PyroMark Q96 ID

For quantitative analysis of genetic or epigenetic DNA modifications using Pyrosequencing technology

Features

  • Sequence context provides built-in quality control of the assay
  • Enables a broad range of analyses, powered by intuitive software
  • Run up to 96 different assays or 96 samples with one assay together
  • Fast delivery of direct and unambiguous sequence data
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PyroMark Q96 ID Software 2.5

Cat. No. / ID: 9022198

Application software for PyroMark Q96 ID systems
SoftwareProduct
PyroMark Q96 ID Software
Pyro Q96 ID, Full Agreement
Pyro Q96 ID, Preventive Subscription
Pyro Q48, Preventive Subscription
The PyroMark Q96 ID Instrument and PyroMark Q96 Vacuum Workstation are intended to be used only in combination with QIAGEN kits indicated for use with the PyroMark Q96 ID for the applications described in the kit handbooks. If the PyroMark Q96 ID Instrument and PyroMark Q96 Vacuum Workstation are used with other than QIAGEN kits, it is the user's responsibility to validate the performance of such product combination for any particular application.

Product Details


PyroMark Q96 ID is a powerful sequencing and quantification platform highly suited for epigenetics, mutation gene expression analysis, and microbial identification and resistance typing. With its 96-well format, automatic base-calling function, and dedicated software solutions for methylation analysis and assay design, the PyroMark Q96 ID can handle any research question requiring true sequence information and quantification of genetic or epigenetic variation.

Performance

Integrating detection and quantification of genetic variation into one powerful system, Pyrosequencing with the PyroMark platform outperforms other sequence-based solutions in the analysis of targeted short DNA sequences. For analysis of methylation in epigenetic studies, Pyrosequencing generates highly reproducible quantification of methylation frequencies at individual or consecutive CpG sites (see figure " Analysis of multiple contiguous CpG sites"), and can detect and quantify even small changes in methylation levels (see figure " Linearity of methylation quantification").

For genetic testing applications, alleles of variable loci are accurately quantified, and heterozygosity is easily resolved (see figure " Analysis of a tri-allelic SNP"). For microbial identification and resistance typing, Pyrosequencing enables the concurrent analysis of multiple samples for common drug resistance mutations (see figure "Analysis of antibacterial resistance in Helicobacter pylori").

See figures

Principle

Pyrosequencing technology, which is based on the principle of sequencing by synthesis, provides quantitative data in sequence context within minutes. PyroMark Q96 ID is a fully integrated system that provides real-time sequence information and is highly suitable for detection of genetic variations, genetic quantification, and short DNA sequencing. The following products are used in combination with PyroMark Q96 ID instrument: PyroMark Q96 Vacuum Workstation, PyroMark CpG SW, PyroMark Assay Design SW, PyroMark IdentiFire SW, PyroMark Gold Q96 Reagents, and PyroMark Control Oligo. Sample preparation solutions are also supplied to enable preparation of single-stranded DNA using the PyroMark Q96 Vacuum Workstation.  

Steps of the Pyrosequencing reaction:

Step 1: A DNA segment is amplified, and the strand to serve as the Pyrosequencing template is biotinylated. After denaturation, the biotinylated single-stranded PCR amplicon is isolated and allowed to hybridize with a sequencing primer. The hybridized primer and single-stranded template are incubated with the enzymes DNA polymerase, ATP sulfurylase, luciferase, and apyrase, as well as the substrates adenosine 5' phosphosulfate (APS) and luciferin (see figure " Principle of Pyrosequencing — step 1"). 

Step 2: The first deoxribonucleotide triphosphate (dNTP) is added to the reaction. DNA polymerase catalyzes the addition of the dNTP to the squencing primer, if it is complementary to the base in the template strand. Each incorporation event is accompanied by release of pyrophosphate (PPi), in a quantity equimolar to the amount of incorporated nucleotide (see figure " Principle of Pyrosequencing — step 2").

Step 3: ATP sulfurylase converts PPi to ATP in the presence of adenosine 5' phosphosulfate (APS). This ATP drives the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP. The light produced in the luciferase-catalyzed reaction is detected by CCD sensors and seen as a peak in the raw data output (Pyrogram). The height of each peak (light signal) is proportional to the number of nucleotides incorporated (see figure " Principle of Pyrosequencing — step 3").

Step 4: Apyrase, a nucleotide-degrading enzyme, continuously degrades unincorporated nucleotides and ATP. When degradation is complete, another nucleotide is added (see figure " Principle of Pyrosequencing — step 4").

Step 5: Addition of dNTPs is performed sequentially. It should be noted that deoxyadenosine alpha-thio triphosphate (dATPαS) is used as a substitute for the natural deoxyadenosine triphosphate (dATP), since it is efficiently used by the DNA polymerase, but not recognized by the luciferase. As the process continues, the complementary DNA strand is elongated, and the nucleotide sequence is determined from the signal peaks in the Pyrogram trace (see figure " Principle of Pyrosequencing — step 5").

Streamlined workflow —  from sample to result

The versatile PyroMark Q96 ID seamlessly integrates into epigenetics and genetic analysis workflows, and complements QIAGEN's advanced technologies for sample preparation, bisulfite conversion, and PCR amplification. This highly reliable instrument enables sequence-based quantification and detection of SNPs, insertion and deletion mutations, CpG sites, as well as generation of sequence information. The streamlined workflow means that results can be achieved faster.

Fast and easy sample preparation

From PCR product to single-stranded template ready for sequencing — up to 96 samples can be prepared in parallel using the PyroMark Q96 Vacuum Workstation, in less than 15 minutes. The workstation ensures easy handling and the actual "hands-on time" is less than 5 minutes.

See figures

Procedure

Fast and easy sample preparation 

From PCR product to single-stranded template ready for sequencing — up to 96 samples can be prepared in parallel using the PyroMark Q96 Vacuum Workstation in less than 15 minutes. The workstation ensures easy handling, and the actual "hands-on time" is less than 5 minutes.

Prior 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 Gold 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.

Applications

Pyrosequencing is becoming increasingly important for research applications in a variety of disciplines. The PyroMark Q96 ID enables powerful and versatile analysis of genetic and epigenetic variation, 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. In addition, because Pyrosequencing integrates sequence detection and quantification, the high analysis resolution can lead to new discoveries.

Software

Easy-to-use PyroMark Q96 ID Software 2.5 

PyroMark Q96 ID software version 2.5 enables comprehensive analysis of your results. The recently updated software now contains four analysis modes: AQ (allele quantification), SNP (analysis of SNPs and InDels), CpG (methylation analysis), and SQA (sequence identification). These four different types of analyses can be performed on the same plate, in the same run.

Flexible and simple Pyrosequencing assay design using PyroMark Assay Design Software

PyroMark Assay Design Software 2.0 ensures easy design of PCR and sequencing primers. The assays are optimized for all PyroMark instruments.

Supporting data and figures

Specifications

FeaturesSpecifications
Overvoltage categoryII
Weight52 kg (114.6 lb)
Kits designed for this instrumentPyroMark Q96 Tests
ApplicationsMethylation analysis, allele quantification (SNP, InDels), sequence analysis
Instrument dimensions490 x 540 x 620 mm (19.3 x 20.5 x 24.4 in)
Operating temperature18–28°C (64–82°F)
Place of operationFor indoor use only
Pollution level2
Power100–120 V AC, 220–240 V AC; 56–60 Hz
Process temperature28°C (82.4°F) ± 1%
Process time10 to 100 minutes for up to 96 samples in parallel
Samples per run (throughput)1–96
SoftwarePyroMark Q96 ID Software 2.5 (included), PyroMark CpG SW 1.0 (supplementary), PyroMark IndentiFire SW 1.0 (supplementary), PyroMark Assay Design SW 2.0 (supplementary)
TechnologyPyrosequencing
AltitudeUp to 2000 m (6500 ft)

Resources

Instrument User Manuals (2)
Kit Handbooks (1)
For performing Pyrosequencing reactions on the PyroMark Q96 ID, PyroMark Q96 MD, and PyroMark Q96 MD Automated
Operating Software (1)
PyroMark Q96 ID Software 2.5
SOFTWARE (253MB)
PyroMark Q96 ID Software 2.5 version 2.5.10.7 is the application software for setting up, performing and analyzing runs on PyroMark Q96 ID instruments. The main feature of this release is 64bit compatibility with Windows 7 operating systems.

This software can only be downloaded by users with a registered PyroMark Q96 ID instrument.

Publications

Genetic diagnostics of functional variants of the human dopamine D2 receptor gene.
Doehring A; Kirchhof A; Lötsch J;
Psychiatr Genet; 2009; 19 (5):259-68 2009 Oct PMID:19512960
Association between Genetic Polymorphism of Multidrug Resistance 1 Gene and Sasang Constitutions.
Kim HJ; Hwang SY; Kim JH; Park HJ; Lee SG; Lee SW; Joo JC; Kim YK;
Evid Based Complement Alternat Med; 2009; 6 Suppl 1 :73-80 2009 Sep PMID:19745014
A pyrosequencing assay for the rapid discrimination of mitochondrial lineages in the Salmo trutta species complex.
Keller I; Taverna A; Seehausen O;
Mol Ecol Resour; 2011; 11 (1):196-9 2011 Jan PMID:21429122
Clinical significance of K-ras and BRAF mutations in Chinese colorectal cancer patients.
Shen H; Yuan Y; Hu HG; Zhong X; Ye XX; Li MD; Fang WJ; Zheng S;
World J Gastroenterol; 2011; 17 (6):809-16 2011 Feb 14 PMID:21390154
Use of Pyromark Q96 ID pyrosequencing system in identifying bacterial pathogen directly with urine specimens for diagnosis of urinary tract infections.
Lu J; Yu R; Yan Y; Zhang J; Ren X;
J Microbiol Methods; 2011; 86 (1):78-81 2011 Apr 5 PMID:21473889

FAQ

Where can I order the Streptavidin Sepharose beads for pyrosequencing?
The recommended Streptavidin Sepharose High Performance beads for pyrosequencing can be ordered at GE healthcare with the catalog 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 catalog no 974203.

FAQ ID -2850
Can I order the nucleotides from PyroMark Gold Reagents separately?
The nucleotides can only be ordered as part of the PyroMark Gold Reagents which also contain enzyme and substrate mix.
FAQ ID -2827
How many nucleotides of a homopolymer can be resolved in pyrosequencing?
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
I need to buy a new computer for my PyroMark Q96. Can I reinstall the PyroMark Q96 software on my new computer or do I need to purchase a new license?

If you need to purchase a new computer for your PyroMark Q96, or if you need to reinstall, upgrade, or replace the operating system (OS), you can reinstall the PyroMark Q96 software without purchasing a new license.

FAQ ID - 3472
What are the corresponding QIAGEN names for former Biotage instruments?

When looking at technical literature, pulse time settings, and other information, the following table can be used to determine the QIAGEN names for former Biotage instruments.

 

Biotage QIAGEN
PSQ 96 PyroMark Q96 ID
PSQ 96 MA PyroMark Q96 ID
PSQ HS 96 PyroMark Q96 MD
PSQ HS 96A PyroMark Q96 MD Automated
FAQ ID -2285
What is a PyroMark instrument method or instrument code?

An instrument method or instrument code encodes the individual pulse time settings of specific cartridge lot batch. These pulse time settings change when e.g. a new batch of capillaries is used with slight variations in the needle diameter. For larger diameters, the pulse settings are lowered to dispense the correct volume of liquid. In addition, the viscosity of enzyme and substrate mixes can change which influences dispensing volumes.

The individual instrument method/code number is printed on the cartridge label. The corresponding methods/code settings can be downloaded as a file from the respective instrument webpage and opened in the PyroMark application software.

FAQ ID -2941
What is the reason for a high substrate peak in the pyrosequencing pyrogram?
Usually pyrophosphate or dATP/ATP contamination in the sample or in the buffer can cause a high substrate peak. Large amounts of pyrophosphate are generated in the PCR reaction and might be carried over to the sequencing reaction. Check the PyroMark buffers and reagents and use new ones.
FAQ ID -2879
What are the features of the new PyroMark Q96 ID software 2.5?

This software will replace the old PyroMark Q96 ID software version 1.0. The PyroMark Q96 ID software version 2.5 contains four assay setup and analysis modes which are AQ, CpG, SNP and SQA. It is based on the Pyromark Q24 application software and is a file based software (runs are not stored in an ORACLE database). It replaces the PyroMark CpG software since a CpG mode is included. There is no SNP multiplexing support and no alignment functionality (as found in software version 1.0).

FAQ ID -2867
Which end of the PCR primer for pyrosequencing should be biotinylated?
In Pyrosequencing, the 5' end should be biotinylated, regardless of whether the forward or reverse primer is biotinylated. You can order pyrosequencing primers here.
FAQ ID -2839
What kind of shaker should be used for the pyrosequencing binding step?
Shaking conditions are 1400 rpm at room temperature. Optimal results are obtained with 2mm orbital diameter.
FAQ ID -2837
Which analyses can be performed with the PyroMark Q96 ID software version 1.0?
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
What should be the single peak height for the PyroMark Control Oligo on the different PyroMark instruments?


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
What is the sensitivity limitation for pyrosequencing?
In general, the standard claim for pyrosequencing sensitivity is about 5% which is also published in many papers. The actual sensitivity limit is assay dependent and has to be determined individually.
FAQ ID -2840
When do I have to change the pulse settings/methods in a pyrosequencing run setup?
Always check for the actual method/code number printed on the cartridge label. Make sure that you choose this method/code number when setting up the pyrorun in the application software. If this method cannot be selected automatically in the application software, you can download the method/code file from the instrument webpage.
FAQ ID -2942
Which PyroMark Gold Q96 Reagent should be used for which instrument and application?

PyroMark Gold Q96 Reagents:

  • PyroMark Gold Q96 SQA Reagents (1 x 96) should be used for performing SQA analyses on the PyroMark Q96 ID. It can also be used to supplement the PyroMark Gold Q96 Reagents (5 x 96) when long runs lead to a shortage of nucleotides.
  • PyroMark Gold Q96 Reagents (5 x 96) should be used for SNP and CpG analyses on the PyroMark Q96 ID and MD instruments. On the PyroMark Q96 MD, the kit contains enough for 15x96 plates.
  • PyroMark Gold Q96 CDT Reagents (6 x 96) should be used only with CDTs on the PyroMark Q96 MD instrument, for performing SNP and CpG anslyses. The nucleotides are pre-diluted for use with CDTs, so this is not to be used on ID instrument or with NDTs on the MD.
  • PyroMark Gold Q96 Reagents (50 x 96) should be used only with the PyroMark Q96 MDA (automated option) for SNP anslyses. Not for use with the PyroMark Q96 ID or non-automated MD.

See the PyroMark Gold Q96 Reagents Handbook for additional information.

FAQ ID -2836
How do I reduce background peaks in the pyrosequencing pyrogram?
There are several reasons for a high assay background; the template can form secondary structures which are extended or the primers itself form dimmers which 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
What is the sample throughput of Pyrosequencing systems?

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
What is the concentration of PyroMark Control Oligo?
PyroMark Control Oligo has a concentration of 20µM and is delivered in a volume of 50µl. Two tubes of 10x dilution buffer (2x 1.7ml) are delivered with the control oligo.
FAQ ID -2846
What is the pyrosequencing data exchange tool for?
This tool is part of the old PyroMark Q96 ID/MD systems that run with the ORACLE database. The tool enables exporting and importing run files from the ORACLE database.
FAQ ID -2864
How many times can vacuum troughs be re-used with the PyroMark Vacuum Preparation Stations?
There is no precise recommendation how many times these troughs on the PyroMark Vacuum Preparation Stations (Q24 and Q96) can be re-used. It depends on the individual handling and cleaning (with water).
FAQ ID -2848
Which heating block is recommended for the pyrosequencing annealing step?
A heating block that can heat up to 80-90 °C is recommended. A solid block is preferable. For the PyroMark Q24 the surface area must be 50mm x 60 mm and for Pyromark Q96 ID/MD 120mm x 80 mm.
FAQ ID -2838
What is the PyroMark Q96 Data Converter?
PyroMark Q96 Data Converter 1.0 is a tool for exporting pyrosequencing runs from PyroMark Q96 ID ORACLE database. It converts the runs into a file format that is compatible with the new PyroMark Q96 ID software version 2.5.
FAQ ID -2868
What concentration should be used for the sequencing primer in pyrosequencing?

Usually the sequencing primer is used at 0.3µM 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 0.3µM and for PyroMark Q96 ID 0.4µM.


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

 

FAQ ID -2826
Where to find explanations to the warning given by the PyroMark software after run data analysis?
The PyroMark Q24 application software contains under Help (Press F1) a Pyromark Q24 Software user guide with all essential information about warnings and software features. The Pyromark ID/MD software also contains a softwareware guide under help. Moreover, the individual instrument user manuals contain helpful information in the troubleshooting section.
FAQ ID -2874
Which purity grade is recommended for pyrosequencing primers?
Only the biotinylated primer needs to be HPLC purified whereas the other primers require standard desalting only.  Pyrosequencing primers can be ordered here.
FAQ ID -2832
What is the reason for split peaks appearing in between dispensations on my pyrosequencing pyrogram?
The PyroMark cartridge needle can be blocked or damaged. Clean the cartridge or exchange with a new one. Check for correct reagent cartridge and cartridge method used in the run. Check if the reagent cartridge cover was closed properly. Make sure that the cartridge was dry after cleaning because nucleotide droplets might be caught at the needle tip and fall down at any time. or exchanged.
FAQ ID -2881
Will dUTP in a PCR reaction affect pyrosequencing?
In general, dUTP/UNG treatment should work for pyrosequencing in order to reduce contamination risk with PCR amplicons from previous PCR’s.
FAQ ID -2843
Is the CpG software included in the PyroMark instruments to study methylation status?
The PyroMark Q24 software and the new PyroMark Q96 ID software version 2.5 support CpG analysis in the CpG mode. The ORACLE-based PyroMark Q96 ID software version and PyroMark Q96 MD software do not support CpG analysis. In this case an independent, additional software is needed which is the PyroMark CpG software version 1.0.
FAQ ID -2842
What is the reason for signals ceasing in the middle of a pyrosequencing run?
The cartridge needle can be blocked or damaged causing a dispensation error. Clean the cartridge following the guidelines or repeat the run with a new cartridge. On the other hand if high amounts of template have been used resulting in very high signals (>100 RLU), the substrate for the sequencing reaction might be depleted. In this case template conditions should be optimized.
FAQ ID -2875
What kind of reading length can I expect when using Pyrosequencing technology for sequence analysis?

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 or more bases can be obtained in just a single reaction with the Q48 PyroMark Autoprep.

 

 

FAQ ID -2216
What is the recommended amplicon size for CpG assays?
The amplicon length should be short (<200bp). This is critical especially for DNA from FFPE tissue which 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
Can unused wells in a pyrosequencing plate be used in the next run?
In principle it’s possible to use so far unused pyrosequencing wells for the next run and leave the already used wells empty. However, due to contamination risk when cleaning and handling plates QIAGEN does not recommend this.
FAQ ID -2872
How do I prevent a drifting baseline in my pyrosequencing pyrogram?
Let the PyroMark instrument warm up (about 60 minutes) to adapt to room temperature before use. Make sure the ambient room temperature is within range 18-28°C.
FAQ ID -2878