PyroMark Q48 Advanced Reagents

PyroMark Q48 Advanced Reagents are designed for use with the new PyroMark Q48 Autoprep and together provide fully automated Pyrosequencing with integrated template preparation. PyroMark Q48 Advanced Reagents are highly suited for analyzing any kind of sequence variation, particularly DNA methylation at CpG or CpN sites and complex mutations, or for de novo sequencing applications such as microbial typing. For long-read Pyrosequencing runs that may require larger volumes of nucleotides, we recommend using the PyroMark Q48 Advanced CpG Reagents.

Advanced technology, software and chemistry for long and reliable sequence runs

PyroMark Q48 Autoprep features improved chemistry and instrument operation algorithms that significantly increase assay read length and accuracy in base calling, mutation analysis and methylation quantification compared to PyroMark Q96 and PyroMark Q24 systems. Assay read length in previous systems was limited by background peaks and reduced light signals in the sequencing reaction. The updated PyroMark “Advanced” chemistry and algorithms reduce this background, 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 reaction.

Increased throughput for more SNP and mutation assays per run

Multiple Primer Dispensation (MPD) is a strategy to increase the capacity of automated sequencing primer dispensation in case more assays are needed than cartridge reservoirs are available. The PyroMark Q48 Autoprep Primer Cartridge has 3 reservoirs, but if more assays are needed per disc, the primers can be mixed and filled into the same reservoir of the primer cartridge. After template preparation, primer mixes are dispensed into the wells automatically. Since only one PCR template is present in each well, only the corresponding sequencing primer will bind to the template. All other primers will stay in solution but will not bind (see  The principle of multiple primer dispensation). Primers in each MPD mix should be designed and checked to avoid formation of primer–dimers or binding to another PCR template.

Compatibility of assays among different PyroMark platforms

Previously designed Pyrosequencing assays are readily compatible with the new PyroMark Q48 Autoprep instrument and the “Advanced” chemistry. Data indicate that the same mutation frequencies and methylation quantification results are obtained when an assay is run on the various PyroMark platforms (see  Compatibility among PyroMark platforms for methylation analysis and  Compatibility among PyroMark platforms for mutation analysis). The cross-platform compatibility is also independent of the distance of the analyzed site away from the sequencing primer (see Compatibility among PyroMark platforms regardless of CpG position). This is particularly important when analyzing multiple sequence variations in a single run, which is typical for complex mutation assays or methylation analysis of consecutive CpG sites.

New disc design enables magnetic template preparation without cross contamination

PyroMark Q48 Discs are specially designed to automate template preparation and Pyrosequencing in the same instrument without manual user interaction. All buffers used for template preparation are efficiently removed from the sample and the disc during the run without the risk of any cross-contamination from well-to-well of one run or from disc-to-disc between subsequent runs (see  Excluding well-to-well and disc-to-disc cross-contamination).

The PyroMark Q48 Autoprep uses proven real-time sequence-based Pyrosequencing technology for detection and quantification in genetic analyses and epigenetic methylation studies. The system can analyze up to 48 samples simultaneously. An easy-to-use, automated protocol prepares single-stranded DNA samples without manual interaction from the user. This protocol uses magnetic streptavidin-coated Sepharose beads (PyroMark Q48 Magnetic Beads), which bind to the biotinylated PCR strand. Annealing of sequencing primers can be automated for up to four different sequencing primers. If more sequencing primers are used, the primers can be manually added to the single-stranded DNA samples.

A run file is created using the PyroMark Q48 Advanced software and is transferred to the instrument via a USB drive or network connection. Reagents, nucleotides and buffers are loaded into the PyroMark Q48 Autoprep cartridges, according to the volumes indicated on the touchscreen. The biotinylated PCR product and magnetic streptavidin-coated Sepharose beads are loaded into the wells of the Q48 Disc, and the disc and an absorber strip are placed into the instrument. Preparation of the single-stranded template, along with sequencing primer annealing are now completely automated and require no additional user interaction. Up to 3 separate sequencing primers or Multiple Primer Dispensation (MPD) mixes can be dispensed automatically. Optional, manual primer addition further increases the number of different assays per run.

Pyrosequencing is 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, PyroMark platforms enable 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.