Genome-wide identification of 5-methylcytosine sites in bacterial genomes by high-throughput sequencing of MspJI restriction fragments.
Genome-wide identification of 5-methylcytosine sites in bacterial genomes by high-throughput sequencing of MspJI restriction fragments.
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DOI：
10.1371/journal.pone.0247541
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发表时间：
2021
期刊：
影响因子：
3.7
通讯作者：
Roberts RJ
中科院分区：
文献类型：
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作者：
Anton BP;Fomenkov A;Wu V;Roberts RJ
单分子实时（SMRT）测序可以很容易地识别DNA序列中的N6-甲基腺嘌呤和N4-甲基胞嘧啶位点，但5-甲基胞嘧啶位点的类似识别并不那么简单。在原核生物DNA中，甲基化通常发生在特定的序列背景或基序中，这是甲基转移酶的一种特性，它“书写”这些表观遗传标记。我们在这里提出了一个简单的，具有成本效益的替代SMRT和亚硫酸氢盐测序的原核5-甲基胞嘧啶甲基化基序的测定。该方法称为MFRE-Seq，依赖于使用MspJI家族限制性酶（MFRE）切除和分离可预测大小的完全甲基化片段，其取决于5-甲基胞嘧啶的存在以进行切割。我们证明MFRE-Seq与Illumina和Ion Torrent测序平台兼容，并且在标准文库制备程序之前仅需要消化步骤和大小选择的消化片段的简单柱纯化。我们将MFRE-Seq应用于许多细菌和古细菌的基因组DNA制备，并成功地证实了已知的基序，并确定了新的基序。这种方法应该是一个有用的补充，现有的方法研究原核甲基化和特征的贡献甲基转移酶。
Single-molecule Real-Time (SMRT) sequencing can easily identify sites of N6-methyladenine and N4-methylcytosine within DNA sequences, but similar identification of 5-methylcytosine sites is not as straightforward. In prokaryotic DNA, methylation typically occurs within specific sequence contexts, or motifs, that are a property of the methyltransferases that “write” these epigenetic marks. We present here a straightforward, cost-effective alternative to both SMRT and bisulfite sequencing for the determination of prokaryotic 5-methylcytosine methylation motifs. The method, called MFRE-Seq, relies on excision and isolation of fully methylated fragments of predictable size using MspJI-Family Restriction Enzymes (MFREs), which depend on the presence of 5-methylcytosine for cleavage. We demonstrate that MFRE-Seq is compatible with both Illumina and Ion Torrent sequencing platforms and requires only a digestion step and simple column purification of size-selected digest fragments prior to standard library preparation procedures. We applied MFRE-Seq to numerous bacterial and archaeal genomic DNA preparations and successfully confirmed known motifs and identified novel ones. This method should be a useful complement to existing methodologies for studying prokaryotic methylomes and characterizing the contributing methyltransferases.
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N6-甲基脱氧腺苷标记了衣原体中的主动转录起始位点。
影响因子：
64.5
作者：
Fu Y;Luo GZ;Chen K;Deng X;Yu M;Han D;Hao Z;Liu J;Lu X;Dore LC;Weng X;Ji Q;Mets L;He C
通讯作者：
He C
影响因子：
48
作者：
Flusberg, Benjamin A.;Webster, Dale R.;Lee, Jessica H.;Travers, Kevin J.;Olivares, Eric C.;Clark, Tyson A.;Korlach, Jonas;Turner, Stephen W.
通讯作者：
Turner, Stephen W.
影响因子：
2.1
作者：
Militello, Kevin T.;Mandarano, Alexandra H.;Simon, Robert D.
通讯作者：
Simon, Robert D.
影响因子：
14.9
作者：
EHRLICH, M;GAMASOSA, MA;GEHRKE, C
通讯作者：
GEHRKE, C
DOI：
10.1038/nrg2719
发表时间：
2010-03
期刊：
Nature reviews. Genetics
影响因子：
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作者：
通讯作者：
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