Grasso Chiara, Trevisan Morena, Fiano Valentina, Tarallo Valentina, De Marco Laura, Sacerdote Carlotta, Richiardi Lorenzo, Merletti Franco, Gillio-Tos Anna
Cancer Epidemiology Unit - C.E.R.M.S, Department of Medical Sciences, University of Turin, Turin, Italy.
Cancer Epidemiology Unit, Department of Medical Sciences, City of Health and Science Hospital, Turin, Italy.
PLoS One. 2016 Mar 2;11(3):e0150483. doi: 10.1371/journal.pone.0150483. eCollection 2016.
Pyrosequencing has emerged as an alternative method of nucleic acid sequencing, well suited for many applications which aim to characterize single nucleotide polymorphisms, mutations, microbial types and CpG methylation in the target DNA. The commercially available pyrosequencing systems can harbor two different types of software which allow analysis in AQ or CpG mode, respectively, both widely employed for DNA methylation analysis.
Aim of the study was to assess the performance for DNA methylation analysis at CpG sites of the two pyrosequencing software which allow analysis in AQ or CpG mode, respectively. Despite CpG mode having been specifically generated for CpG methylation quantification, many investigations on this topic have been carried out with AQ mode. As proof of equivalent performance of the two software for this type of analysis is not available, the focus of this paper was to evaluate if the two modes currently used for CpG methylation assessment by pyrosequencing may give overlapping results.
We compared the performance of the two software in quantifying DNA methylation in the promoter of selected genes (GSTP1, MGMT, LINE-1) by testing two case series which include DNA from paraffin embedded prostate cancer tissues (PC study, N = 36) and DNA from blood fractions of healthy people (DD study, N = 28), respectively.
We found discrepancy in the two pyrosequencing software-based quality assignment of DNA methylation assays. Compared to the software for analysis in the AQ mode, less permissive criteria are supported by the Pyro Q-CpG software, which enables analysis in CpG mode. CpG mode warns the operators about potential unsatisfactory performance of the assay and ensures a more accurate quantitative evaluation of DNA methylation at CpG sites.
The implementation of CpG mode is strongly advisable in order to improve the reliability of the methylation analysis results achievable by pyrosequencing.
焦磷酸测序已成为核酸测序的一种替代方法，非常适合许多旨在表征目标DNA中的单核苷酸多态性、突变、微生物类型和CpG甲基化的应用。市售的焦磷酸测序系统可搭载两种不同类型的软件，分别允许以AQ模式或CpG模式进行分析，这两种模式都广泛用于DNA甲基化分析。
本研究的目的是评估两种分别允许以AQ模式或CpG模式进行分析的焦磷酸测序软件在CpG位点进行DNA甲基化分析的性能。尽管CpG模式是专门为CpG甲基化定量而生成的，但许多关于该主题的研究都是使用AQ模式进行的。由于尚无证据证明这两种软件在这类分析中具有同等性能，本文的重点是评估目前用于焦磷酸测序的CpG甲基化评估的两种模式是否会给出重叠的结果。
我们通过测试两个病例系列来比较这两种软件在定量选定基因（GSTP1、MGMT、LINE-1）启动子中DNA甲基化方面的性能，这两个病例系列分别包括来自石蜡包埋前列腺癌组织的DNA（PC研究，N = 36）和来自健康人血液组分的DNA（DD研究，N = 28）。
我们发现在基于两种焦磷酸测序软件的DNA甲基化检测质量评估中存在差异。与用于AQ模式分析的软件相比，能够以CpG模式进行分析的Pyro Q-CpG软件支持的宽松标准较少。CpG模式会向操作人员警告检测可能存在的不理想性能，并确保对CpG位点的DNA甲基化进行更准确的定量评估。
强烈建议采用CpG模式，以提高焦磷酸测序可实现的甲基化分析结果的可靠性。
