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

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

Author information

1
Ludwig Institute for Cancer Research, Nuffield Department of Medicine, University of Oxford, Oxford, UK.
2
Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Oxford, UK.
3
Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Peking University, Beijing, China.
4
College of Chemistry, Nankai University, Tianjin, China.
5
Center for Mitochondrial Biology and Medicine and Center for Translational Medicine, School of Life Science and Technology, Xi'an Jiaotong University, Xi'an, China.
6
Ludwig Institute for Cancer Research, Nuffield Department of Medicine, University of Oxford, Oxford, UK. benjamin.schuster-boeckler@ludwig.ox.ac.uk.
7
Ludwig Institute for Cancer Research, Nuffield Department of Medicine, University of Oxford, Oxford, UK. chunxiao.song@ludwig.ox.ac.uk.
8
Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Oxford, UK. chunxiao.song@ludwig.ox.ac.uk.

Abstract

Bisulfite sequencing has been the gold standard for mapping DNA modifications including 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) for decades1-4. However, this harsh chemical treatment degrades the majority of the DNA and generates sequencing libraries with low complexity2,5,6. 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.

Comment in

PMID:
30804537
DOI:
10.1038/s41587-019-0041-2
[Indexed for MEDLINE]

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