Microbiome analysis has become a crucial tool for basic and translational research due to its potential for translation into clinical practice. However, there is ongoing controversy regarding the comparability of different bioinformatic analysis platforms and a lack of recognized standards, which might have an impact on the translational potential of results. This study investigates how the performance of different microbiome analysis platforms impacts the final results of mucosal microbiome signatures. Across five independent research groups, we compared three distinct and frequently used microbiome analysis bioinformatic packages (DADA2, MOTHUR, and QIIME2) on the same subset of fastQ files. The source data set encompassed 16S rRNA gene raw sequencing data (V1-V2) from gastric biopsy samples of clinically well-defined gastric cancer (GC) patients (