Sequencing 5-formyluracil in genome DNA at single-base resolution

Albeit with low content, in genome DNA, the 5-formyluracil has been an important modification. The 5-formyluracil was found to be widely distributed amongst living bodies (prokaryotes and mammalian cells). Capable of binding to proteins to prevent the gene expressions, the 5-formyluracil could also induce mismatch in the DNA. In certain cancerous tissues, the content of 5-formyluracil was found to be higher than the normal tissues adjacent to the tumor. Nonetheless, the lack of a higher resolution mapping technique, has been hampering the studies of 5-formyluracil. Hence, we adopted the single-base resolution analysis method to profile 5-formyluracil at the genome scale. We analyzed the distribution of 5-formyluracil in the genome. This technique enabled us to have a better understanding of 5-formyluracil. The Azi-BIAN not only selectively labelled and enriched the 5-formyluracil in the genome DNA, rather also influenced the electro-density. Our results established that the azi-BIAN labelled 5-formyluracil could mismatch with guanine under pH 8.8 and match with adenine under pH 7.2, without any interference of the other canonical nucleobases. We used azi-BIAN to enrich the 5-formyluracil in the genome DNA of the human thyroid carcinoma cells. In the preperation of library, we used two different pH. In pH 7.2, the 5-formyluracil sites was read as thymine (e.g., Cal62_output_nonmismatch1 and Cal62_output_nonmismatch2), and the 5-formyluracil sites was read as cytosine (e.g., Cal62_output_mismatch1 and Cal62_output_mismatch2) in pH 8.8. By comparison the inputs (e.g., Cal62_input1 and Cal62_input2) and outputs (e.g., Cal62_output_nonmismatch1, Cal62_output_nonmismatch2, Cal62_output_mismatch1 and Cal62_output_mismatch2), we got the the 5-formyluracil peaks in the human thyroid carcinoma cells. Therefore, in the 5-formyluracil peaks, we got the 5-formyluracil sites by comparison the mutations in two libraries in the human thyroid carcinoma cells.