Competitive-quantification sequencing for misincorporated ribonucleoside monophosphates in genome

Misincorporated ribonucleoside-monophosphates (rNMPs) by DNA polymerases into genomic DNA are the most endogenous damages that have profound relationship with genome instability. To measure the detail profiles of incorporated and residual rNMP that link to genome repair in normal cells, this project developed RisCQ-seq, a high-throughput sequencing method for absolute quantification of global rNMP level. In the method, unseparated-sequencing libraries of RNaseH digested- and undigested-DNA fragments allow competitive sequencing to estimate frequency of rNMP misincorporation with synthetic-DNA/rNMP standards. The RisCQ-seq anaysis of wild-type and RNaseH2 knockout (rnh201delta) cells in the Saccharomyces cerevisiae model, provided ribonucleotide excision repair (RER) efficiency at each genomic regions by simple subtraction between the rNMP frequencies.