PyroMark Q24 Advanced Software

将PyroMark Q24升级至PyroMark Q24 Advanced的分析软件

Features

先进的技术、软件和化学试剂，可用于长序列分析
连续CpG或CpN位点的定量甲基化分析
任意序列位置的改进的序列差异定量
简单且更佳的碱基检出功能
同一仪器和同一运行中的Assay通用性

Product Details

PyroMark Q24 Advanced改进了焦磷酸测序技术，可提供比以往更佳的实时测序检测和定量。用户可使用PyroMark Q24 Advanced Software升级现有的PyroMark Q24，充分利用更加先进的技术和功能。该软件有4个分析模块：AQ：等位基因定量；SNP：基因分型；CpG：CpG或CpN位点的甲基化；SEQ：未知序列的碱基检出。

Performance

CpN甲基化定量

PyroMark Q24 Advanced的CpN分析模式能够实现对不属于CpG位点的胞嘧啶残基进行甲基化分析。CpN和CpG位点的分析可在一次焦磷酸测序反应中进行。可在反应设置阶段，分别选择待分析的位点（参见）。

便利的分析工具

PyroMark Q24 Advanced是一款直观、友好的分析软件，能够进行便利的分析。如果在运行中出现错误，或系统检测到试剂有异，会自动报错，提示出错的孔（参见）。“Warning Info（警告信息）”按钮可提供更多信息，以及纠错的推荐方案，防止后续反应中再次出现问题（参见）。

较长的焦磷酸测序运行和更高的准确度

PyroMark Q24 Advanced采用了改进的化学试剂和仪器操作算法，大大增加了读取长度和碱基检出功能的准确度，可轻松实现较长的从头测序运行分析。此前，读取长度受到测序反应中的背景峰和较低的光信号的限制。已升级的PyroMark Q24 Advanced化学试剂和算法可降低该背景峰，从而提高读取长度和可靠性。根据待分析序列的不同，只需一次PyroMark Q24 Advanced反应即可获得高度准确的140个或更多碱基的读取长度（参见）。

任意位置的更先进的甲基化分析

PyroMark Q24 Advanced可在任意序列位置实现改进的长片段序列甲基化定量。以前，远离测序引物的甲基化位点分析是无法确定的，但现在利用更长的读取长度和更高的准确度，可在整个测序运行中实现高度可靠的甲基化定量（参见）。

改进的同聚物测序准确度

DNA甲基化分析中的亚硫酸氢盐转化可导致核苷酸序列中频繁的poly T延伸，以前，由于这些位点的光信号定量的不确定性，在这种T同聚物之后直接进行CpG位点分析面临诸多难题。PyroMark Q24 Advanced具有更高的准确度，可在8个T核苷酸链之后甚至之中实现可靠的CpG甲基化定量（参见）。

长片段序列的突变分析

PyroMark Q24 Advanced还可利用单个Assay实现可靠的多态性定量。由于单核苷酸多态性（SNP）及其他突变通常不相邻，因此传统的焦磷酸测序化学方法一般需要分析针对每个突变位点的单独的Assay。全新的PyroMark Q24 Advanced化学方法可实现更长片段分析，在一次运行中实现多个突变或SNP的可靠分析（参见）。

See figures

Quantification of CpN methylation.

Additional warning information.

Troubleshooting recommendations.

Long de novo sequencing runs.

Analysis of 16 CpG sites in a long sequence run.

Improved methylation quantification in homopolymers.

Quantitative mutation analysis in long sequence runs.

Principle

用户可使用PyroMark Q24 Advanced Software升级现有的PyroMark Q24，充分利用更加先进的技术和功能。该软件与使用运行固件3.0以上的PyroMark Q24仪器兼容。请参阅“相关资料”选项卡下的《Upgrade to PyroMark Q24 Advanced》文件，了解升级的相关介绍。

Applications

焦磷酸测序在各学科的研究应用中发挥了愈来愈重要的作用。不论是检查病原体中的抗药性的产生、基因表达调控中表观遗传学DNA甲基化的作用、家畜中特定表型的遗传标志物，还是线粒体DNA法医样本中的多态性，PyroMark Q24 Advanced均可提供功能强大且通用的基因和表观遗传学变异分析。此外，由于焦磷酸测序整合了序列检测和定量，提高的分析分辨率可促进新发现的诞生。

产品对比
	PyroMark Q24 Advanced	PyroMark Q24
通量	1–24 个样本	1–24个样本
体积	25 µl	25 µl
PCR产物需要量	5–10 µl (~0.5–3 pmol产物)	5–10 µl (~0.5–3 pmol产物)
读长（取决于Assay和序列）	10–140 bp以上	10–80 bp
应用软件	PyroMark Q24 Advanced SW （需要固件1.5.6903或更高）	PyroMark Q24 SW 2.0
软件功能	SEQ（从头测序） CpG/CpN甲基化 SNP AQ	SQA （从头测序） CpG甲基化 AQ/SNP
主要应用	复杂突变分析表观遗传学（CpG和CpN分析）耐药性分析和微生物鉴定	突变分析耐药性分析
兼容试剂	PyroMark Q24 Advanced Reagents PyroMark Q24 Advanced CpG Reagents	PyroMark Q24 Gold Reagents
灵敏度	2%的突变 98% wt	2%的突变 98% wt

Supporting data and figures

Quantification of CpN methylation.

The new CpN mode of Pyromark Q24 Advanced enables methylation analysis of cytosine residues that are not part of CpG sites. Each of these positions can be selected individually. This example shows the analysis of a CpN site (CpA in this case) in the first position, followed by 2 classical CpG sites.

Resources

Download Safety Data Sheets for QIAGEN product components.

FAQ

The new PyroMark Q24 Advanced Software (version 3.0.1) is compatible with Windows XP, Vista (32 and 64 bit), Windows 7 (32 and 64 bit), and Windows 10 (64 bit).

FAQ ID -3164

If you need to install the PyroMark Q24 software on a new computer replacing your old one, or after you reinstall or upgrade the operating system, you can reinstall the PyroMark Q24 software without purchasing a new license.

FAQ ID - 3473

For PyroMark Q24, PyroMark Q24 Advanced, PyroMark Q96 ID v2.5, and PyroMark CpG software, data are file-based. The original run files can be attached to email and sent to QIAGEN Technical Service.

For PyroMark Q96 ID 1.0, PyroMark Q96 MD/MDA 1.0, PSQ MA, and PSQ HS/HSA software, data are stored in an Oracle database and need exporting (See Instruction for run file export). Exported data can be attached to email and sent to QIAGEN Technical Service.

The log files from the PyroMark Q96 ID, PyroMark Q96 MD/MDA, PSQ MA, and PSQ HS/HSA instruments provide valuable information. See Instruction for log file retrieval) for log file retrieval instruction

Please send the log file along with the original run file to QIAGEN Technical Service.

FAQ ID -9051

Do not remove the USB stick while data are being copied. Corrupted data files are usually due to the interrupted copying process. Follow these steps to retrieve recently saved runs.

1. When the instrument is not processing, insert a USB stick into the USB port on the instrument.

2. Use the Up and Down buttons to select “Administration” in the menu and press OK.

3. Select “Copy Recently Saved Runs” and press OK.

4. Use the Up and Down buttons to select the run file(s) for retrieval and press Select.

5. When the instrument confirms that the run file(s) has been saved to the USB stick, press Close and remove the USB stick.

In addition, repeat the process with a different USB stick. If the issue persists, please contact QIAGEN Technical Service for further assistance.

FAQ ID -9053

Please see PyroMark software operating system compatibility and hardware requirements.

FAQ ID -9054

PyroMark instruments dispense the correct volume of enzyme mix, substrate mix, and nucleotides using variable pulse times and dispensing pressures. The settings for pulse times and dispensing pressure are specific for each lot of PyroMark dispensing tips and cartridges and need to be updated in the PyroMark software being used to operate the instrument.

Dispensing pressures and pulse time settings must be checked and, if necessary, updated every time a cartridge from a new lot number is used. Check the details at Managing PyroMark Instrument Methods.

FAQ ID -9055

The histogram is the theoretical peak pattern based on the Sequence to Analyze that you enter into the software, while the pyrogram is the experimental peak pattern that is detected by PyroMark instruments. The peak pattern in the pyrogram has to be in agreement with that in the histogram for a successful run. Overlaying the histogram on the pyrogram provides an invaluable tool for trouble-shooting Pyrosequencing runs. To do so, right click anywhere on the pyrogram and select Show Histogram on the pop-up menu.

FAQ ID -9056

Yes. The PyroMark Q24, PyroMark Q24 Advanced, and PyroMark Q96 v2.5 software provide multiple types of assay modes, which enable analyzing different types of samples at the same time.

FAQ ID -9057

A single peak height is the signal produced from a non-variable single nucleotide. The single peak height is an important criterion for trouble-shooting.

The optimal single peak height is 30 RLU (minimum 20 RLU) (Relative Light Unit) for PyroMark Q24 and PyroMark Q24 Advanced, 20 RLU (minimum 10 RLU) for PyroMark Q96 ID, and 100 RLU (minimum 50 RLU) for PyroMark Q96 MD instrument, respectively.

FAQ ID -9058

The PyroMark Q96 ID 1.0, PyroMark Q96 MD/MDA 1.0, PSQ MA, and PSQ HS/HSA software requires a functional Oracle database, which cannot be installed on an office computer. You can install the PyroMark software on an office computer and access the Oracle database on the PyroMark operator’s computer via network. The operator’s computer needs to be powered on while you are accessing the Oracle database remotely. Your IT department is responsible for the network setup.

FAQ ID -9059

This can result from very high signal in a well which results in cross-talk between neighbouring wells. It could also be that the PyroMark instrument's camera is misaligned (contact: QIAGEN Technical service).

FAQ ID -9060

Sodium hydroxide carry-over, too much template, and incorrectly stored reagent are the most common causes of wide peaks. Ensure correct volume of denaturation buffer is used on the vacuum prep station. Use 5–20 µl of PCR product depending on instrument models. Store reagent as described in the handbook.

If the issue persists, please send the run files (see Instruction for run file export) to QIAGEN Technical Service for further assistance.

FAQ ID -9062