Technical adequacy of bisulfite sequencing and pyrosequencing for detection of mitochondrial DNA methylation: Sources and avoidance of false-positive detection.
Technical adequacy of bisulfite sequencing and pyrosequencing for detection of mitochondrial DNA methylation: Sources and avoidance of false-positive detection.
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DOI：
10.1371/journal.pone.0192722
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发表时间：
2018
期刊：
影响因子：
3.7
通讯作者：
Shioda T
中科院分区：
文献类型：
--
作者：
Owa C;Poulin M;Yan L;Shioda T
哺乳动物线粒体DNA中是否存在胞嘧啶甲基化是一个有争议的问题。由于DNA甲基化的检测取决于5 '-修饰的胞嘧啶对亚硫酸氢盐催化转化为尿嘧啶的抗性，因此检查了影响mtDNA甲基化分析技术充分性的参数。通过长距离PCR扩增缺乏胞嘧啶甲基化的阴性对照扩增子（NCA）以覆盖整个人或小鼠mtDNA。当焦磷酸测序模板扩增子在亚硫酸氢盐转化后进行凝胶纯化时，NCA的亚硫酸氢盐焦磷酸测序在ND 1（7个CpG位点）或CYTB（8个CpG位点）基因处未检测到显著水平的亚硫酸氢盐抗性胞嘧啶（brC）（CI 95 = 0%-0.94%）;在没有凝胶纯化的情况下，从NCA中检测到显著的假阳性brC（CI 95 = 4.2%-6.8%）。当测序引物对亚硫酸氢盐转化和未转化的模板无选择性时，从人iPS细胞或小鼠肝脏中分离的高度纯化的线性化mtDNA的亚硫酸氢盐焦磷酸测序在人ND 1基因中检测到显著的brC（~30%）。然而，使用在亚硫酸氢盐转化的模板中具有选择性的测序引物的重复实验几乎完全（< 0.8%）抑制了brC检测，支持使用非选择性引物检测brC的假阳性性质。对线性化、凝胶纯化的人mtDNA进行Bisulte-seq深度测序，发现ND 1基因9个CpG位点的brC为9.4%~ 14.8%。然而，由于所有这些brC与相邻的非CpG brC相关，显示出相同程度的亚硫酸氢盐抗性，因此该mtDNA编码基因中的DNA甲基化未得到证实。在没有线性化的情况下，通过亚硫酸氢盐焦磷酸测序或纯化的mtDNA模板的深度测序产生的数据不通过质量控制标准。鸟枪亚硫酸氢盐测序人类mtDNA检测到极低水平的CpG甲基化（<0.65%）超过非CpG甲基化（<0.55%）。综上所述，我们的研究表明，充分的mtDNA甲基化分析使用依赖于亚硫酸氢盐转化的方法需要建立每个实验，考虑不完全的亚硫酸氢盐转化和模板杂质或拓扑结构的影响。
The existence of cytosine methylation in mammalian mitochondrial DNA (mtDNA) is a controversial subject. Because detection of DNA methylation depends on resistance of 5’-modified cytosines to bisulfite-catalyzed conversion to uracil, examined parameters that affect technical adequacy of mtDNA methylation analysis. Negative control amplicons (NCAs) devoid of cytosine methylation were amplified to cover the entire human or mouse mtDNA by long-range PCR. When the pyrosequencing template amplicons were gel-purified after bisulfite conversion, bisulfite pyrosequencing of NCAs did not detect significant levels of bisulfite-resistant cytosines (brCs) at ND1 (7 CpG sites) or CYTB (8 CpG sites) genes (CI95 = 0%-0.94%); without gel-purification, significant false-positive brCs were detected from NCAs (CI95 = 4.2%-6.8%). Bisulfite pyrosequencing of highly purified, linearized mtDNA isolated from human iPS cells or mouse liver detected significant brCs (~30%) in human ND1 gene when the sequencing primer was not selective in bisulfite-converted and unconverted templates. However, repeated experiments using a sequencing primer selective in bisulfite-converted templates almost completely (< 0.8%) suppressed brC detection, supporting the false-positive nature of brCs detected using the non-selective primer. Bisulfite-seq deep sequencing of linearized, gel-purified human mtDNA detected 9.4%-14.8% brCs for 9 CpG sites in ND1 gene. However, because all these brCs were associated with adjacent non-CpG brCs showing the same degrees of bisulfite resistance, DNA methylation in this mtDNA-encoded gene was not confirmed. Without linearization, data generated by bisulfite pyrosequencing or deep sequencing of purified mtDNA templates did not pass the quality control criteria. Shotgun bisulfite sequencing of human mtDNA detected extremely low levels of CpG methylation (<0.65%) over non-CpG methylation (<0.55%). Taken together, our study demonstrates that adequacy of mtDNA methylation analysis using methods dependent on bisulfite conversion needs to be established for each experiment, taking effects of incomplete bisulfite conversion and template impurity or topology into consideration.
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DOI：
10.1073/pnas.1012311108
发表时间：
2011-03-01
影响因子：
11.1
作者：
Shock, Lisa S.;Thakkar, Prashant V.;Taylor, Shirley M.
通讯作者：
Taylor, Shirley M.
影响因子：
5.3
作者：
Wong M;Gertz B;Chestnut BA;Martin LJ
通讯作者：
Martin LJ
影响因子：
25
作者：
Guo, Junjie U.;Su, Yijing;Shin, Joo Heon;Shin, Jaehoon;Li, Hongda;Xie, Bin;Zhong, Chun;Hu, Shaohui;Le, Thuc;Fan, Guoping;Zhu, Heng;Chang, Qiang;Gao, Yuan;Ming, Guo-li;Song, Hongjun
通讯作者：
Song, Hongjun
分析污染物诱导的线粒体DNA甲基化变化。
DOI：
10.1007/978-1-4939-2288-8_19
发表时间：
2015
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
--
作者：
Byun, Hyang-Min;Barrow, Timothy M
通讯作者：
Barrow, Timothy M
在脐带血和胎盘中线粒体 - 凝结仪轴上与年龄相关靶标的DNA甲基化的变化。
影响因子：
3.8
作者：
Janssen BG;Byun HM;Cox B;Gyselaers W;Izzi B;Baccarelli AA;Nawrot TS
通讯作者：
Nawrot TS