Standardization of laboratory practices for the study of the human gut microbiome

Technical advances in next-generation sequencing (NGS) have made it more accessible to study the human microbiome, resulting in more available data and knowledge. As a result of this expansion of data, the need to obtain comparable and reproducible data has become one of the most important challenges facing microbiome research nowadays. In this study, we aim to contribute to existing knowledge to promote high quality microbiome data and minimize bias introduced by technical variation throughout studies, from sample collection, storage, to sequencing strategies. While immediate freezing upon sampling has been the "golden standard" in the field, this method is often logistically difficult and expensive, becoming a limiting factor when conducting large scale studies or in regions where maintenance of the cold-chain presents difficulties. Therefore, we compared the immediately frozen method to storage at room temperature for 3 - 5 days in two commercially available stabilization solutions (Omnigene gut and Zymo Research) as well as without buffer. Other important aspects were tested, such as DNA extraction, bacterial DNA input or number of PCR cycles. Method choice for cell disruption resulted in the biggest difference in compositional profiles. The changes observed in microbiome profiles in samples stored at RT without stabilization solution was prevented by the use of these. For library preparation and sequencing, we found the highest heterogeneity in the DNA extraction step, followed by the use of different Illumina barcodes, indicating that both of these steps have an impact during library preparation. We did not observe a batch effect between the different sequencing runs. Standardized methods are important to allow comparison of results between different research groups worldwide and reliably expand microbiome data to a broad range of diseases, ethnical backgrounds and geographic locations. A more global perspective will increase our understanding of the human microbiome around the world. ### Competing Interest Statement The authors have declared no competing interest.