Duplexed direct RNA sequencing protocol using polyadenylation and polyuridylation

Oxford Nanopore Technologies provides multiplexing options for DNA and cDNA sequencing, but not for direct RNA sequencing. Here we describe a duplexing approach and validate it by simultaneously sequencing the Saccharomyces cerevisiae rRNA from wild type and knockout that have differential rRNA modifications, successfully demultiplexing the data using bioinformatics approaches.