Table 1.

Common Methods for 5-hmC detection

Type	Detection Level	Detection Principle	Pre-modification of DNA1	Processing Time2	Specialized Instrument3
Global	Whole Sample	ELISA4	Required	6–8 hrs	Not Required,
Loci Specific	Loci	GSRE, TET1- oxidation, PCR and sequencing	Required	1–3 days	Not Required
Whole Genome	Loci	Massive Parallel Sequencing	Required	Variable	Required5

¹Antibody binding (anti-5-hydroxymethylcytosine) or enzyme modifications (i.e. GT, GSRE, TET1)

²Time for completion of ELISA is usually less than one working day and depends on the commercial vendor’s manual. For loci 5-hmC detection, it requires 1–3 days for pretreatment of DNA, PCR and/or sequencing. Whole genome massive parallel sequencing requires library construction, massive sequencing, data analysis, data mining and gene validation. Time for completion varies between facilities.

³Instruments other than the thermocycler and plate reader.

⁴Techniques using chromatography and mass spectroscopy to detect global hydroxymethylation are available but not commonly used.

⁵Massive parallel sequencing on different types of sequencers is available in core facilities or commercial vendors.