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Abstract

Both genome and transcriptome carry a wide range of different chemical modifications. Among these modifications, methylation is the most abundant, exerting important functions in multiple biological processes in both DNA and RNA related pathways in prokaryotes and eukaryotes. The recent discoveries of N6-methyladenosine (m6A) in transcriptome with reversible dynamics and regulatory roles and N6-methyladenine (6mA) in eukaryotic genome as an epigenetic mark have drawn considerable attentions of the scientific community. The development of highthroughput sequencing (next generation sequencing) provides us a powerful tool to study this chemical modification in genome-wide and/or transcriptome-wide level. The high resolution map, hence, is of great necessity for further investigation in its biological meanings. To meet the requirement, two major strategies are designed and will be discussed in the thesis. The photo-crosslinking-assisted strategy introduces covalent bond between antibody and nucleic acid and improves the efficiency and specificity of immunoprecipitation; the nuclease digestion further narrows down the detection region, thus significantly increasing the mapping resolution. The strategy was applied to the detection of m6A in bacterial mRNA, the study of relative positions of methylation and RNA binding proteins, and the first glimpse of 6mA pattern and methylation motif in Chlamydomonas genome. The deamination based m6A sequencing approach, inspired by deamination based bisulfite sequencing to distinguish 5-methylcytosine from cytosine, is designed to achieve the single nucleotide resolution m6A map. The model study and pilot experiment have demonstrated NGS detectable A-to-G conversion is introduced by deamination treatment, which is dependent on methylation level, indicating the potential of this method to transcriptome-wide differentiate and quantify m6A.

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