PyroMark Q48 Autoprep: Instrument, reagents and accessories

Advantages of the PyroMark Q48 over other Pyrosequencing instruments

• Automated protocol requires less manual interaction
• Compact instrument size
• Advanced Reagents for longer read lengths
• Enabled for connectivity
• Touch screen display that guides users through the process

Users of PyroMark Q96 ID, and the PyroMark Q24 and PyroMark Q24 Advanced instruments should be aware that reagents for these instruments will be discontinued after 2023 and 2025 respectively. Assays should be migrated to the PyroMark Q48 Autoprep instrument to improve the Pyrosequencing workflow with automation and advanced methylation, mutation and SNP quantification.

* Read length varies, depending on the specific assay and sequence being analyzed.

** The PyroMark Q48 Autoprep System uses a file sharing network protocol (SMB version 1). Please consult with your IT team to safely connect the system to your network.

Improved workflow

Unlike other Pyrosequencing instruments, the PyroMark Q48 automates the Pyrosequencing workflow by using discs and magnetic beads for the Pyrosequencing protocol. The instrument touch screen guides users through each step thereby reducing errors.

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

The PyroMark “Advanced” chemistry and algorithms have lower backgrounds than the older “Gold” chemistry such as used by the PyroMark Q96 ID. This results in increased read length and reliability. Depending on the sequence to be analyzed, accurate read lengths of 140 or more bases can be obtained in just a single reaction.

Compatibility of assays among different PyroMark platforms

QIAGEN provides best practice recommendations on how to transfer assays to the PyroMark Q48 Autoprep Instrument. With some optimization, previously designed Pyrosequencing assays using other PyroMark instruments can be made compatible with the new PyroMark Q48 Autoprep Instrument. When optimized, 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 mutation analysis and  Compatibility among PyroMark platforms for methylation analysis). The cross-platform compatibility is also independent of the distance of the analyzed site away from the sequencing primer. This is particularly important when analyzing multiple sequence variations in a single run, typical for complex mutation assays or methylation analysis of consecutive CpG sites.

Contact us for assay transfer support.

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).

Increased throughput for more SNP and mutation assays per run

Multiple Primer Dispension (MPD) is a strategy to increase the capacity of automated sequencing primer dispension in cases where 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 and therefore 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.

PyroMark PCR and RT-PCR kits

The PyroMark PCR and PyroMark RT-PCR Kits are provided in a master mix format and are specifically optimized for Pyrosequencing analysis. The RT-PCR mix contains Omniscript and Sensiscript reverse transcriptases, a combination that provides highly efficient and sensitive reverse transcription from 1 pg to 2 μg of starting RNA. Both the PyroMark PCR and PyroMark RT-PCR Kits use HotStarTaq DNA polymerase. This enzyme is activated by a 15 minute, 95°C incubation, a step that often increases the yield of the specific PCR product.

The PyroMark PCR and PyroMark RT-PCR Kits also use Q-Solution, an innovative additive that facilitates and improves PCR amplification of difficult templates by modifying the melting behavior of DNA. CoralLoad Concentrate is also included, and its use has been linked to an improvement in yields. CoralLoad PCR Buffer provides the same high PCR specificity and minimal reaction optimization as the conventional QIAGEN PCR Buffer. Additionally, it contains two gel marker dyes for improved pipetting visibility and enhances convenience by enabling direct loading of PCR products onto a gel (see CoralLoad Concentrate for convenient gel loading of DNA).

PyroMark CpG assays

The PyroMark CpG assays use a design algorithm specific for CpG results that have a high success rate. The assay database includes over 84,000 assays for gene-specific human, mouse and rat CpG sites. We also offer predesigned assays for validating results from methylation arrays, such as the Infinium HumanMethylation450k and MethylationEPIC BeadChip arrays, through the provision of CpG loci identification numbers (CG#). (see  High Pyrosequencing success rate of predesigned PyroMark CpG assays).