Single base resolution analysis of 5-hydroxymethylcytosine in 188 human genes: implications for hepatic gene expression.

Ivanov M, Kals M, Lauschke V, Barragan I, Ewels P, Käller M, Axelsson T, Lehtiö J, Milani L, Ingelman-Sundberg M

Nucleic Acids Res. 44 (14) 6756-6769 [2016-08-19; online 2016-04-29]

To improve the epigenomic analysis of tissues rich in 5-hydroxymethylcytosine (hmC), we developed a novel protocol called TAB-Methyl-SEQ, which allows for single base resolution profiling of both hmC and 5-methylcytosine by targeted next-generation sequencing. TAB-Methyl-SEQ data were extensively validated by a set of five methodologically different protocols. Importantly, these extensive cross-comparisons revealed that protocols based on Tet1-assisted bisulfite conversion provided more precise hmC values than TrueMethyl-based methods. A total of 109 454 CpG sites were analyzed by TAB-Methyl-SEQ for mC and hmC in 188 genes from 20 different adult human livers. We describe three types of variability of hepatic hmC profiles: (i) sample-specific variability at 40.8% of CpG sites analyzed, where the local hmC values correlate to the global hmC content of livers (measured by LC-MS), (ii) gene-specific variability, where hmC levels in the coding regions positively correlate to expression of the respective gene and (iii) site-specific variability, where prominent hmC peaks span only 1 to 3 neighboring CpG sites. Our data suggest that both the gene- and site-specific components of hmC variability might contribute to the epigenetic control of hepatic genes. The protocol described here should be useful for targeted DNA analysis in a variety of applications.

Bioinformatics Compute and Storage [Service]

Clinical Proteomics Mass spectrometry [Collaborative]

NGI Stockholm (Genomics Applications) [Service]

NGI Stockholm (Genomics Production) [Collaborative]

NGI Uppsala (SNP&SEQ Technology Platform) [Collaborative]

QC bibliography QC xrefs

PubMed 27131363

DOI 10.1093/nar/gkw316

Crossref 10.1093/nar/gkw316


pmc PMC5001587