PyroMark Q24 Advanced Reagents
For advanced methylation and mutation quantification in long sequence runs using Pyrosequencing
- Advanced technology, software, and chemistry for long sequence runs
- Quantitative methylation analysis at consecutive CpG or CpN sites
- Improved quantification of sequence variations at any sequence position
PyroMark Q24 Advanced Reagents feature advanced Pyrosequencing chemistry to provide even better real-time sequence-based detection and quantification than before. PyroMark Q24 Advanced Reagents are optimized for use with PyroMark Q24 Advanced Systems and are highly suited for analyzing any kind of sequence variation, particularly DNA methylation at CpG or CpN sites, complex mutations, or for de novo sequencing applications such as microbial typing. For long-read Pyrosequencing runs which may require larger volumes of nucleotides, such as de novo sequencing or methylation analyses we recommend using the PyroMark Q24 Advanced CpG Reagents.
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PyroMark Q24 Advanced Reagents (4 x 24)
Enzyme mix, substrate mix, nucleotides, and buffers for Pyrosequencing assays; intended for use with PyroMark Q24 Advanced
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970902
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PyroMark Q24 Advanced CpG Reagents (4 x 24)
Enzyme mix, substrate mix, nucleotides, and buffers for CpG and long read Pyrosequencing assays; intended for use with PyroMark Q24 Advanced
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970922
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PyroMark Q24 Advanced Reagents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。
长片段从头测序。|在长片段测序中分析16个CpG位点。|对均聚物进行更好的甲基化定量分析。|对长片段进行定量突变分析。|
序列读长是对未知序列进行分析的关键因素,通常仅可对40–80各碱基进行可靠分析。PyroMark Q24 Advanced利用改善的试剂和算法,显著提高了序列读长,并提供更高的准确性。使用PyroMark Q24(上方)和PyroMark Q24 Advanced(下方)和相同的试剂进行从头测序。蓝色柱形表示可靠检测到的碱基;黄色表示可能正确检测的碱基,但用户应检查确定;红色表示不可靠的检测结果。|PyroMark Q24 Advanced提高了测序长度和较靠后位点的甲基化分析可靠性。图中展示了PyroMark Q24 Advanced进行CpG分析时对135个核苷酸的序列中16个不同CpG位点进行的准确分析。如使用PyroMark Q24则需分别进行3次反应。|对紧邻T均聚物之后的、或T均聚物之间的CpG位点进行甲基化分析十分具有挑战性。甲基化程度由亚硫酸盐处理后,转换的C碱基与未转换的C碱基的比例决定,因为C到T的转换往往导致长T均聚物在扩增子中伸展,这种情况经常发生。 PyroMark Q24 Advanced能够对紧邻T均聚物之后的、或T均聚物之间的CpG位点进行准确的定量分析。图中例子显示了对8个T碱基内的CpG位点的分析。|由于突变和单核苷酸多态性(SNP)很少在直接邻近位置发生,因此常用的焦磷酸测序试剂在分析一个以上的突变位点时,可能需要不同试剂。PyroMark Q24 Advanced利用新型试剂,具有更长的序列读长,能够在一次运行中可靠分析多个突变。图中例子显示了10:90的EGFR野生型和突变型混合物的分析。即使在分析了60个核苷酸之后,分析仍是准确的。|
性能
Longer Pyrosequencing runs and improved accuracy
PyroMark Q24 Advanced features improved chemistry and instrument operation algorithms that significantly increase the assay read length and accuracy of the base calling functionality, enabling easy analysis of long de novo sequencing runs. Assay read length was previously limited by background peaks and reduced light signals in the sequencing reaction. The updated chemistry and algorithms of PyroMark Q24 Advanced reduce these background peaks, thereby increasing read length and reliability. Depending on the sequence to be analyzed, highly accurate read lengths of 140 or more bases can be obtained in just a single PyroMark Q24 Advanced reaction (see figure Long de novo sequencing runs).
Improved methylation analysis at any position
PyroMark Q24 Advanced enables improved methylation quantification in long sequence runs at any sequence position. Previously, analysis of methylation sites further away from the sequencing primer could be uncertain, but now with longer read lengths and higher accuracy, methylation quantification is highly reliable throughout the entire sequencing run (see figure Analysis of 16 CpG sites in a long sequence run).
Improved sequencing accuracy in homopolymers
Bisulfite conversion in DNA methylation analysis leads to frequent poly T stretches in the nucleotide sequence, and analysis of CpG sites directly after such T homopolymers has previously been challenging due to uncertain quantification of the light signal at these sites. The increased accuracy of PyroMark Q24 Advanced enables reliable quantification of CpG methylation behind and even within a stretch of 8 T nucleotides (see figure Improved methylation quantification in homopolymers).
Analysis of mutations over long sequences
PyroMark Q24 Advanced also provides reliable quantification of multiple polymorphisms in a single assay. Since single nucleotide polymorphisms (SNPs) and other mutations are often not located close to one another, traditional Pyrosequencing chemistry usually requires separate assays for each mutation site to be analyzed. The new chemistry of PyroMark Q24 Advanced allows much longer runs, enabling reliable analysis of more than one mutation or SNP in the same run (see figure Quantitative mutation analysis in long sequence runs).
原理
Pyrosequencing technology, which is based on the principle of sequencing by synthesis, provides quantitative data in sequence context within minutes. PyroMark Q24 Advanced Reagents and PyroMark Q24 Advanced CpG Reagents feature updated Pyrosequencing chemistry to enable analysis of complex mutations, methylation analysis in epigenetic research, resistance typing, and microbial identification. These reagents are optimized for use with PyroMark Q24 Advanced Systems.
操作流程
Fast and easy sample preparation
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 Q24 Advanced Reagents or PyroMark Q24 Advanced CpG 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.
应用
Pyrosequencing is becoming increasingly important for research applications in a variety of disciplines. 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, the PyroMark Q24 Advanced enables powerful and versatile analysis of genetic and epigenetic variation. In addition, because Pyrosequencing integrates sequence detection and quantification, the enhanced analysis resolution can lead to new discoveries.
| Throughput |
1–24 samples |
1–24 samples |
| Running volume |
25 µl |
25 µl |
| PCR requirements |
5–10 µl
(~0.5–3 pmol of product) |
5–10 µl
(~0.5–3 pmol of product) |
Read length (assay- and
sequence-dependent) |
10–140 bp or more |
10–80 bp |
| Application software |
PyroMark Q24 Advanced SW
(requires firmware 1.5.6903 or higher) |
PyroMark Q24 SW 2.0 |
| Software functionality |
SEQ (de novo sequencing)
CpG/CpN methylation
SNP
AQ |
SQA (de novo sequencing)
CpG methylation
AQ/SNP
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| Main applications |
Complex mutation analysis
Epigenetics (CpG and CpN analysis)
Resistance typing and microbial ID |
Mutation analysis
Resistance typing
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| Compatible reagents |
PyroMark Q24 Advanced Reagents
PyroMark Q24 Advanced CpG Reagents |
PyroMark Q24 Gold Reagents |
| Sensitivity |
2% mutation
98% wt |
2% mutation
98% wt |
Important: PyroMark Q24 Advanced Reagents and PyroMark Q24 Advanced CpG Reagents cannot be used with PyroMark Q24 Software. PyroMark Gold Q24 Reagents cannot be used with PyroMark Q24 Advanced Software. Using the incorrect reagent and software combination will result in incorrect reagent dispensations and lead to inaccurate results.
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A Pyrosequencing method for the forensic identification of tissue source using QIAGEN’s PyroMark Q24 Advanced system.
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FAQ ID -3160
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浏览
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FAQ ID -3165
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浏览
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Instrument methods file for use with PyroMark Q24 Advanced Software, PyroMark Q24 Advanced Reagents or PyroMark Q24 Advanced CpG Reagents, and PyroMark Q24 Cartridges marked with method 0003
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Instrument methods file for use with PyroMark Q24 Advanced Software, PyroMark Q24 Advanced Reagents or PyroMark Q24 Advanced CpG Reagents, and PyroMark Q24 Cartridges marked with method 0013
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显示更多
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Instrument methods file for use with PyroMark Q24 Advanced Software, PyroMark Q24 Advanced Reagents or PyroMark Q24 Advanced CpG Reagents, and PyroMark Q24 Cartridges marked with method 0005
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显示更多
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Instrument methods file for use with PyroMark Q24 Advanced Software, PyroMark Q24 Advanced Reagents or PyroMark Q24 Advanced CpG Reagents, and PyroMark Q24 Cartridges marked with method 0008
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显示更多
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Instrument methods file for use with PyroMark Q24 Advanced Software, PyroMark Q24 Advanced Reagents or PyroMark Q24 Advanced CpG Reagents, and PyroMark Q24 Cartridges marked with method 0006
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显示更多
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Instrument methods file for use with PyroMark Q24 Advanced Software, PyroMark Q24 Advanced Reagents or PyroMark Q24 Advanced CpG Reagents, and PyroMark Q24 Cartridges marked with method 0011
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显示更多
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Instrument methods file for use with PyroMark Q24 Advanced Software, PyroMark Q24 Advanced Reagents or PyroMark Q24 Advanced CpG Reagents, and PyroMark Q24 Cartridges marked with method 0004
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显示更多
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Instrument methods file for use with PyroMark Q24 Advanced Software, PyroMark Q24 Advanced Reagents or PyroMark Q24 Advanced CpG Reagents, and PyroMark Q24 Cartridges marked with method 0009
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显示更多
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Instrument methods file for use with PyroMark Q24 Advanced Software, PyroMark Q24 Advanced Reagents or PyroMark Q24 Advanced CpG Reagents, and PyroMark Q24 Cartridges marked with method 0007
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显示更多
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Instrument methods file for use with PyroMark Q24 Advanced Software, PyroMark Q24 Advanced Reagents or PyroMark Q24 Advanced CpG Reagents, and PyroMark Q24 Cartridges marked with method 0012
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显示更多
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Instrument methods file for use with PyroMark Q24 Advanced Software, PyroMark Q24 Advanced Reagents or PyroMark Q24 Advanced CpG Reagents, and PyroMark Q24 Cartridges marked with method 0010
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显示更多
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图片
长片段从头测序。
序列读长是对未知序列进行分析的关键因素,通常仅可对40–80各碱基进行可靠分析。PyroMark Q24 Advanced利用改善的试剂和算法,显著提高了序列读长,并提供更高的准确性。使用PyroMark Q24(上方)和PyroMark Q24 Advanced(下方)和相同的试剂进行从头测序。蓝色柱形表示可靠检测到的碱基;黄色表示可能正确检测的碱基,但用户应检查确定;红色表示不可靠的检测结果。
在长片段测序中分析16个CpG位点。
PyroMark Q24 Advanced提高了测序长度和较靠后位点的甲基化分析可靠性。图中展示了PyroMark Q24 Advanced进行CpG分析时对135个核苷酸的序列中16个不同CpG位点进行的准确分析。如使用PyroMark Q24则需分别进行3次反应。
对均聚物进行更好的甲基化定量分析。
对紧邻T均聚物之后的、或T均聚物之间的CpG位点进行甲基化分析十分具有挑战性。甲基化程度由亚硫酸盐处理后,转换的C碱基与未转换的C碱基的比例决定,因为C到T的转换往往导致长T均聚物在扩增子中伸展,这种情况经常发生。 PyroMark Q24 Advanced能够对紧邻T均聚物之后的、或T均聚物之间的CpG位点进行准确的定量分析。图中例子显示了对8个T碱基内的CpG位点的分析。
对长片段进行定量突变分析。
由于突变和单核苷酸多态性(SNP)很少在直接邻近位置发生,因此常用的焦磷酸测序试剂在分析一个以上的突变位点时,可能需要不同试剂。PyroMark Q24 Advanced利用新型试剂,具有更长的序列读长,能够在一次运行中可靠分析多个突变。图中例子显示了10:90的EGFR野生型和突变型混合物的分析。即使在分析了60个核苷酸之后,分析仍是准确的。
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