PyroMark Q96 Systems

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.

PyroMark Gold Q24 reagent and the PyroQ24 Advanced reagent can only be used on the intended instrument model. The chemistry in the PyroMark Q24 Advanced reagent has been improved. Using PyroMark Q24 reagent on a PyroMark Q24 Advanced instrument can lead to much lower peaks; while using PyroMark Q24 Advanced reagent on a PyroMark Q24 instrument can result in increased background and running out of enzyme and substrate prematurely.

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.

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.

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.

All new assays have to be validated by the user. Interaction between primers or loops formed on single-strangled DNA can serve as priming sites for base incorporation by DNA polymerase. The following controls should be included when an assay is analyzed for the first time:

 1. PCR without template DNA —shows if the primers interact to give a background signal in Pyrosequencing reactions

2. PCR with template DNA but with no sequencing primer — shows if the template can loop back on itself and give a background signal in Pyrosequencing reactions

3. Sequencing primer without any PCR product — shows if the sequencing primer can form duplexes or hairpins and give background signal in Pyrosequencing reactions

4. Biotinylated primer without any PCR product — shows if the biotinylated primer can form duplexes or hairpins and give background signal in Pyrosequencing reactions

5. Sequencing primer and biotinylated primer together without PCR product —shows if the sequencing primer and the biotinylated primer can form duplexes and give background signal in Pyrosequencing reactions

Programs from these controls ought not to show any significant peaks after any nucleotide addition.

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.

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.

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.

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).

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.

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

In the case of mistakes and instrument failure, the single stranded DNA can be re-used without re-run PCR. Perform the vacuum prep steps with the streptavidin beads and release them to a new Pyrosequencing plate for a fresh run.

Mutation assays designed  by QIAGEN detect the most common mutations. In addition, QIAGEN provides plug-ins to analyze more mutations and convenient reports for PyroMark KRAS, PyroMark EGFR, and PyroMark BRAF kits. The plug-ins can be obtained by emailing pyro.plugin@qiagen.com

If the mutation cannot be analyzed successfully, please send the original run files to QIAGEN Technical Service for further assistance.

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.

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.

Pyrosequencing assay can detect unexpected/unknown mutations. Modify the Sequence to Analyze to analyze unexpected/unknown mutations. See How can I modify the Sequnce to Analyze post a run.

If the mutation cannot be analyzed successfully, please send the original run files to QIAGEN Technical Service for further assistance.

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.

Insufficient amount of PCR template, loss of streptavidin bead during vacuum prep steps, incorrect instrument method, and incorrect reagent are the common causes of low peak signals.

Check the PCR product with QIAxcel instrument or agarose gel electrophoresis and determine the appropriate amount of PCR product to be used. Perform vacuum prep function test. Determine the vacuum prep station functionality using the PyroMark Control Oligo with and without the vacuum prep steps. Ensure the correct instrument method is used. Refer to Managing PyroMark Instrument Methods. Ensure the correct reagent is used for the specific instrument model.

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.

Please see the Pyrosequencing Technology and Platform Overview.

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.

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.

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.

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.

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.

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.