Unveiling microbial communities with EasyAmplicon: A user‐centric guide to perform amplicon sequencing data analysis

Abstract The advent of next‐generation sequencing has revolutionized microbiome research, enabling in‐depth exploration of microbial communities through amplicon sequencing. The widespread adoption of sequencing across diverse fields, coupled with decreasing costs, underscores the critical need for validated, fully automated, reproducible, and adaptable analysis pipelines. However, analyzing these high‐throughput datasets often necessitates extensive bioinformatics expertize, hindering accessibility for many researchers. To address this challenge, in 2023 we developed EasyAmplicon, a comprehensive, user‐friendly pipeline that integrates popular tools such as USEARCH and VSEARCH, offering a streamlined workflow from raw data to results. Remarkably, EasyAmplicon has garnered significant recognition within a year, as evidenced by 127 citations to date. To further facilitate the researchers and enhance usability, we present a detailed protocol with a video recording that guides users through each step of the pipeline, including data preprocessing (quality filtering, chimera removal), amplicon sequence variant analysis, diversity analysis, and data visualization. The protocol is designed for ease of use, with each step documented, allowing researchers to execute the workflow without requiring complex scripting skills. The EasyAmplicon pipeline is freely available on GitHub ( https://github.com/YongxinLiu/EasyAmplicon ).