In our recent effort of developing methylation-dependent endonuclease mediated methylation sequencing technology, efficiency and reproducibility were restrained by adapters' self-ligation. In the present study, three strategies to prevent self-ligation of adapters used in enzyme mediated methylation sequencing have been developed. Our data have demonstrated that these strategies can either inhibit or eliminate the adapters' self-ligation. These strategies are crucial in enzyme-mediated methylation sequencing and may be useful in some other genomic sequencing technologies.