PyroMark Q24 Advanced Software

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).

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 sequence-dependent)	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 SNP AQ	SQA (de novo sequencing)CpG methylation AQ/SNP
Main applications	Complex mutation analysisEpigenetics (CpG and CpN analysis)Resistance typing and microbial ID	Mutation analysisResistance typing
Compatible reagents	PyroMark Q24 Advanced ReagentsPyroMark Q24 Advanced CpG Reagents	PyroMark Q24 Gold Reagents
Sensitivity	2% mutation98% wt	2% mutation98% 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.