Robust bisulfite free SMRT Sequencing of mdC based on a novel hpTet3-enzyme

Genomic DNA contains next to the four canonical nucleosides dA, dC, dG and T the additional base 5-methyldeoxycytidine (mdC). The presence of this methylated cytidine nucleoside in promoter regions or gene bodies has an instrumental effect on the transcriptional activity of the corresponding gene. The methylation patterns of genes are therefore responsible for either silencing or activation of genes. Sequencing of mdC positions in the genome is consequently of paramount importance for early cancer diagnostics in order to determine incorrect expression of genes. Today, the bisulfite method is the gold standard for mdC-sequencing, which has however the caveat that the majority of the input DNA is degraded during the bisulfite treatment. In addition, bisulfite sequencing is rather error prone. Here we report a benign, bisulfite free mdC sequencing method based on 3rd generation single molecule SMRT sequencing. The fundament of the technology is a new Tet3 enzyme that oxidizes mdCs with high efficiency to 5-carboxycytidine (cadC). caC in turn provides and excellent read out by SMRT sequencing using specially trained AI-based algorithms. Overall design: We developed a novel TET3 enzyme that oxidizes mdCs with high efficiency to 5-carboxycytidine (cadC) for biochemical and molecular biological application. We created three different synthetic model genomes from whole-genome amplified (WGA) lambda genomes: (1) unmodifed DNA obtained from WGA, (2) M.SssI methylated DNA containing 5mC in CpG context, (3) hpTet3 oxidized M.SssI-DNA containing only 5caC in a CpG context. Each DNA was subjected to UHPLC-QQQ-MS for qc of the individual enzymatic steps. Subsequently, each DNA was sequenced on a Sequel IIe system. We evaluated and compared the kinetic porfiles from the Sequel IIe in the close proximity of a CpG site. In addition we used the individual data sets to train deep-learning algorithms for 5mC and 5caC and compared there performance with respect to recalling of CpG sites, accuracy and percision.