Consistent microbial insights across sequencing methods in soil studies: the role of reference taxonomies.

Microbes play an important role in soil functioning, underpinning food production systems and delivering an array of essential ecosystem services. To elucidate how these microbes relate to ecosystem functions, accurate identification and classification of soil microorganisms are important. We evaluated the comparability of shotgun and amplicon sequencing approaches by profiling soil microbiota from 131 diverse temperate grassland soils across Ireland. We assessed method comparability in terms of (i) detection and classification of the most abundant phyla, (ii) their capacity to differentiate samples based on their microbial community, and (iii) their capacity to link microbial communities to measured nitrogen cycle functions. Our findings reveal that both methods offer moderately similar outcomes, providing consistent detection of major phyla, similar microbial community differentiation patterns, and largely identifying the same relationships between the phyla and nitrogen functions. The variations observed between the two methods were mostly associated with differences in the choice of reference taxonomy. Amplicon sequencing represents a cost-effective, less computationally demanding option, while shotgun sequencing provides deeper taxonomic resolution and access to the latest databases, making it suitable for detailed microbial profiling. Our study underscores the need for careful method selection based on project requirements, database availability, and financial resources.IMPORTANCEStudying the microorganisms in soil remains a challenge as soils are one of the most complex and diverse environments. Compounding these challenges is the lack of culturable representatives in soil, with over 99% of soil microorganisms yet to be cultivated in a laboratory setting. Leveraging next-generation sequencing technologies, which bypass traditional culture-dependent methods, scientists are now able to attain low-cost, high-throughput DNA sequencing that can detect even the rarest microorganisms within samples. The present study rigorously compares amplicon and shotgun sequencing techniques in profiling microbial communities across diverse temperate grassland soil samples, focusing on how different databases, classifiers, and sequencing methods influence the results. Our study underscores the crucial need for a harmonized taxonomic database that could greatly enhance comparability and accuracy in the understanding of soil microbiomes.