used to operate the instrument.


Dispensing pressures and pulse time settings must be checked and, if necessary, updated every time a cartridge from a new lot number is used. Check the details at Managing PyroMark Instrument Methods.

FAQ ID -9055

The histogram is the theoretical peak pattern based on the Sequence to Analyze that you enter into the software, while the pyrogram is the experimental peak pattern that is detected by PyroMark instruments. The peak pattern in the pyrogram has to be in agreement with that in the histogram for a successful run. Overlaying the histogram on the pyrogram provides an invaluable tool for trouble-shooting Pyrosequencing runs. To do so, right click anywhere on the pyrogram and select Show Histogram on the pop-up menu.

FAQ ID -9056
Can I run multiple types of assays in one run?

Yes. The PyroMark Q24, PyroMark Q24 Advanced, and PyroMark Q96 v2.5 software provide multiple types of assay modes, which enable analyzing different types of samples at the same time.

FAQ ID -9057
What is a single peak height? What are the optimal single peak heights on various PyroMark instruments?

A single peak height is the signal produced from a non-variable single nucleotide.  The single peak height is an important criterion for trouble-shooting.


The optimal single peak height is 30 RLU (minimum 20 RLU) (Relative Light Unit) for PyroMark Q24 and PyroMark Q24 Advanced, 20 RLU (minimum 10 RLU) for PyroMark Q96 ID, and 100 RLU (minimum 50 RLU) for PyroMark Q96 MD instrument, respectively.

FAQ ID -9058
Can I install the PyroMark Q96 ID 1.0, PyroMark Q96 MD/MDA 1.0, PSQ MA, and PSQ HS/HSA software on an office computer for data analysis?

The PyroMark Q96 ID 1.0, PyroMark Q96 MD/MDA 1.0, PSQ MA, and PSQ HS/HSA software requires a functional Oracle database, which cannot be installed on an office computer. You can install the PyroMark software on an office computer and access the Oracle database on the PyroMark operator’s computer via network. The operator’s computer needs to be powered on while you are accessing the Oracle database remotely. Your IT department is responsible for the network setup.


FAQ ID -9059
What causes peaks to appear between dispensations on the pyrogram?

This can result from very high signal in a well which results in cross-talk between neighbouring wells. It could also be that the PyroMark instrument's camera is misaligned (contact: QIAGEN Technical service).

FAQ ID -9060
What causes wide peaks?

Sodium hydroxide carry-over, too much template, and incorrectly stored reagent are the most common causes of wide peaks. Ensure correct volume of denaturation buffer is used on the vacuum prep station. Use 5–20 µl of PCR product depending on instrument models. Store reagent as described in the handbook.


If the issue persists, please send the run files (see Instruction for run file export) to QIAGEN Technical Service for further assistance.

FAQ ID -9062