A subset of regulatory CD4+ T cells (Tregs) is characterized by the stable constitutive expression of the transcription factor FOXP3. Demethylation at a conserved region within intron 1 of FOXP3, the Tregspecific demethylated region (TSDR), is exclusive to this subset of Tregs: other immune cells that do not express FOXP3 or express the transcription factor transiently, such as activated effector T cells or TGF-β-treated CD4+ T cells, are methylated at the TSDR [1–4]. Hence, the TSDR provides a specific target to enumerate Tregs within biological samples; developing methods that enable robust Treg-cell quantitation with small numbers of cells from clinical samples remains an important goal. Current methods to measure FOXP3 demethylation include qPCR [5, 6] and epigenetic sequencing methylation analysis [7] of Sanger sequencing traces [8], neither of which capture the methylation information at each CpG site on a single piece of DNA, but rather produce an average methylation over the whole region or an average at each site. Although several studies have used a PCR cloning and sequencing method to assess the methylation of each CpG site within the TSDR, this is a laborious method and normally less than 50 clones per sample are analyzed [3, 9, 10].

We therefore developed a nextgeneration sequencing (NGS) method to assess at single-base resolution the methylation status of the FOXP3 TSDR. The method, which we originally developed to genotype rs1800521 single nucleotide polymorphism in the gene AIRE (Methods detailed as Supporting Information), reports at single base resolution the methylation of each DNA amplicon, provides hundreds to thousands of reads per replicate and can be multiplexed to analyze several DNA regions simultaneously.