Principle of selective chemical labeling of 5-hmC followed by SMRT DNA sequencing.

Effects of 5-hmC modifications on polymerase kinetics in SMRT DNA sequencing. (a–c) Using synthetic DNA templates with known positions of 5-hmC (triangles), the graphs show IPD ratios, at each template position, of the modified DNA template over a control DNA template of identical sequence but lacking 5-hmC. Conditions were 5-hmC untreated (a), upon coupling with glucose azide (b) and upon additional coupling with the disulfide-containing biotin linker, followed by cleavage of the disulfide bond (c).

Example of 5-hmC detection by SMRT sequencing from mESC genomic DNA. (a) The raw SMRT sequencing read. A.u., arbitrary units. (b) Sequencing subreads from the SMRTbell template, mapped onto mouse chromosome 1 over the sequencing time course. Pauses appear as discontinuities as the polymerase temporarily stops progressing along the DNA template. F, forward strand reads; R, reverse strand reads. (c) Subreads are grouped and annotated by IPD values in a heat-map scale, identifying a hemi-hydroxymethylated CG position in this DNA molecule. Arrows indicate consistent pausing of the polymerase (that is, large IPD value) at the same genomic position across multiple intramolecular subreads, indicating the presence of a 5-hmC adduct.