show Abstracthide AbstractA critical issue in microbiome research is the selection of reliable laboratory and bioinformatics pipelines. The present scenario is marked by an absence of standard and optimal technical workflow, making it difficult to compare data from various studies that utilize different methodologies. Our study, the most comprehensive so far, encompasses every step of processing from DNA extraction to sequencing. We utilized a single stool sample and implemented five DNA purification methods, seven library construction techniques, and four sequencing approaches. Additionally, we employed a ZymoBIOMICS Microbial Community Standard to verify the effectiveness of the applied methods. Our data serve as a valuable resource for exploring how experimental processes influence outcomes and for identifying the best combinations of various methods. Additionally, this dataset acts as a strong foundation for evaluating microbiome bioinformatics software. Furthermore, our data can be applied to meta-analyses concerning the gut microbiome of dogs.