PyroMark Q24, PyroMark Q24 Advanced and PyroMark Q48 Autoprep

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.

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.

The PyroMark Control Oligo is a self-priming oligo which can be used directly in the pyrosequencing reaction without an extra sequencing primer. It was designed for installation of the different PyroMark systems and can be used for general troubleshooting.

It can be used directly for sequencing without previous PCR setup and sample preparation by the vacuum prep tool. By this test, the performance of the instrument can be checked.

In addition, it can also be processed through the vacuum sample preparation to check the performance of this procedure. The control oligo run results in a defined pyrograms with certain peak heights for the individual instruments which are described in the handbook.

The PyroMark Control Oligo can be used on all PyroMark systems including the Q24, Q24 advanced, Q96 ID, and Q96 MD.

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.

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.

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.

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 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.