Christopher Oakes from The Ohio State University spoke at London Calling 2019 on “Discerning the origin of Epstein-Barr virus in patients using nanopore-derived DNA methylation signatures.” Oakes spoke about how they study tumor viruses and cancer. The Epstein-Barr virus is common, the “prototypical cancer virus,” according to Oakes. Immune cells suppress the virus, but the virus can also go into a dormant phase and survive. Gaining genetic mutation, the Epstein-Barr virus (EBV) can proliferate and cause tumors. Tumor viruses can be exploited in screening to detect tumors early. Tumor viruses can also be used to classify and diagnose different cancer types. EBV is typically detected as plasma DNA (cell-free). Oakes and team are interested in DNA methylation of EBV. DNA methylation shifts, Oakes explained, can help determine the lytic induction of EBV. Oakes analyzed EBV DNA methylation by extracting virus DNA, performing whole genome amplification (WGA) which results in unmethylated DNA and then methylating with SssI (CpG) to create a percent methylation standard curve. The team prepared DNA with the LSK 109 library kit and sequenced on MinION MIN 106 flow cells. They used fast basecalling, PoreChop, and minimap2 to align to the EBV reference genome. They then re-basecalled with high accuracy basecalling and used Nanopolish to index and call CpG methylation. Because they have a standard curve, they could perform some comparisons and calculate error rates. Nanopore sequencing can be used to monitor patients on different therapies and lytic or dormant stages. Binning methylation as high or low is just the beginning, noted Oakes. There are complex methylation patterns during EBV in different tumor types. EBV methylation and gene expression patterns could be used for work on therapies and the development of antibodies, for example. Oakes explained that they want to improve the sensitivity of their approach with cell-free DNA, focusing on enrichment and detection of methylation. I thought the approach Oakes and team used to develop a method and standard curve was unique and could help study phage methylation patterns, for example.
