PyroMark Q48 Autoprep

通过整合模板制备功能进行自动化焦磷酸测序，提升甲基化、突变和SNP定量分析能力

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PyroMark Q48 Autoprep Instrument

Cat. No. / ID: 9002471

PyroMark Q48 Instrument, multistep pipet, software and documentation

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PyroMark Q48 Autoprep

PyroMark Q48 Cartridge

PyroMark Q48 Discs

PyroMark Q48 Absorber Strips

RNeasy Protect Kit

PyroMark Q48 AutoPrep Starter Kit

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PyroMark Q48 Autoprep适用于分子生物学应用。该产品不适用于疾病的诊断、预防或治疗。

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Features

全自动实验流程，减少手工操作
直观的设备和分析软件，简单易用
通量更高，提高单次运行的样本检测数量和每天运行轮次
一流的焦磷酸测序性能，提高测序读长和结果可靠性
使用多引物分配系统，可实现更高的自动化通量
可通过定量DNA甲基化分析，获得更多的研究结果

Product Details

PyroMark Q48 Autoprep的一大亮点是在焦磷酸测序操作流程中整合了全自动模板制备功能。凭借崭新的设计和软件，该产品将通量在PyroMark Q24 Advanced的基础上提升一倍，同时性能更优异，非常适合各类功能性研究以及NGS和芯片检测实验中的大批量样本验证。

Performance

先进的技术、软件和化学法提高测序读长和结果可靠性

PyroMark Q48 Autoprep进一步改进了试剂和仪器运行算法，同PyroMark Q96和PyroMark Q24系统相比，大幅提高了检测读长和碱基检出、突变分析和甲基化定量分析精确度。以往系统的检测读长会受本底峰值限制，导致测序反应的光信号会有所下降。升级后的PyroMark “Advanced”化学试剂和仪器算法可以减少本底干扰，进而增加读长和结果的可靠性。根据待分析的序列，一次反应可获得140 bp以上的高精度读长。

样本通量更高，提高单次运行处理的SNP和突变检测数量

Multiple Primer Dispensation (MPD) 技术是一种在检测体系数目超过可用卡夹储液槽数目时提高测序引物自动分配能力的策略。PyroMark Q48 Autoprep Primer Cartridge有3个储液槽，但是如果一块测序板上需要进行的检测体系多于3个，那么可以将不同引物混合起来放入同一个引物卡夹内。当模板制备完成后，引物混合物将被自动分配到每个孔内。因为每个孔内只有一种PCR模板，所以只有与其对应的引物可以和模板结合。所有其它引物将留在溶液中不发生结合。MPD混合物中的每个引物都要单独设计和验证，避免其形成引物二聚体或与其它模板结合。

兼容各种PyroMark检测平台

新的PyroMark Q48 Autoprep仪器和“Advanced”化学法直接兼容以往设计的焦磷酸测序检测体系。数据表明，使用不同的PyroMark平台处理同一检测体系时，所获得的突变频率和甲基化分析结果相同。这种跨平台兼容性也不受测序引物中的分析位点距离的影响。这在通过单次反应分析多种序列变异时尤为重要，比如典型的复杂突变体系检测或连续CpG位点甲基化分析。

新的测序disc孔盘设计避免了磁性模板制备交叉污染

PyroMark Q48 Disc无需用户手动干预即可在同一仪器上完成自动化模板制备和焦磷酸测序。模板制备所需的缓冲液在反应过程中即从样本和disc孔盘中有效地去除，因而每次运行、每个轮次都没有孔间、disc孔盘间的交叉污染风险。

Principle

PyroMark Q48 Autoprep使用基于实时测序的焦磷酸测序技术，在遗传学和表观遗传学研究中进行检测和定量。系统可以同时分析多达48个样本，简单易用的自动化流程制备单链DNA测序模板，无需人工操作。这一流程使用磁性链霉亲和素包被的Sepharose微珠（PyroMark Q48 Magnetic Beads）来结合生物素标记的PCR产物链。系统可以同时自动进行四对测序引物的退火。如果需要用到更多的测序引物，则可以人工将这些引物加入到单链DNA样本中。

焦磷酸测序反应步骤：

第一步：一个DNA片段被扩增，同时作为焦磷酸测序模板的DNA链被生物素标记。经过变性之后，生物素标记的单链PCR扩增产物被分离出来并于一段测序引物杂交。杂交后的引物和单链模板与DNA聚合酶、ATP硫化酶、荧光素酶、腺苷三磷酸双磷酸酶，以及作为底物的腺苷-5’-磷酸硫酸酐(APS)和荧光素进行孵育。

第二步：将第一个脱氧核苷酸三磷酸(dNTP)加入到反应体系中。如果它和模板链的碱基配对，则在DNA聚合酶催化作用下，该dNTP被加到测序引物末端。每次合成事件均伴随一个焦磷酸(PPi)的释放，释放的焦磷酸摩尔数等于合成的碱基数。

第三步：在存在APS的条件下，ATP硫化酶将PPi转化为ATP。生成的ATP驱动萤光素酶将荧光素转化为氧化荧光素，同时产生与ATP数量正相关的可见光。在荧光素酶催化反应中产生的可见光被PMT探测器检测到并记录原始数据中的一个峰(Pyrogram)。每个峰（即光信号）的高度与合成的碱基数正相关。

第四步：腺苷三磷酸双磷酸酶（一种核苷酸降解酶）持续降解未结合的核苷酸和ATP。当降解完成后，另一个核苷酸被加入到反应体系中。

第五步：顺序依次加入dNTP。需要注意的是在反应体系中使用脱氧腺苷α-硫代磷酸(dATPαS)代替了天然的脱氧腺苷磷酸(dATP)，因为dATP虽然能被DNA聚合酶有效使用，但不能被荧光素酶识别。随着这一过程的进行，互补DNA链逐渐延长，同时Pyrogram轨迹记录的信号峰值反映出核酸的序列。

Procedure

PyroMark Q48 Advanced软件生成一个运行文件并通过USB接口或网络连接传输到仪器中。试剂、核苷酸和缓冲液按照触摸屏上显示的体积被加入到PyroMark Q48 Autoprep测序槽中。生物素标记的PCR产物和磁性链霉亲和素包被的Sepharose微珠被加入到Q48 Disc孔盘的孔内，然后将孔盘和吸收条放入仪器内。单链模板的制备和测序引物的退火现在已经完全自动化，不需要额外的人工操作。仪器可自动分配３种不同测序引物或Multiple Primer Dispensation (MPD)混合物。可选择性的人工加入额外的测序引物，进一步提升了单次运行可检测的样本数。

Applications

焦磷酸测序在多个学科的研究中的应用价值日益突出。无论是检测病原体的抗药性演化，DNA甲基化对基因表达的表观遗传调控，家畜中特定表型的遗传标记，还是法医样本中的线粒体DNA多态性，PyroMark平台都能提供强大而灵活的遗传和表观遗传变异分析。此外，焦磷酸测序整合了序列检测和定量功能，其强大的分析能力更易促成新的发现。

Software

PyroMark Q48 Autoprep Software安装于PC上，可对您的结果进行全面的分析。该软件包括４种分析模式——AQ, SNP, CpG和SEQ。AQ模式可用于对多种突变SNP和InDel进行定量研究。它适用于分析单个或多个突变位点，以及双拷贝、三拷贝或四拷贝等位基因突变。SNP模式提供对SNP和InDel的基因型分析。CpG模式可以分析单个或多个CpG或CpN位点，并提供针对亚硫酸氢盐处理的内参。SEQ用于对未知序列的碱基序列进行解读。

PyroMark Q48 Autoprep Software是一款用户友好、直观的软件，提供方便易用的先进分析方法。如果运行中出现问题，或系统检测到实验的不一致性，软件或对每个孔提出针对性的警告信息。点击"Warning Info"按钮可以获得有关警告的进一步信息和排除故障的建议以及如何避免在后续实验中再次出现该问题。

Services

Have your throughput demands increased? Do you want to expand your application range or further streamline workflows? Do you need to manage more complex samples? Do you need improved connectivity?

If the answer to any of these questions is yes, then QIAGEN’s trade-in/trade-up program is just right for you!

QIAGEN makes it easy to keep up to speed with the latest automation and sample processing technologies. Simply exchange an old instrument for a new one. Improve your efficiency by trading in a competitor instrument or trading up with an older QIAGEN instrument.

Learn more about trade-in/trade-up opportunities.

Supporting data and figures

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

Compatibility among PyroMark platforms for mutation analysis.

The principle of multiple primer dispension (MPD).

Compatibility among PyroMark platforms for methylation analysis.

Specifications

Features	Specifications
Applications	Simultaneous running of multiple assays and assay types including SNP, AQ, CpG and SQA to achieve methylation analysis, de novo sequencing, mutation characterization including In/Dels, speciation, quantitative allele sequencing and SNP genotyping
Instrument dimensions	W X D X H: 250 mm (9.8 in.) X 300 mm (11.8 in.) X 300 mm (11.8 in.) with chamber lid and injector cover closed; 250 mm (9.8 in.) X 560 mm (22 in.) X 390 mm (15.4 in.) with chamber lid and injector cover open
Connections	One USB port, Ethernet connection
Humidity	Relative humidity of 20–80% (noncondensing)
Kits designed for this instrument	PyroMark Q48 Advanced Reagents, PyroMark Q48 Advanced CpG Reagents
Operating temperature	18–30ºC (64–86ºF)
Overvoltage category	II
Place of operation	For indoor use only in a draft-free location. No exposure to direct sunlight.
Pollution level	2
Power	100–240V AC, 50/60 Hz. Mains supply voltage fluctuations are not to exceed ±10% of the nominal supply voltages.
Process temperature	28°C ± 0.5°C
Process time	Depends on the number of dispensations
Samples per run (throughput)	1–48
Software	PyroMark Q48 Advanced Software (to be installed on PC)
Technology	Pyrosequencing
Weight	8.5 kg (18.7 lb)
Altitude	Up to 2000 m (6500 ft)

Resources

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

Download Safety Data Sheets for QIAGEN product components.

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.

Shortage of PyroMark Q48 Autoprep Absorber Strips

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

FAQ

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

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

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

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

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

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 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

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

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

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

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

FAQ ID -2823

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

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

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

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

FAQ ID -2832

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

FAQ ID -2839

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

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

FAQ ID -2843

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

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

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

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 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 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

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

FAQ ID - 3340