亚硫酸氢盐
亚硫酸氢盐测序
DNA甲基化
5-甲基胞嘧啶
甲基化DNA免疫沉淀
生物
DNA
结扎测序
基因组文库
照明菌甲基化试验
基因组DNA
计算生物学
DNA测序
Illumina染料测序
DNA纳米球测序
遗传学
分子生物学
基因
基序列
基因表达
作者
Romualdas Vaisvila,V. K. Chaithanya Ponnaluri,Zhiyi Sun,Bradley W. Langhorst,Lana Saleh,Shengxi Guan,Nan Dai,Matthew A. Campbell,Brittany S. Sexton,Katherine Marks,Mala Samaranayake,James C. Samuelson,Heidi E. Church,Esta Tamanaha,Ivan R. Corrêa,Sriharsa Pradhan,Eileen T. Dimalanta,Thomas C. Evans,Louise Williams,Theodore B. Davis
出处
期刊:Genome Research
[Cold Spring Harbor Laboratory]
日期:2021-06-17
卷期号:31 (7): 1280-1289
被引量:163
标识
DOI:10.1101/gr.266551.120
摘要
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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