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Background: Biological mutagens (such as transposon) with sequences inserted, play a crucial role to link observed phenotype and genotype in reverse genetic studies. For this reason, accurate and efficient software tools for identifying insertion sites based on the analysis of sequencing reads are desired. Results: We developed a bioinformatics tool, a Finder, to identify genome-wide Insertions in Mutagenesis (named as “InMut-Finder”), based on target sequences and flanking sequences from long reads, such as Oxford Nanopore Sequencing. InMut-Finder succeeded in identify > 100 insertion sites in Medicago truncatula and soybean mutants based on sequencing reads of whole-genome DNA or enriched insertion-site DNA fragments. Insertion sites discovered by InMut-Finder were validated by PCR experiments. Conclusion: InMut-Finder is a comprehensive and powerful tool for automated insertion detection from Nanopore long reads. The simplicity, efficiency, and flexibility of InMut-Finder make it a valuable tool for functional genomics and forward and reverse genetics. InMut-Finder was implemented with Perl, R, and Shell scripts, which are independent of the OS. The source code and instructions can be accessed at https:// github. com/ jsg20 0830/ InMut- Finder.