Oxidative bisulfite sequencing of 5-methylcytosine and 5-hydroxymethylcytosine

Abstract

To uncover the function of and interplay between the mammalian cytosine modifications 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), new techniques and advances in current technology are needed. To this end, we have developed oxidative bisulfite sequencing (oxBS-seq), which can quantitatively locate 5mC and 5hmC marks at single-base resolution in genomic DNA. In bisulfite sequencing (BS-seq), both 5mC and 5hmC are read as cytosines and thus cannot be discriminated; however, in oxBS-seq, specific oxidation of 5hmC to 5-formylcytosine (5fC) and conversion of the newly formed 5fC to uracil (under bisulfite conditions) means that 5hmC can be discriminated from 5mC. A positive readout of actual 5mC is gained from a single oxBS-seq run, and 5hmC levels are inferred by comparison with a BS-seq run. Here we describe an optimized second-generation protocol that can be completed in 2 d.

Figure 1

Structures of 5mC, 5hmC, 5fC and 5caC. R, DNA backbone.

Figure 2

After bisulfite treatment of DNA, C reads as T, whereas 5mC and 5hmC read as C. With a specific oxidation of 5hmC to 5fC followed by bisulfite treatment, C and 5hmC read as T, whereas only 5mC reads as C, thereby giving a positive readout of 5mC. 5hmC can then be ascertained by the difference of these two outputs.

Figure 3

Workflow of oxBS-seq. NGS, next-generation sequencing.

Figure 4

Efficacy of PCR amplification. (a–c) Gel electrophoresis experiments (in 2% (wt/vol) agarose gel) of PCR of Illumina libraries prepared from 1 μg of sonicated genomic DNA after undergoing the BS-only workflow (a); the oxBS-seq workflow described in the present protocol (b); and the previous version of the oxBS-seq workflow (c)¹⁴. From each 50-μl PCR solution prepared for use with Agilent Pfu Turbo C_x DNA polymerase, 5-μl aliquots of each PCR solution were removed at cycles 0, 3, 6, 9, 12, 15, 18 and 21 and placed in the wells of the agarose gels. Libraries analyzed in a and b show similar amplification, whereas three additional cycles are required for the library analyzed in c to achieve the same DNA intensity as a and b. The ladder (L) is the Thermo Scientific GeneRuler 100-bp DNA ladder.