A novel two-step metabarcoding approach improves soil microbiome biodiversity assessment.

The foundation of microbial ecology research is Next-Generation Sequencing (NGS), which allows for reconstruction of the soil microbiome taxonomical structure and the calculation of biodiversity metrics. However, obtaining reliable data on soil biodiversity poses several challenges, with accurate primer selection being one of the most critical. While 16S rDNA primers are widely used for their ability to broadly target bacterial communities, they can introduce biases. These primers may preferentially amplify certain bacterial groups, leading to a skewed representation of the microbial diversity in soil samples. To overcome the bias, we developed a novel, Two-Step Metabarcoding (TSM) approach to obtain more accurate and detailed data on soil microbiome structure and biodiversity. The first step involved sequencing of amplicons generated using universal 16S rDNA primers, provided an initial overview of the microbial community, and allowed the identification of key taxonomical groups. In the second step, we employed sequencing of amplicons generated with taxa-specific primers designed for the most abundant phyla in the community. We used the obtained data for a more reliable reconstruction of microbiome taxonomic structure and biodiversity. This two-step approach ensures a thorough exploration of the soil microbiome and promises to enhance our understanding of soil microbial dynamics and ecology.