Bisulfite-Free, Nanoscale Analysis of 5-Hydroxymethylcytosine at Single Base Resolution

Abstract

High-resolution detection of genome-wide 5-hydroxymethylcytosine (5hmC) sites of small-scale samples remains challenging. Here, we present hmC-CATCH, a bisulfite-free, base-resolution method for the genome-wide detection of 5hmC. hmC-CATCH is based on selective 5hmC oxidation, chemical labeling and subsequent C-to-T transition during PCR. Requiring only nanoscale input genomic DNA samples, hmC-CATCH enabled us to detect genome-wide hydroxymethylome of human embryonic stem cells in a cost-effective manner. Further application of hmC-CATCH to cell-free DNA (cfDNA) of healthy donors and cancer patients revealed base-resolution hydroxymethylome in the human cfDNA for the first time. We anticipate that our chemical biology approach will find broad applications in hydroxymethylome analysis of limited biological and clinical samples.

Tissue-specific 5-hydroxymethylcytosine landscape of the human genome

“…including affinity-based methods (5hmC-DIP-Seq, CMS-Seq, GLIB and hMe-Seal) and high-resolution methods (oxBS-Seq, TAB-Seq, hmC-CATCH, TAPS and ACE-Seq) 20,21 . For instance, we previously developed hmC-CATCH 22 , which is a bisulfitefree method for genome-wide detection of 5hmC. hmC-CATCH couples selective chemical labeling and biotin pulldown [22][23][24][25] , thus allowing 5hmC enrichment and detection at single-base resolution.…”

“…For instance, we previously developed hmC-CATCH 22 , which is a bisulfitefree method for genome-wide detection of 5hmC. hmC-CATCH couples selective chemical labeling and biotin pulldown [22][23][24][25] , thus allowing 5hmC enrichment and detection at single-base resolution. While hydroxymethylome maps have been obtained for mammalian cell lines and mouse tissues, 5hmC profiles in human tissues are poorly characterized thus far.…”

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

“…However, its utility in detecting genome‐wide 5hmC was not demonstrated. We recently developed a bisulfite‐free, genome‐wide, and base‐resolution 5hmC sequencing method termed chemical‐assisted C‐to‐T conversion of 5hmC sequencing (hmC‐CATCH) . hmC‐CATCH is based on the selective oxidation of 5hmC to 5fC, and subsequent labeling of newly generated 5fC by using derivatives of 1,3‐indandione (Scheme ); endogenous 5fC is first blocked before the oxidation reaction.…”

“…We recently developed ab isulfite-free, genome-wide, and base-reso-lution 5hmC sequencing method termed chemical-assisted Cto-T conversion of 5hmC sequencing (hmC-CATCH). [45] hmC-CATCH is based on the selectiveoxidation of 5hmC to 5fC, and subsequentl abeling of newly generated 5fCb yu sing derivatives of 1,3-indandione( Scheme 6);e ndogenous 5fCi sf irst blockedb efore the oxidation reaction. Importantly,w es howed that K 2 RuO 4 ,b ut not KRuO 4 ,m ediated very mild oxidation of DNA.…”

Advances in the Profiling of Single‐Cell DNA Modifications

“…Our lab reported another bisulfite‐free, base‐resolution 5hmC detection method called hmC‐CATCH seq . hmC‐CATCH combines a mild, selective 5hmC oxidation reaction with an established 5fC labeling and detection method, allowing 5hmC detection in limited amount of biological and clinical samples.…”