부산대학교 도서관

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.