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With the gut microbiome being implicated in many diseases and even offering the potential for future therapies, its study has been gaining interest over the years. However, to gain more in-depth insight and understanding of the impact of the microbiome on our health, it is critical to have accurate microbial data that is reproducible and comparable across studies. Variation in experimental techniques, particularly DNA extraction and sequencing methods, may result in variability and the inability to compare results. In this chapter, we describe in detail our microbiome analysis methodology focusing on stool samples., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)