Spliced leader (SL) trans-splicing occurs in a wide range of eukaryotes and plays a critical role in processing mRNAs derived from operon structures. However, current research on this mechanism remains limited, partly due to the difficulty in accurately identifying genuine SL trans-splicing events. The advent of long-read RNA sequencing technologies, such as direct RNA sequencing by Oxford Nanopore Technologies, offers a more promising avenue for detecting these events with greater resolution. Here, we present SLRanger, an integrated tool to detect SL sequences and predict operon structures in eukaryotic transcriptomes. SLRanger improves upon the traditional Smith-Waterman (SW) alignment framework by incorporating an optimized scoring scheme tailored to SL detection in native long RNA reads. We primarily validated our method using direct RNA sequencing data from Caenorhabditis elegans, a well-established model organism for studying trans-splicing. Through a dynamic cutoff strategy, SLRanger robustly identified high-confidence SL-carrying reads. Leveraging the SL information, SLRanger achieved over 80% accuracy in operon gene prediction, recovering more than 70% of known operon genes in C. elegans. SLRanger was also applied to detect SL from cDNA long RNA reads and another trans-spliced species. Our results demonstrate that SLRanger not only provides a reliable approach for characterizing SL trans-splicing events but also serves as an effective framework for operon discovery, enabling transcriptomic analysis for operons and facilitating downstream data-mining applications.

MOTIVATION: Primer design is a routine practice for modern molecular biology labs. Bioinformatics tools like primer3 and primer-blast have standardized the primer design for a specific region. However, large-scale primer design, especially for genome-wide screening, is still a labor-intensive job for most wet-lab researchers using these pipelines. RESULTS: Here, we present the primerdiffer pipeline, which can be used to batch design primers that differentiate haplotypes on a large scale with precise false priming checking. This command-line interface (CLI) pipeline includes greedy primer search, local and global in silico PCR-based false priming checking, and automated best primer selection. The local CLI application provides flexibility to design primers with the user's own genome sequences and specific parameters. Some species-specific primers designed to genotype the hybrid introgression strains from Caenorhabditis briggsae and Caenorhabditis nigoni have been validated using single-worm PCR. This pipeline provides the first CLI-based large-scale primer design tool to differentiate haplotypes in any targeted region. AVAILABILITY AND IMPLEMENTATION: The open-source python modules are available at github (https://github.com/runsheng/primerdiffer, https://github.com/runsheng/primervcf) and Python package index (https://pypi.org/project/primerdiffer/, https://pypi.org/project/primervcf/).