We have engineered an RT-active DNA polymerase variant called RT-KTq I614Y that produces error RT-signatures specific for pseudouridine (?) without prior chemical treatment of the RNA samples. These signatures are amplified during DNA amplicon library preparation and are detected by NGS. This method was applied to distinguish U from ? in RNA oligonucleotides (modified or unmodified) used in the previous polymerase screening and oligonucleotides which are designed from the human 18S rRNA at the position around 1445. Finally, RT-KTq I614Y was used to detect ?55 in tRNAGly(GCC) from Saccharomyces cerevisiae wildtype compared to data from tRNAGly(GCC) from S. cerevisiae pus4?. Overall design: RNA oligonucleotides (modified or unmodified) used in the enzyme screening and oligonucleotides (modified or unmodified) designed from the human 18S rRNA were reverse transcribed by RT-KTq I614Y. Respective cDNA was used to prepare DNA amplicon libraries which were analyzed by NGS. Furthermore, purified total tRNA fractions from S. cerevisiae wt and pus4? deletion strain were employed to detect ?55.