PyroMark Q48 Autoprep: Instrument, reagents and accessories

For automated Pyrosequencing with advanced methylation, mutation and SNP quantification

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PyroMark Q48 Autoprep Instrument

Cat. No. / ID: 9002471

PyroMark Q48 Instrument, multistep pipet, software and documentation

InstrumentReagentKitAssayAccessoriesSoftware

PyroMark Q48 Autoprep

Q48 Reagents

PyroMark Kits

Assays and Tests

Consumables

PyroMark Software

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✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Features

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

Product Details

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.

Performance

Feature	PyroMark Q48 Autoprep	PyroMark Q24/Q24 Advanced	PyroMark Q96 ID
Samples per run	48	24	96
Automation of protocol	Automated template preparation with magnetic beads within a single instrument	No automation; separate vacuum station required for managing Sepharose beads	No automation; separate vacuum station required for managing Sepharose beads
Sequencing chemistry; read length	Advanced reagents: Up to 140 bp*	Gold reagent: Up to 80 bp* Advanced reagents: Up to 140 bp*	Gold reagent: up to 80 bp*
User interface	Protocols programmed with a remote PC; the instrument is accessed through various connectivity options and a touch screen**	Protocols programmed with the instrument’s screen buttons	Protocols programmed with a local PC
Support	Ongoing	Ends 2025	Ends 2023

* 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.

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).

See figures

Compatibility among PyroMark platforms for methylation analysis

Excluding well-to-well and disc-to-disc cross-contamination

The principle of multiple primer dispension (MPD)

High Pyrosequencing success rate of predesigned PyroMark CpG Assays

Supporting data and figures

Compatibility among PyroMark platforms for methylation analysis

ER-alpha methylation was measured in defined mixtures of methylated and unmethylated control DNA, representing methylation degrees of 0, 50, and 100%, using four different PyroMark platforms. The methylation measured for one CpG site was plotted against the expected percentage of methylated DNA. All four platforms gave the same results, showing that previously designed assays can be transferred among various PyroMark platforms.

Service Plans

Pyro Q48, Core Agreement

Cat. No. / ID: 9245255

One-time instrument repair performed at a regional repair center including shipping, labor and spare parts for a period of one year. Instrument repair turnaround time of 7–10 business days. Includes one on-site Preventive Maintenance or Inspection Service and a 10% discount on additional repair service during the Core Agreement period.

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Pyro Q48, Preventive Subscription

Cat. No. / ID: 9244448A

One on-site Preventive Maintenance or Inspection Service visit for the PyroMark Q48, including travel, labor and parts. Includes a 10% discount on repair services during the Preventive Subscription period.

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Resources

Download Safety Data Sheets for QIAGEN product components.

Scientific poster

Automated Pyrosequencing with integrated template preparation for advanced methylation, mutation and SNP quantification

パイロシークエンス（Pyrosequencing）解析用に至適化された感度と精度の高い1 ステップRT-PCR

Shortage of PyroMark Q48 Autoprep Absorber Strips

For use with PyroMark Q24 Advanced, PyroMark Q24, PyroMark Q96 ID and PyroMark Q96 MD systems

For use with PyroMark Q48 Autoprep, PyroMark Q24 Advanced, PyroMark Q24, PyroMark Q96 ID and PyroMark Q96 MD systems

A Pyrosequencing method for the forensic identification of tissue source using QIAGEN’s PyroMark Q24 Advanced system.

This is the latest version of the PyroMark Q48 Autoprep instrument software. The software may only be downloaded by registered users with a registered PyroMark Q48 Autoprep instrument. For updating please follow instructions provided in the user manual chapter 5.4 Upgrading the instrument software. The user manual can be downloaded from the PyroMark Q48 Autoprep webpage.

PyroMark Assay Design Software version 2.0.2 is compatible with Windows 7 and Windows 10 (64 bit) operating systems. This software may only be downloaded by registered users with a valid PyroMark Assay Design Software license. If you do not have a valid software license, contact your QIAGEN sales representative.

FAQ

PyroMark IdentiFire Software is compatible with Windows 2000, Windows XP, and Windows 7 (32 bit).

FAQ ID - 3340

PyroMark CpG Assay can be ordered in tubes or on 96-well plates. PyroMark CpG Assays, 96 wells, require a minimum order of 24 assays per plate.

FAQ ID -2824

There is no specific PyroMark Assay Design Software user manual available but a so-called Quick Guide can be downloaded from the PyroMark instrument webpage. Furthermore, the software contains a comprehensive online help (accessible via the Help menu or by pressing the “F1” key).

FAQ ID -2851

The recommended Streptavidin Sepharose High Performance beads for pyrosequencing can be ordered at GE Healthcare with the cat. no. 17-5113-01.

The PyroMark Q48 Autoprep protocol uses magnetic streptavidin-coated Sepharose® beads (PyroMark Q48 Magnetic Beads), which bind to the biotinylated PCR strand.

PyroMark Q48 Magnetic Beads can be ordered at QIAGEN with the cat. no. 974203.

FAQ ID -2850

The nucleotides can only be ordered as part of the PyroMark Gold Reagents which also contain enzyme and substrate mix.

FAQ ID -2827

Verify that the environmental temperature did not change (e.g., air conditioner cycling on or off) and remains under 32°C during the run. Ensure that reagents have been adapted to room temperature prior to the run.

FAQ ID - 3843

In the range of 3−5 bases can be resolved depending on the sequence context and base. If it is possible, sequencing of a homopolymer of more than 3−5 nucleotides should be avoided by resetting the sequencing primer.

FAQ ID -2871

All new assays have to be validated by the user. Interaction between primers or loops formed on single-strangled DNA can serve as priming sites for base incorporation by DNA polymerase. The following controls should be included when an assay is analyzed for the first time:

1. PCR without template DNA —shows if the primers interact to give a background signal in Pyrosequencing reactions

2. PCR with template DNA but with no sequencing primer — shows if the template can loop back on itself and give a background signal in Pyrosequencing reactions

3. Sequencing primer without any PCR product — shows if the sequencing primer can form duplexes or hairpins and give background signal in Pyrosequencing reactions

4. Biotinylated primer without any PCR product — shows if the biotinylated primer can form duplexes or hairpins and give background signal in Pyrosequencing reactions

5. Sequencing primer and biotinylated primer together without PCR product —shows if the sequencing primer and the biotinylated primer can form duplexes and give background signal in Pyrosequencing reactions

Programs from these controls ought not to show any significant peaks after any nucleotide addition.

FAQ ID -9066

The PyroMark Assay Design and application software do not support PCR setup with a pipetting scheme or PCR cycling conditions. General recommendations on how to setup and optimization of the PCR reaction are contained in the PyroMark PCR Handbook.

FAQ ID -2862

In pyrosequencing, the 5' end should be biotinylated, regardless of whether the forward or reverse primer is biotinylated.

FAQ ID -2839

The PyroMark Q96 ID software version 1.0 contains a SNP mode for simplex and multiplex entries (and Allele Quantification analyses can be performed for simplex entries) and an SQA mode for de novo sequencing analysis. This software version does not contain a CpG mode for methylation analysis, however the PyroMark CpG software v1.0 can be used.

FAQ ID -2845

PyroMark Q24: The mean single peak height is 95 +/- 55 RLU.
PyroMark Q48: The mean single peak height is 70 +/- 40 RLU.
PyroMark Q96 ID: The mean single peak height is 35 +/- 10 RLU.
Pyromark Q96 MD: The mean single peak height should be at least 350 RLU.

FAQ ID -2852

The PyroMark Q96 MD software contains a SNP mode for simplex and multiplex entries (and Allele Quantification analyses can be performed for simplex entries). This software version does not contain a CpG mode for methylation analysis, however the PyroMark CpG software v1.0 can be used. SQA analysis for de novo sequencing is not possible.

See our Product Selection Guide for additional information on CpG supplementary software.

FAQ ID -2866

The PyroMark Custom Assay includes a 10x PCR Primer Set (mixture of forward and reverse PCR Primer) and 10x Sequencing Primer. Reagents for performing PCR and pyrosequencing reaction are not included.

FAQ ID -2815

PyroMark CpG Assays are genome-wide, pre-designed methylation assays for pyrosequencing analysis. An optimized design algorithm was used for highly specific assay design and advanced CpG methalytion results.

FAQ ID -2821

In general, the standard claim for pyrosequencing sensitivity is approximately 5%, which is also published in many papers. The actual sensitivity limit is assay dependent and has to be determined individually.

FAQ ID -2840

QIAamp/DNeasy Kits can be used for DNA isolation, EpiTect Bisulfite Kits for DNA conversion, PyroMark PCR Kit for PCR amplification, EpiTect Control DNA Set for PCR controls, and PyroMark Gold Q24 Reagents or PyroMark Gold Q96 Reagents for the sequencing reaction.

Depending on the platform used, the following reagent kits are required for pyrosequencing:

PyroMark Q96 ID and MD: PyroMark Gold Q96 Reagents
PyroMark Q24: PyroMark Gold Q24 Reagents
PyroMark Q24 Advanced: PyroMark Q24 Advanced Reagents and PyroMark Q24 Advanced CpG Reagents
PyroMark Q48 Autoprep: PyroMark Q24 Advanced Reagents and PyroMark Q24 Advanced CpG Reagents

FAQ ID -2822

In the case of mistakes and instrument failure, the single stranded DNA can be re-used without re-run PCR. Perform the vacuum prep steps with the streptavidin beads and release them to a new Pyrosequencing plate for a fresh run.

FAQ ID -9063

There are several reasons for a high assay background; the template can form secondary structures that are extended or the primers itself form dimmers that serve as template. Perform accurate sequencing controls (e.g., PCR or sequencing primer only) as recommended in the PyroMark User Manual to observe this kind of background. In addition, an unspecific priming of primer to template or unspecific annealing of sequencing primer to template might also be a background cause. Please check your complete primer design and, if needed, perform a redesign. Try to lower the primer concentration as possible to avoid excess of primer.

FAQ ID -2877

PyroMark instruments offer a range of throughput scales. The PyroMark Q24 can process 1–24 samples in parallel, the PyroMark Q48 Autoprep, 1–48; the PyroMark Q96 ID, 1–96; and the PyroMark Q96 MD, 1–96; or the automation option enables automated processing of ten 96-well plates. The sample processing speed depends on the number of nucleotide dispensations necessary for the programmed analysis. Twenty dispensations take approximately 24 minutes on all instruments; thus, 96 samples are typically processed in 10–100 minutes.

FAQ ID -2215

PyroMark Control Oligo has a concentration of 20 µM and is delivered in a volume of 50 µl. Two tubes of 10x dilution buffer (2x 1.7 ml) are delivered with the control oligo.

FAQ ID -2846

PyroMark CpG Assays are shipped lyophilized at ambient temperatures (20−25°C) and should be stored at −20°C either reconstituted or lyophilized. Repeated freeze−thaw cycles should be avoided. When stored under these conditions, the reconstituted product can be kept for at least 18 months from the date of receipt without reduction in performance.

FAQ ID -2816

The PyroMark CpG Assay is reconstituted as a 10x PCR Primer Set in 550 µl TE, pH 8.0 and the 10x Sequencing Primer is reconstituted in 1175 µl Annealing Buffer if using the PyroMark Q24, 880 µl if using the PyroMark Q96 ID, and 1175 µl if using the PyroMark Q96 MD.

FAQ ID -2817

Pyrosequencing assay can detect unexpected/unknown mutations. Modify the Sequence to Analyze to analyze unexpected/unknown mutations. See How can I modify the Sequnce to Analyze post a run.

If the mutation cannot be analyzed successfully, please send the original run files to QIAGEN Technical Service for further assistance.

FAQ ID -9064

Usually the sequencing primer is used at 0.3 µM in annealing buffer but some assays might require additional optimization of the sequencing primer concentration.

For PyroMark Q24 and PyroMark Q96 MD, the final concentration of the sequencing primer is 0.3 µM and, for PyroMark Q96 ID, 0.4 µM.

The PyroMark Q48 Autoprep dispenses the sequencing primers for annealing. The final concentration of sequencing primers in a well is 0.8 µM but may be adapted to optimize assays.

FAQ ID -2826

No, QIAGEN does not design any Custom PyroMark CpG Assay. Customers have the possibility to order pre-designed, genome-wide PyroMark CpG Assays or order a user-designed assay (e.g., with the PyroMark Assay Design Software or assays known from previous projects or from the literature).

FAQ ID -2818

The new PyroMark Assay Design Software Version 2.0 contains new algorithms to facilitate CpG assay design and enables PCR and sequencing primer design for all pyroseqeuncing applications and PyroMark instruments.

FAQ ID -2849

Primer sequences for PyroMark CpG Assays are not provided; they are proprietary.

FAQ ID -2823

Only the biotinylated primer needs to be HPLC purified, whereas the other primers require standard desalting only.

FAQ ID -2832

In general, dUTP/UNG treatment should work for pyrosequencing to reduce contamination risk with PCR amplicons from previous PCRs.

FAQ ID -2843

The unmethylated and unconverted human control DNA of the EpiTect PCR Control DNA Set allows to check that primers designed for the specific detection of unmethylated and converted DNA (U-converted DNA), and for methylated, converted DNA (M-converted DNA) does not bind to untreated genomic DNA.*

In case bisulfite conversion was not complete, leaving certain unmethylated C residues unconverted, false positives would result if the primer specific for M-converted DNA binds to untreated gDNA.

This control DNA can also be used to check conversion efficiency during bisulfite treatment.

*Summary of principle: Methylation of DNA occurs on cytosine residues, especially on CpG dinucleotides enriched in small regions of DNA. Incubation of target DNA with sodium bisulfite, using, for example, EpiTect Bisulfite Kits, results in conversion of unmethylated cytosine residues into uracil, leaving methylated cytosines unchanged.

FAQ ID -2007

Typical reading length using pyrosequencing technology is 40−60 bases. However, as with any sequencing technology, the maximum read length will depend on template secondary structure, base content, quality of PCR-product, and other parameters.

Depending on the sequence to be analyzed, highly accurate read lengths of 140 bases or more can be obtained in just a single reaction with the Q48 PyroMark Autoprep.

FAQ ID -2216

The amplicon length should be short (<200 bp). This is critical especially for DNA from FFPE tissue that is often degraded by the fixation so that short fragments are easier to amplify. Moreover, the DNA suffers from harsh bisulfite treatment and might receive further double strand breaks. Therefore, the amplicon size should be kept as short as possible.

FAQ ID -2825

Reconstiuted enzyme and substrate of PyroMark Gold Reagents, should not be vortexed since this could lead to conformational changes which affect the activity.

FAQ ID -2844

All relevant information regarding PyroMark CpG Assay setup can be found on the GeneGlobe website. For the Q24 and Q96, the "Sequence to Analyze" and dispensation order should not be copied manually to create a new assay. Instead, the assay file should be downloaded from the web and opened in the PyroMark CpG software, PyroMark Q96 ID v2.5 (or higher) software, and PyroMark Q24 Software to keep important software settings.

When using the PyroMark CpG assays with the PyroMark Q48, use the "Sequence to Analyze" provided in the "product specification" section to create an assay setup file in the PyroMark Q48 software. This is done by selecting New CpG assay and pasting in the Sequence to Analyze (not the "sequence after bisulfite treatment") into the Sequence Before Bisulfite Treatment field and pressing Create Dispensation order.

FAQ ID -2814