PRJNA563770
Detection of Base Analogs Incorporated During DNA Replication by Nanopore Sequencing
DNA synthesis is a fundamental requirement for cell proliferation and DNA repair, but no single method can identify the location, direction and speed of replication forks with high resolution. Mammalian cells have the ability to incorporate thymidine analogs along with the natural A, T, G and C bases during DNA synthesis, which allows for labelling of replicating or repaired DNA. Here, we demonstrate the use of the Oxford Nanopore Technologies MinION to detect 11 different thymidine analogs including CldU, BrdU, IdU, as well as, EdU alone or coupled to Biotin and other bulky adducts in synthetic DNA templates. We also show that the large adduct Biotin can be distinguished from the smaller analog IdU, which opens the possibility of using analog combinations to identify the location and direction of DNA synthesis. Furthermore, we detect IdU label on single DNA molecules, incorporated during DNA replication in the genome of mouse pluripotent stem cells. We conclude that this novel method has the potential for genome-wide examination of DNA replication in stem cell differentiation or cell transformation.