QutRNA2: robust tRNA modification discovery from Nanopore direct tRNA sequencing.

Transfer RNAs (tRNAs) are essential for protein synthesis and are extensively modified to ensure their structure and function. Direct RNA sequencing with Oxford Nanopore Technologies enables positional modification analysis but is challenged by tRNAs' short length, redundancy, and dense modifications. We present QutRNA2, a scalable workflow that includes GPU-accelerated local alignment, statistical filtering, pairwise error profile comparison, and customizable visualization. Achieving up to 25-fold speed gains over CPU methods, QutRNA2 identifies enzyme-dependent modifications in nuclear- and mitochondrial-encoded tRNAs, demonstrated in high-volume human and mouse samples. This open-source solution provides a comprehensive, multiplexing-compatible framework for tRNA analysis, addressing a key gap in current tools. QutRNA2 is released under Apache-2.0 license and is available at https://github.com/dieterich-lab/QutRNA2.