Detection of 5mC and 5hmC in DNA templates generated by PCR using modified dCTPs by linear PCR using KTq DNA polymerase variant RIV A8

We have engineered the thermostable KlenTaq DNA polymerase variant called RIV A8 that produces error signatures specific for 5-methylcytosine (5mC) and 5-hydroxycytosine (5hmC) without prior chemical treatment of the DNA samples. These signatures are amplified during DNA amplicon library preparation and are detected by NGS. This method was applied to distinguish C from 5mC and C from 5hmC in DNA templates generated by PCR using modified dCTPs (unmodified by using dCTP, methylated by using d5mCTP, hydroxymethylated by using d5hmCTP). Overall design: DNA templates generated by PCR using modified dCTPs (unmodified by using dCTP, methylated by using d5mCTP, hydroxymethylated by using d5hmCTP) were linear amplified by RIV A8. Respective ssDNA product was used to prepare DNA amplicon libraries which were analyzed by NGS to detect 5mC and 5hmC by reading an increased error rate.