Precise genomic mapping of 5-hydroxymethylcytosine via covalent tether-directed sequencing

Abstract

5-hydroxymethylcytosine (5hmC) is the most prevalent intermediate on the oxidative DNA demethylation pathway and is implicated in regulation of embryogenesis, neurological processes, and cancerogenesis. Profiling of this relatively scarce genomic modification in clinical samples requires cost-effective high-resolution techniques that avoid harsh chemical treatment. Here, we present a bisulfite-free approach for 5hmC profiling at single-nucleotide resolution, named hmTOP-seq (5hmC-specific tethered oligonucleotide-primed sequencing), which is based on direct sequence readout primed at covalently labeled 5hmC sites from an in situ tethered DNA oligonucleotide. Examination of distinct conjugation chemistries suggested a structural model for the tether-directed nonhomologous polymerase priming enabling theoretical evaluation of suitable tethers at the design stage. The hmTOP-seq procedure was optimized and validated on a small model genome and mouse embryonic stem cells, which allowed construction of single-nucleotide 5hmC maps reflecting subtle differences in strand-specific CG hydroxymethylation. Collectively, hmTOP-seq provides a new valuable tool for cost-effective and precise identification of 5hmC in characterizing its biological role and epigenetic changes associated with human disease.

Selective Chemical Labeling and Sequencing of 5-Hydroxymethylcytosine in DNA at Single-Base Resolution

“…The previous base-resolution mapping of 5hmC allowed for the determination of the surrounding base composition of 5hmC sites in mouse ESCs. 22 We aligned our 5hmC sites in CG context and examined the base compositions (Fig. S1).…”

“…Generally, there are two strategies, which are the selective enrichment-based profiling or deamination based single-base resolution sequencing methods. [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22] While the current applications of these methods have provided key information about the distribution of 5hmC and its functional insights, there are major shortage for both strategies. The lack of single-base resolution information of the profiling strategy limited its application in detailed 5hmC location context while the single-base resolution method is limited by its high sequencing cost.…”

Identification of fetal unmodified and 5-hydroxymethylated CG sites in maternal cell-free DNA for non-invasive prenatal testing

“…The second important milestone of our study is the first known to date demonstration that 5hmC profiling in maternal cfDNA can accurately inform on fetal karyotype. The hmTOP-seq method [25], which covalently targets 5hmC residues, enabled the construction of genome-wide 5hmC maps of relatively low 5hmC levels in CV samples and cfDNA. Most importantly, we observed that hmTOP-seq was most discriminatory in detection of T21 fetuses independently of fetal fraction.…”

“…For profiling of uCGs and 5hmCGs we employed our recently developed uTOP-seq and hmTOP-seq strategies, respectively, which assess the modification status of genomic CG sites through selective covalent coupling of a priming oligonucleotide to azide-modified CGs and their subsequent sequencing [24,25]. By leveraging the high robustness of covalent derivatization and the sensitivity of such targeted sequencing we successfully adapted both strategies for the analysis of nanogram quantities of cfDNA.…”

“…In recent years, covalent derivatization has been adapted for epigenome-wide studies of various cytosine modifications [22][23][24][25]. Here, we applied for the first time the covalent derivatization of unmodified CG sites (uCGs) in maternal cfDNA for identification of fetal-derived genomic regions.…”