SpikeSeq: A rapid, cost efficient and simple method to identify SARS-CoV-2 variants of concern by Sanger sequencing part of the spike protein gene.

In 2020, the novel coronavirus, SARS-CoV-2, caused a pandemic, which is still raging at the time of writing this. Here, we present results from SpikeSeq, the first published Sanger sequencing-based method for the detection of Variants of Concern (VOC) and key mutations, using a 1 kb amplicon from the recognized ARTIC Network primers. The proposed setup relies entirely on materials and methods already in use in diagnostic RT-qPCR labs and on existing commercial infrastructure offering sequencing services. For data analysis, we provide an automated, open source, and browser-based mutation calling software (https://github.com/kblin/covid-spike-classification, https://ssi.biolib.com/covid-spike-classification). We validated the setup on 195 SARS-CoV-2 positive samples, and we were able to profile 85% of RT-qPCR positive samples, where the last 15% largely stemmed from samples with low viral count. We compared the SpikeSeq results to WGS results. SpikeSeq has been used as the primary variant identification tool on > 10.000 SARS-CoV-2 positive clinical samples during 2021. At approximately 4€ per sample in material cost, minimal hands-on time, little data handling, and a short turnaround time, the setup is simple enough to be implemented in any SARS-CoV-2 RT-qPCR diagnostic lab. Our protocol provides results that can be used to choose antibodies in a clinical setting and for the tracking and surveillance of all positive samples for new variants and known ones such as Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1) Delta (B.1.617.2), Omicron BA.1(B.1.1.529), BA.2, BA.4/5, BA.2.75.x, and many more, as of October 2022.

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.

Connecting structure to function with the recovery of over 1000 high-quality metagenome-assembled genomes from activated sludge using long-read sequencing.

Microorganisms play crucial roles in water recycling, pollution removal and resource recovery in the wastewater industry. The structure of these microbial communities is increasingly understood based on 16S rRNA amplicon sequencing data. However, such data cannot be linked to functional potential in the absence of high-quality metagenome-assembled genomes (MAGs) for nearly all species. Here, we use long-read and short-read sequencing to recover 1083 high-quality MAGs, including 57 closed circular genomes, from 23 Danish full-scale wastewater treatment plants. The MAGs account for ~30% of the community based on relative abundance, and meet the stringent MIMAG high-quality draft requirements including full-length rRNA genes. We use the information provided by these MAGs in combination with >13 years of 16S rRNA amplicon sequencing data, as well as Raman microspectroscopy and fluorescence in situ hybridisation, to uncover abundant undescribed lineages belonging to important functional groups.

Partitioning the metabolic scope: the importance of anaerobic metabolism and implications for the oxygen- and capacity-limited thermal tolerance (OCLTT) hypothesis.

Ongoing climate change is predicted to affect the distribution and abundance of aquatic ectotherms owing to increasing constraints on organismal physiology, in particular involving the metabolic scope (MS) available for performance and fitness. The oxygen- and capacity-limited thermal tolerance (OCLTT) hypothesis prescribes MS as an overarching benchmark for fitness-related performance and assumes that any anaerobic contribution within the MS is insignificant. The MS is typically derived from respirometry by subtracting standard metabolic rate from the maximal metabolic rate; however, the methodology rarely accounts for anaerobic metabolism within the MS. Using gilthead sea bream (Sparus aurata) and Trinidadian guppy (Poecilia reticulata), this study tested for trade-offs (i) between aerobic and anaerobic components of locomotor performance; and (ii) between the corresponding components of the MS. Data collection involved measuring oxygen consumption rate at increasing swimming speeds, using the gait transition from steady to unsteady (burst-assisted) swimming to detect the onset of anaerobic metabolism. Results provided evidence of the locomotor performance trade-off, but only in S. aurata. In contrast, both species revealed significant negative correlations between aerobic and anaerobic components of the MS, indicating a trade-off where both components of the MS cannot be optimized simultaneously. Importantly, the fraction of the MS influenced by anaerobic metabolism was on average 24.3 and 26.1% in S. aurata and P. reticulata, respectively. These data highlight the importance of taking anaerobic metabolism into account when assessing effects of environmental variation on the MS, because the fraction where anaerobic metabolism occurs is a poor indicator of sustainable aerobic performance. Our results suggest that without accounting for anaerobic metabolism within the MS, studies involving the OCLTT hypothesis could overestimate the metabolic scope available for sustainable activities and the ability of individuals and species to cope with climate change.