Insights into the eukaryotic gene regulation networks have improved due to the advent of diverse classes of non-coding RNAs. The transfer RNA (tRNA)-derived non-coding RNAs or tncRNAs is a novel class of non-coding RNAs, shown to regulate gene expression at transcription and translation levels. Here, we present a pipeline 'tncRNA Toolkit' for accurately identifying tncRNAs using small RNA sequencing (sRNA-seq) data. Previously, we identified tncRNA in six major angiosperms by utilizing our pipeline and highlighted the significant points regarding their generation and functions. The 'tncRNA Toolkit' is available at the URL: http://www.nipgr.ac.in/tncRNA. The scripts are written in bash and Python3 programming languages. The program can be efficiently run as a standalone command-line tool and installed in any Linux-based Operating System (OS). The user can run this program by providing the input of sRNA-seq data and genome file.The various features of the 'tncRNA Toolkit' are as follows:•Major tncRNA classes identified by this tool include tRF-5, tRF-3, tRF-1, 5'tRH, 3'tRH, and leader tRF. Also, it categorizes miscellaneous tncRNAs as other tRF.•It provides the following information for each identified tncRNA viz. tncRNA class, raw and normalized read count (RPM), read length, progenitor tRNA information (amino acid, anticodon, locus, strand), tncRNA sequence, and tRNA modification sites.•We hope to facilitate quick and reliable tncRNA identification, which will boost the exploration of this novel class of non-coding RNAs and their relevance in the living world, including plants.

Non-coding RNAs; Pipeline; Transcription regulation; sRNA-seq; tRFs; tncRNA Toolkit: A pipeline for identification of tRNA-derived non-coding RNAs; tncRNAs