High-throughput DNA extraction and cost-effective miniaturized metagenome and amplicon library preparation of soil samples for DNA sequencing.

Reductions in sequencing costs have enabled widespread use of shotgun metagenomics and amplicon sequencing, which have drastically improved our understanding of the microbial world. However, large sequencing projects are now hampered by the cost of library preparation and low sample throughput, comparatively to the actual sequencing costs. Here, we benchmarked three high-throughput DNA extraction methods: ZymoBIOMICS™ 96 MagBead DNA Kit, MP BiomedicalsTM FastDNATM-96 Soil Microbe DNA Kit, and DNeasy® 96 PowerSoil® Pro QIAcube® HT Kit. The DNA extractions were evaluated based on length, quality, quantity, and the observed microbial community across five diverse soil types. DNA extraction of all soil types was successful for all kits, however DNeasy® 96 PowerSoil® Pro QIAcube® HT Kit excelled across all performance parameters. We further used the nanoliter dispensing system I.DOT One to miniaturize Illumina amplicon and metagenomic library preparation volumes by a factor of 5 and 10, respectively, with no significant impact on the observed microbial communities. With these protocols, DNA extraction, metagenomic, or amplicon library preparation for one 96-well plate are approx. 3, 5, and 6 hours, respectively. Furthermore, the miniaturization of amplicon and metagenome library preparation reduces the chemical and plastic costs from 5.0 to 3.6 and 59 to 7.3 USD pr. sample. This enhanced efficiency and cost-effectiveness will enable researchers to undertake studies with greater sample sizes and diversity, thereby providing a richer, more detailed view of microbial communities and their dynamics.

Introduction and transmission of SARS-CoV-2 lineage B.1.1.7, Alpha variant, in Denmark.

BACKGROUND: In early 2021, the SARS-CoV-2 lineage B.1.1.7 (Alpha variant) became dominant across large parts of the world. In Denmark, comprehensive and real-time test, contact-tracing, and sequencing efforts were applied to sustain epidemic control. Here, we use these data to investigate the transmissibility, introduction, and onward transmission of B.1.1.7 in Denmark. METHODS: We analyzed a comprehensive set of 60,178 SARS-CoV-2 genomes generated from high-throughput sequencing by the Danish COVID-19 Genome Consortium, representing 34% of all positive cases in the period 14 November 2020 to 7 February 2021. We calculated the transmissibility of B.1.1.7 relative to other lineages using Poisson regression. Including all 1976 high-quality B.1.1.7 genomes collected in the study period, we constructed a time-scaled phylogeny, which was coupled with detailed travel history and register data to outline the introduction and onward transmission of B.1.1.7 in Denmark. RESULTS: In a period with unchanged restrictions, we estimated an increased B.1.1.7 transmissibility of 58% (95% CI: [56%, 60%]) relative to other lineages. Epidemiological and phylogenetic analyses revealed that 37% of B.1.1.7 cases were related to the initial introduction in November 2020. The relative number of cases directly linked to introductions varied between 10 and 50% throughout the study period. CONCLUSIONS: Our findings corroborate early estimates of increased transmissibility of B.1.1.7. Both substantial early expansion when B.1.1.7 was still unmonitored and continuous foreign introductions contributed considerably to case numbers. Finally, our study highlights the benefit of balanced travel restrictions and self-isolation procedures coupled with comprehensive surveillance efforts, to sustain epidemic control in the face of emerging variants.

MiDAS 4: A global catalogue of full-length 16S rRNA gene sequences and taxonomy for studies of bacterial communities in wastewater treatment plants.

Microbial communities are responsible for biological wastewater treatment, but our knowledge of their diversity and function is still poor. Here, we sequence more than 5 million high-quality, full-length 16S rRNA gene sequences from 740 wastewater treatment plants (WWTPs) across the world and use the sequences to construct the 'MiDAS 4' database. MiDAS 4 is an amplicon sequence variant resolved, full-length 16S rRNA gene reference database with a comprehensive taxonomy from domain to species level for all sequences. We use an independent dataset (269 WWTPs) to show that MiDAS 4, compared to commonly used universal reference databases, provides a better coverage for WWTP bacteria and an improved rate of genus and species level classification. Taking advantage of MiDAS 4, we carry out an amplicon-based, global-scale microbial community profiling of activated sludge plants using two common sets of primers targeting regions of the 16S rRNA gene, revealing how environmental conditions and biogeography shape the activated sludge microbiota. We also identify core and conditionally rare or abundant taxa, encompassing 966 genera and 1530 species that represent approximately 80% and 50% of the accumulated read abundance, respectively. Finally, we show that for well-studied functional guilds, such as nitrifiers or polyphosphate-accumulating organisms, the same genera are prevalent worldwide, with only a few abundant species in each genus.

There it is! Fusarium pseudograminearum did not lose the fusaristatin gene cluster after all.

Fusarium pseudograminearum is a significant pathogen of cereals in arid regions worldwide and has the ability to produce numerous bioactive secondary metabolites. The genome sequences of seven F. pseudograminearum strains have been published and in one of these strains, C5834, we identified an intact gene cluster responsible for biosynthesis of the cyclic lipopeptide fusaristatin A. The high level of sequence identity of the fusaristatin cluster remnant in strains that do not produce fusaristatin suggests that the absence of the cluster evolved once, and subsequently the resulting locus with the cluster fragments became widely dispersed among strains of F. pseudograminearum in Australia. We examined a selection of 99 Australian F. pseudograminearum isolates to determine how widespread the ability to produce fusaristatin A is in F. pseudograminearum. We identified 15 fusaristatin producing strains, all originating from Western Australia. Phylogenetic analyses could not support a division of F. pseudograminearum into fusaristatin producing and nonproducing populations, which could indicate the loss has occurred relatively recent.