Comparison of DNA extraction methods on different sample matrices within the same terrestrial ecosystem.

Metataxonomic studies of ecosystem microbiotas require the simultaneous processing of samples with contrasting physical and biochemical traits. However, there are no published studies of comparisons of different DNA extraction kits to characterize the microbiotas of the main components of terrestrial ecosystems. Here, and to our knowledge for the first time, five DNA extraction kits were used to investigate the composition and diversity of the microbiota of a subset of samples typically studied in terrestrial ecosystems such as bulk soil, rhizosphere soil, invertebrate taxa and mammalian feces. DNA extraction kit was associated with changes in the relative abundance of hundreds of ASVs, in the same samples, resulting in significant differences in alpha and beta diversity estimates of their microbiotas. Importantly, the impact of DNA extraction kit on sample diversity varies according to sample type, with mammalian feces and soil samples showing the most and least consistent diversity estimates across DNA extraction kits, respectively. We show that the MACHEREY-NAGEL NucleoSpin® Soil kit was associated with the highest alpha diversity estimates, providing the highest contribution to the overall sample diversity, as indicated by comparisons with computationally assembled reference communities, and is recommended to be used for any large-scale microbiota study of terrestrial ecosystems.

Microbial perspective of inhibited carbon turnover in Tangel humus of the Northern Limestone Alps.

Tangel humus primarily occurs in montane and subalpine zones of the calcareous Alps that exhibit low temperatures and high precipitation sums. This humus form is characterized by inhibited carbon turnover and accumulated organic matter, leading to the typical thick organic layers. However, the reason for this accumulation of organic matter is still unclear, and knowledge about the microbial community within Tangel humus is lacking. Therefore, we investigated the prokaryotic and fungal communities along with the physical and chemical properties within a depth gradient (0-10, 10-20, 20-30, 30-40, 40-50 cm) of a Tangel humus located in the Northern Limestone Alps. We hypothesized that humus properties and microbial activity, biomass, and diversity differ along the depth gradient and that microbial key players refer to certain humus depths. Our results give the first comprehensive information about microbiota within the Tangel humus and establish a microbial zonation of the humus. Microbial activity, biomass, as well as microbial alpha diversity significantly decreased with increasing depths. We identified microbial biomarkers for both, the top and the deepest depth, indicating different, microbial habitats. The microbial characterization together with the established nutrient deficiencies in the deeper depths might explain reduced C-turnover and Tangel humus formation.

Mock community as an in situ positive control for amplicon sequencing of microbiotas from the same ecosystem.

Metataxonomy has become the standard for characterizing the diversity and composition of microbial communities associated with multicellular organisms and their environment. Currently available protocols for metataxonomy assume a uniform DNA extraction, amplification and sequencing efficiency for all sample types and taxa. It has been suggested that the addition of a mock community (MC) to biological samples before the DNA extraction step could aid identification of technical biases during processing and support direct comparisons of microbiota composition, but the impact of MC on diversity estimates of samples is unknown. Here, large and small aliquots of pulverized bovine fecal samples were extracted with no, low or high doses of MC, characterized using standard Illumina technology for metataxonomics, and analysed with custom bioinformatic pipelines. We demonstrated that sample diversity estimates were distorted only if MC dose was high compared to sample mass (i.e. when MC > 10% of sample reads). We also showed that MC was an informative in situ positive control, permitting an estimation of the sample 16S copy number, and detecting sample outliers. We tested this approach on a range of sample types from a terrestrial ecosystem, including rhizosphere soil, whole invertebrates, and wild vertebrate fecal samples, and discuss possible clinical applications.