The MinION system (Oxford Nanopore Technologies, United Kingdom) was used to directly sequence genomic DNAs of two recombinant Escherichia coli strains. One strain carried a plasmid with the gene for M.HpaII DNA methyltransferase, which methylates the second cytosine in a CCGG site. The other strain contained M.HspAI DNA methyltransferase, which modifies the central cytosine in a GCGC sequence. Either enzyme methylates cytosine to produce 5-methylcytosine. The presence of 5-methylcytosine was found to be detectable with high accuracy when DNA is sequenced at high coverage. In particular, 98.9% of the GCGC sites were identified as methylated at the first cytosine when DNA from the strain carrying the cloned M.HspAI gene was sequenced at 1300× coverage. Only 0.09% of the remaining tetranucleotides with a central CpG dinucleotide were false positives and were identified as methylated at the central cytosine. For the strain with M.HpaII, 91.3% of all CCGG sites were identified as methylated in a set of positions covered by more than 700 reads and only 0.13% of the other tetranucleotides with a central CpG dinucleotide were identified as sites with 5-methylcytosine in the second position. Thus, coverage of at least 700–1000× is necessary for accurately measuring 5-methylcytosine and eliminating false-positive results when using the method.

MinION系统（英国牛津纳米孔技术公司）用于直接测序两个重组大肠杆菌的基因组DNA。株。一株带有带有M.HpaII DNA甲基转移酶基因的质粒，该质粒甲基化CCGG位点中的第二个胞嘧啶。另一株含有M.HspAI DNA甲基转移酶，其修饰了GCGC序列中的中央胞嘧啶。两种酶都可以使胞嘧啶甲基化，生成5-甲基胞嘧啶。当以高覆盖率对DNA进行测序时，发现可以高精度检测到5-甲基胞嘧啶的存在。特别地，当来自携带克隆的M.HspAI基因的菌株的DNA以1300x覆盖率测序时，在第一胞嘧啶中鉴定出98.9％的GCGC位点是甲基化的。仅有0.09％的带有中央CpG二核苷酸的四核苷酸为假阳性，并在中央胞嘧啶处被鉴定为甲基化。对于带有M.HpaII的菌株，为91。所有CCGG位点中有3％被鉴定为在700个以上的读数所覆盖的一组位置中被甲基化，而只有0.13％的具有中央CpG二核苷酸的其他四核苷酸被鉴定为第二位置中具有5-甲基胞嘧啶的位点。因此，使用该方法时，至少要覆盖700-1000倍才能准确测量5-甲基胞嘧啶并消除假阳性结果。