GESV: a bioinformatic pipeline for genome editing sequence variants analysis

Rapid and accurate identification of sequence modifications is essential in genome editing, yet user-friendly bioinformatic tools for processing amplicon data remain limited. Inspired by amplicon-data processing strategies in microbiomics, we developed GESV (Genome-Edited Sequence Variants), a simple and flexible pipeline for the direct extraction of sequence variants from amplicon data of genome-edited organisms. Unlike conventional methods that adopt traditional single-nucleotide variant calling pipelines to analyze editing outcomes of genome-edited organisms, the GESV workflow directly extract sequence variants through the following core steps: merging paired-end reads, removing low-quality and short reads, dereplicating sequences to generate unique sequences with abundance, filtering out potential PCR/sequencing errors, and finally calling sequence modifications to profile edited sequence variants (ESVs). We demonstrate that this pipeline simplifies amplicon-based genotyping while maintaining high accuracy and flexibility. Both online and standalone versions of the GESV tool are available at http://crispr.hzau.edu.cn/gesv. Together, this study presents a novel and flexible data analysis pipeline for amplicon-based genotyping of gene-edited organisms, which will facilitate the application of gene editing in crop breeding.

The online version contains supplementary material available at https://doi.org/10.1007/s11032-026-01736-8.