PyroMark Q24 Advanced Reagents
For advanced methylation and mutation quantification in long sequence runs using Pyrosequencing
PyroMark Q24 Advanced Reagents feature advanced Pyrosequencing chemistry to provide even better real-time sequence-based detection and quantification than before. PyroMark Q24 Advanced Reagents are optimized for use with PyroMark Q24 Advanced Systems and are highly suited for analyzing any kind of sequence variation, particularly DNA methylation at CpG or CpN sites, complex mutations, or for de novo sequencing applications such as microbial typing. For long-read Pyrosequencing runs which may require larger volumes of nucleotides, such as de novo sequencing or methylation analyses we recommend using the PyroMark Q24 Advanced CpG Reagents.
Longer Pyrosequencing runs and improved accuracyPyroMark 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 positionPyroMark 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 homopolymersBisulfite 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 sequencesPyroMark 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).
Pyrosequencing technology, which is based on the principle of sequencing by synthesis, provides quantitative data in sequence context within minutes. PyroMark Q24 Advanced Reagents and PyroMark Q24 Advanced CpG Reagents feature updated Pyrosequencing chemistry to enable analysis of complex mutations, methylation analysis in epigenetic research, resistance typing, and microbial identification. These reagents are optimized for use with PyroMark Q24 Advanced Systems.
Fast and easy sample preparationPrior to Pyrosequencing, a biotinylated PCR product is generated. This biotinylated PCR product is bound to Streptavidin-coated Sepharose beads, and the beads are captured with the Vacuum Tool on the Vacuum Workstation, where they are thoroughly washed and subsequently denatured, generating single-stranded DNA suitable for Pyrosequencing. This template DNA is released into the Pyrosequencing reaction plate containing the sequencing primer, and after primer annealing, the plate is placed into the PyroMark instrument. PyroMark Q24 Advanced Reagents or PyroMark Q24 Advanced CpG Reagents contain the enzymes, nucleotides, and substrate for the Pyrosequencing reaction; these are pipetted into the dispensing cartridge, according to the volumes provided by the software, and are also placed into the instrument for the Pyrosequencing run.
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.
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