New developments in next-generation sequencing technologies and massive data received from this approach open wide prospects for personalised medicine and nutrition studies. Metagenomic analysis of the gut microbiota is paramount for the characterization of human health and wellbeing. Despite the intensive research, there is a huge gap and inconsistency between different studies due to the non-standardised and biased pipeline. Methodical and systemic understanding of every stage in the process is necessary to overcome all bottlenecks and grey zones of gut microbiota studies, where all details and interactions between processes are important.

Here we show that an inexpensive, but reliable iSeq 100 platform is an excellent tool to perform the analysis of the human gut microbiota by amplicon sequencing of the 16 S rRNA gene. Two commercial DNA extraction kits and different starting materials performed similarly regarding the taxonomic distribution of identified bacteria. DNA/RNA Shield reagent proved to be a reliable solution for stool samples collection, preservation, and storage, as the storage of faecal material in DNA/RNA Shield for three weeks at different temperatures and thawing cycles had a low impact on the bacterial distribution.

Altogether, a thoroughly elaborated pipeline with close attention to details ensures high reproducibility with significant biological but not technical variations.