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