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

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

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Technical Information (0)
Kit Handbooks (0)
Operating Software (0)
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Genotyping (0)
Scientific Posters (0)

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

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 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
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
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