Enzymatic methyl sequencing detects DNA methylation at single-base resolution from picograms of DNA.
Enzymatic methyl sequencing detects DNA methylation at single-base resolution from picograms of DNA.
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DOI：
10.1101/gr.266551.120
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发表时间：
2021-07
期刊：
影响因子：
7
通讯作者：
Davis TB
中科院分区：
文献类型：
--
作者：
Vaisvila R;Ponnaluri VKC;Sun Z;Langhorst BW;Saleh L;Guan S;Dai N;Campbell MA;Sexton BS;Marks K;Samaranayake M;Samuelson JC;Church HE;Tamanaha E;Corrêa IR Jr;Pradhan S;Dimalanta ET;Evans TC Jr;Williams L;Davis TB
亚硫酸酯测序在单碱基分辨率下检测5mC和5hmC。然而，亚硫酸氢盐处理会损害DNA，导致片段化、DNA丢失和有偏差的测序数据。为了克服这些问题，开发了酶甲基序列法（EM-seq）。该方法通过两组酶促反应检测5mC和5hmC。在第一个反应中，TET2和T4-BGT将5mC和5hmC转化为不能被APOBEC3A降解的产物。在第二个反应中，APOBEC3A通过将未修饰的胞嘧啶转化为尿嘧啶来脱氨。因此，这三种酶能够识别5mC和5hmC。将EM-seq文库与亚硫酸转化的DNA进行比较，每种文库类型在转化前连接到Illumina适配器上。使用NA12878基因组DNA、无细胞DNA和FFPE DNA在一系列DNA输入上构建文库。EM-seq文库中检测到的5mC和5hmC与亚硫酸盐文库相似。然而，使用EM-seq制作的文库在所有检查的具体措施（覆盖率、复制、灵敏度等）中都优于亚硫酸转换文库。EM-seq文库显示出均匀的GC分布，更好的DNA输入相关性，基因组特征中CpGs数量的增加，以及胞嘧啶甲基化呼叫的准确性。EM-seq仅使用100 pg DNA就有效，并且这些文库保持了上述亚硫酸盐测序的优势。EM-seq文库建设，使用具有挑战性的样品和较低的DNA输入，为研究和临床应用开辟了新的途径。
Bisulfite sequencing detects 5mC and 5hmC at single-base resolution. However, bisulfite treatment damages DNA, which results in fragmentation, DNA loss, and biased sequencing data. To overcome these problems, enzymatic methyl-seq (EM-seq) was developed. This method detects 5mC and 5hmC using two sets of enzymatic reactions. In the first reaction, TET2 and T4-BGT convert 5mC and 5hmC into products that cannot be deaminated by APOBEC3A. In the second reaction, APOBEC3A deaminates unmodified cytosines by converting them to uracils. Therefore, these three enzymes enable the identification of 5mC and 5hmC. EM-seq libraries were compared with bisulfite-converted DNA, and each library type was ligated to Illumina adaptors before conversion. Libraries were made using NA12878 genomic DNA, cell-free DNA, and FFPE DNA over a range of DNA inputs. The 5mC and 5hmC detected in EM-seq libraries were similar to those of bisulfite libraries. However, libraries made using EM-seq outperformed bisulfite-converted libraries in all specific measures examined (coverage, duplication, sensitivity, etc.). EM-seq libraries displayed even GC distribution, better correlations across DNA inputs, increased numbers of CpGs within genomic features, and accuracy of cytosine methylation calls. EM-seq was effective using as little as 100 pg of DNA, and these libraries maintained the described advantages over bisulfite sequencing. EM-seq library construction, using challenging samples and lower DNA inputs, opens new avenues for research and clinical applications.
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影响因子：
4.5
作者：
de Koning AP;Gu W;Castoe TA;Batzer MA;Pollock DD
通讯作者：
Pollock DD
DNA 亚硫酸氢盐转化中的错误：调节不适当和失败的转化频率。
影响因子：
14.9
作者：
Genereux DP;Johnson WC;Burden AF;Stöger R;Laird CD
通讯作者：
Laird CD
DOI：
10.1007/978-1-4939-7481-8_9
发表时间：
2018-01-01
期刊：
DNA METHYLATION PROTOCOLS, 3 EDITION
影响因子：
--
作者：
Peat, Julian R.;Smallwood, Sebastien A.
通讯作者：
Smallwood, Sebastien A.
影响因子：
12.3
作者：
Akalin A;Kormaksson M;Li S;Garrett-Bakelman FE;Figueroa ME;Melnick A;Mason CE
通讯作者：
Mason CE
早期人类发育过程中DNA甲基化动力学的全基因组分析。
影响因子：
4.5
作者：
Okae H;Chiba H;Hiura H;Hamada H;Sato A;Utsunomiya T;Kikuchi H;Yoshida H;Tanaka A;Suyama M;Arima T
通讯作者：
Arima T