Fig. 1: Overview of TEMPT for deep methylation sequencing of EV-DNA samples.

A schematic illustration of the whole process for methylation profiling of EV-DNA samples. The whole process includes patient sample collection, EV extraction by ultracentrifugation and lysis, library construction by TEMPT, biomarker identification, model prediction, and gene oncology analysis. In brief, the first step in TEMPT involved fragmenting EV-DNA (gray) using single-adapter Tn5 tagmentation to add a 5’ adapter (orange), dark gray indicates the Mosaic End of the Tn5 adapter. Then, fragmented EV-DNA is enzymatically converted by TET, T4-βGT, APOBEC enzymes. Gray, blue, black, green, red balls indicate C, 5mC, 5hmC, 5caC/5ghmC, and U, respectively. After conversion, TdT adds dC nucleotides to the 3’ end of the DNA fragments, creating poly-C tail at the 3’ end. Then, a splinted adapter (light and dark blue) with a 5’ poly dG end is ligated using E. coli ligase. Finally, DNA fragments with adapters on both ends are amplified by PCR and sequenced. Purple and yellow indicate i5 and i7 indexes; green and amaranth correspond to i5 and i7 flow cell primers for amplification and sequencing. Raw data are processed for downstream analysis, including biomarker identification, model prediction, and gene oncology analysis.