Bisulfite-free, Base-resolution, and Quantitative Sequencing of Cytosine Modifications

Abstract

The deamination of unmodified cytosine to uracil by treatment with bisulfite has for decades been the gold standard for sequencing epigenetic DNA modifications including 5methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). However, this harsh chemical reaction degrades the majority of the DNA and generates sequencing libraries with low complexity. Here, we present a novel bisulfite-free and base-resolution sequencing method, TET Assisted Pic-borane Sequencing (TAPS), for detection of 5mC and 5hmC. TAPS relies on mild reactions, detects modifications directly without affecting unmodified cytosines and can be adopted to detect other cytosine modifications. Compared with bisulfite sequencing, TAPS results in higher mapping rates, more even coverage and lower sequencing costs, enabling higher quality, more comprehensive and cheaper methylome analyses. One Sentence Summary:A bisulfite-free and base-resolution method to directly sequence epigenetically modified cytosine.

Compilation of Modern Technologies To Map Genome‐Wide Cytosine Modifications in DNA

“…Recently, Liu et al. reported a bisulfite‐free and base‐resolution cytosine modification sequencing method, termed TET assisted 2‐picoline borane (pic‐borane) sequencing (TAPS) . The approach was based on 5caC‐to‐T transition chemistry, which was enabled by the conversion of 5caC to dihydrouracil (DHU) using pic‐borane.…”

“…Combining the 5caC‐to‐T transition chemistry with TET oxidation of 5mC, the method realized whole‐genome, base‐resolution detection of 5mC (Scheme A). The reaction can also be used to detect 5hmC because pic‐borane can also convert 5fC into DHU and 5hmC can be oxidized to 5fC (Scheme B) …”

“…[46] Recently,L iu et al reported ab isulfite-free and base-resolution cytosine modification sequencing method, termed TET assisted 2-picoline borane (pic-borane) sequencing (TAPS). [47] The approach wasb ased on 5caC-to-T transition chemistry, which was enabled by the conversion of 5caC to dihydrouracil (DHU)u sing pic-borane.C ombining the 5caC-to-T transition chemistry with TET oxidation of 5mC, the methodr ealized whole-genome, base-resolution detection of 5mC (Scheme 7A). The reaction can also be used to detect 5hmC because pic-borane can also convert 5fCi ntoD HU and 5hmC can be oxidized to 5fC( Scheme 7B).…”

“…The reaction can also be used to detect 5hmC because pic-borane can also convert 5fCi ntoD HU and 5hmC can be oxidized to 5fC( Scheme 7B). [47] 4. Direct Sequencing of Unamplified DNA Currently,d evelopments in third-generation sequencing technologies, such as single-molecule, real-time sequencing (SMRT) and nanopore-based sequencing, have allowed new opportu-Scheme5.Chemical labeling methodsfor single-base-resolution detection of 5-5fC.…”

Dynamic regulation of DNA methylation in human T-cell biology

“…These techniques typically rely on enzymatic-or chemical conversion of modified bases coupled with bisulfite conversion. However, bisulfite treatment is known to degrade DNA, especially long fragments, therefore recent efforts have focused on the development of bisulfite-free methods for base-resolution detection of DNA modifications [109,110]. Furthermore, as bisulfite conversion largely reduces the complexity of the DNA, especially for rare modifications, care has to be taken to minimize PCR amplification biases [111].…”