Cellular ncRNA pools are known to be dynamically regulated and can have high degrees of variation between different cell types and developmental stages. Dysregulation of transfer RNAs (tRNAs), a type of ncRNA, has been linked to a diverse array of detrimental physiological conditions including neurological diseases and cancer. While characterization of transfer RNA (tRNAs) diversity is relevant to disease, current library preparation approaches are limited in their ability to capture highly modified tRNA bases, which block reverse transcriptase and interfere with the production of full-length cDNA intermediates needed for sequencing. To date, several cDNA library preparation techniques, including OTTR, have attempted to overcome these problems and improve our ability to characterize ncRNA pools.