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无亚硫酸盐直接检测碱基分辨率下的 5-甲基胞嘧啶和 5-羟甲基胞嘧啶。

Bisulfite-free direct detection of 5-methylcytosine and 5-hydroxymethylcytosine at base resolution.

机构信息

Ludwig Institute for Cancer Research, Nuffield Department of Medicine, University of Oxford, Oxford, UK.

Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Oxford, UK.

出版信息

Nat Biotechnol. 2019 Apr;37(4):424-429. doi: 10.1038/s41587-019-0041-2. Epub 2019 Feb 25.


DOI:10.1038/s41587-019-0041-2
PMID:30804537
Abstract

Bisulfite sequencing has been the gold standard for mapping DNA modifications including 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) for decades. However, this harsh chemical treatment degrades the majority of the DNA and generates sequencing libraries with low complexity. Here, we present a bisulfite-free and base-level-resolution sequencing method, TET-assisted pyridine borane sequencing (TAPS), for detection of 5mC and 5hmC. TAPS combines ten-eleven translocation (TET) oxidation of 5mC and 5hmC to 5-carboxylcytosine (5caC) with pyridine borane reduction of 5caC to dihydrouracil (DHU). Subsequent PCR converts DHU to thymine, enabling a C-to-T transition of 5mC and 5hmC. TAPS detects modifications directly with high sensitivity and specificity, without affecting unmodified cytosines. This method is nondestructive, preserving DNA fragments over 10 kilobases long. We applied TAPS to the whole-genome mapping of 5mC and 5hmC in mouse embryonic stem cells and show that, compared with bisulfite sequencing, TAPS results in higher mapping rates, more even coverage and lower sequencing costs, thus enabling higher quality, more comprehensive and cheaper methylome analyses.

摘要

亚硫酸氢盐测序几十年来一直是 DNA 修饰(包括 5-甲基胞嘧啶(5mC)和 5-羟甲基胞嘧啶(5hmC))图谱绘制的金标准。然而,这种苛刻的化学处理会降解大部分 DNA,并产生低复杂度的测序文库。在这里,我们提出了一种无亚硫酸盐和碱基分辨率的测序方法,即 TET 辅助的吡啶硼烷测序(TAPS),用于检测 5mC 和 5hmC。TAPS 将 5mC 和 5hmC 的十 - 十一易位(TET)氧化为 5-羧基胞嘧啶(5caC),与 5caC 的吡啶硼烷还原为二氢尿嘧啶(DHU)相结合。随后的 PCR 将 DHU 转化为胸腺嘧啶,使 5mC 和 5hmC 发生 C 到 T 的转变。TAPS 以高灵敏度和特异性直接检测修饰物,而不会影响未修饰的胞嘧啶。这种方法是非破坏性的,可保留超过 10kb 长的 DNA 片段。我们将 TAPS 应用于小鼠胚胎干细胞中 5mC 和 5hmC 的全基因组图谱绘制,结果表明,与亚硫酸氢盐测序相比,TAPS 可获得更高的图谱绘制率、更均匀的覆盖度和更低的测序成本,从而实现更高质量、更全面和更经济的甲基组分析。

相似文献

[1]
Bisulfite-free direct detection of 5-methylcytosine and 5-hydroxymethylcytosine at base resolution.

Nat Biotechnol. 2019-2-25

[2]
High-Resolution Analysis of 5-Hydroxymethylcytosine by TET-Assisted Bisulfite Sequencing.

Methods Mol Biol. 2021

[3]
Subtraction-free and bisulfite-free specific sequencing of 5-methylcytosine and its oxidized derivatives at base resolution.

Nat Commun. 2021-1-27

[4]
Methylation-assisted bisulfite sequencing to simultaneously map 5fC and 5caC on a genome-wide scale for DNA demethylation analysis.

Nat Protoc. 2016-6-9

[5]
Tet-Assisted Bisulfite Sequencing (TAB-seq).

Methods Mol Biol. 2018

[6]
Comprehensive evaluation of genome-wide 5-hydroxymethylcytosine profiling approaches in human DNA.

Epigenetics Chromatin. 2017-4-20

[7]
Quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution.

Science. 2012-4-26

[8]
Oxidative Bisulfite Sequencing: An Experimental and Computational Protocol.

Methods Mol Biol. 2021

[9]
Base-resolution profiling of active DNA demethylation using MAB-seq and caMAB-seq.

Nat Protoc. 2016-6

[10]
Bisulfite-free, base-resolution analysis of 5-formylcytosine at the genome scale.

Nat Methods. 2015-11

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[3]
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[4]
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[10]
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