Multicenter benchmarking of short and long read wet lab protocols for clinical viral metagenomics

Metagenomics is gradually being implemented for diagnosing infectious diseases. However, in-depth protocol comparisons for viral detection have been limited to individual sets of experimental workflows and laboratories. In this study, we present a benchmark of metagenomics protocols used in clinical diagnostic laboratories initiated by the European Society for Clinical Virology (ESCV) Network on NGS (ENNGS). A mock viral reference panel was designed to mimic low biomass clinical specimens. The panel was used to assess the performance of twelve metagenomic wet lab protocols currently in use in the diagnostic laboratories of participating ENNGS member institutions. Both Illumina and Nanopore, shotgun and targeted capture probe protocols were included. Performance metrics sensitivity, specificity, and quantitative potential were assessed using a central bioinformatics pipeline. Overall, viral pathogens with loads down to 104 copies/ml (corresponding to CT values of 31 in our PCR assays) were detected by all the evaluated metagenomic wet lab protocols. In contrast, lower abundant mixed viruses of CT values of 35 and higher were detected only by a minority of the protocols. Considering the reference panel as the gold standard, optimal thresholds to define a positive result were determined per protocol, based on the horizontal genome coverage. Implementing these thresholds, sensitivity and specificity of the protocols ranged from 67 to 100 % and 87 to 100 %, respectively. A variety of metagenomic protocols are currently in use in clinical diagnostic laboratories. Detection of low abundant viral pathogens and mixed infections remains a challenge, implying the need for standardization of metagenomic analysis for use in clinical settings.</p

Multicenter benchmarking of short and long read wet lab protocols for clinical viral metagenomics

Metagenomics is gradually being implemented for diagnosing infectious diseases. However, in-depth protocol comparisons for viral detection have been limited to individual sets of experimental workflows and laboratories. In this study, we present a benchmark of metagenomics protocols used in clinical diagnostic laboratories initiated by the European Society for Clinical Virology (ESCV) Network on NGS (ENNGS). A mock viral reference panel was designed to mimic low biomass clinical specimens. The panel was used to assess the performance of twelve metagenomic wet lab protocols currently in use in the diagnostic laboratories of participating ENNGS member institutions. Both Illumina and Nanopore, shotgun and targeted capture probe protocols were included. Performance metrics sensitivity, specificity, and quantitative potential were assessed using a central bioinformatics pipeline. Overall, viral pathogens with loads down to 104 copies/ml (corresponding to CT values of 31 in our PCR assays) were detected by all the evaluated metagenomic wet lab protocols. In contrast, lower abundant mixed viruses of CT values of 35 and higher were detected only by a minority of the protocols. Considering the reference panel as the gold standard, optimal thresholds to define a positive result were determined per protocol, based on the horizontal genome coverage. Implementing these thresholds, sensitivity and specificity of the protocols ranged from 67 to 100 % and 87 to 100 %, respectively. A variety of metagenomic protocols are currently in use in clinical diagnostic laboratories. Detection of low abundant viral pathogens and mixed infections remains a challenge, implying the need for standardization of metagenomic analysis for use in clinical settings.</p

Emerging mechanisms to fine-tune receptor kinase signaling specificity

Organisms need to constantly inform their cellular machinery about the biochemical and physical status of their surroundings to adapt and thrive. While some external signals are also sensed intracellularly, a considerable share of external information is registered already at the plasma membrane (PM). Receptor kinases (RKs) are crucial for plant cells to integrate such cues from the environment, from microbes, or from other cells to coordinate their physiological response and their development. Early studies on RK signaling depicted the path from external signal to internal response in a linear fashion, but recent findings show that these cellular information highways are highly interconnected and pass signals through molecular intersections. In this review, we first discuss how individual RKs simultaneously contribute to the transduction and deconvolution of a multitude of signals by controlled assembly into diverse RK complexes, exemplified by FERONIA signaling versatility. We then elaborate on how cells can exert highly localized control over the assembly, interaction and composition of such complexes in order to attain essential cellular output specificity

Community dynamics and metagenomic analyses reveal Bacteroidota's role in widespread enzymatic Fucus vesiculosus cell wall degradation

Enzymatic degradation of algae cell wall carbohydrates by microorganisms is under increasing investigation as marine organic matter gains more value as a sustainable resource. The fate of carbon in the marine ecosystem is in part driven by these degradation processes. In this study, we observe the microbiome dynamics of the macroalga Fucus vesiculosus in 25-day-enrichment cultures resulting in partial degradation of the brown algae. Microbial community analyses revealed the phylum Pseudomonadota as the main bacterial fraction dominated by the genera Marinomonas and Vibrio. More importantly, a metagenome-based Hidden Markov model for specifc glycosyl hydrolyses and sulphatases identifed Bacteroidota as the phylum with the highest potential for cell wall degradation, contrary to their low abundance. For experimental verifcation, we cloned, expressed, and biochemically characterised two α-L-fucosidases, FUJM18 and FUJM20. While protein structure predictions suggest the highest similarity to a Bacillota origin, protein–protein blasts solely showed weak similarities to defned Bacteroidota proteins. Both enzymes were remarkably active at elevated temperatures and are the basis for a potential synthetic enzyme cocktail for large-scale algal destruction

Differential accumulation of callose, arabinoxylan and cellulose in nonpenetrated versus penetrated papillae on leaves of barley infected with Blumeria graminis f. sp. hordei

First published: 20 August 2014• In plants, cell walls are one of the first lines of defence for protecting cells from successful invasion by fungal pathogens and are a major factor in basal host resistance. For the plant cell to block penetration attempts, it must adapt its cell wall to withstand the physical and chemical forces applied by the fungus. • Papillae that have been effective in preventing penetration by pathogens are traditionally believed to contain callose as the main polysaccharide component. Here, we have re-examined the composition of papillae of barley (Hordeum vulgare) attacked by the powdery mildew fungus Blumeria graminis f. sp. hordei (Bgh) using a range of antibodies and carbohydrate-binding modules that are targeted to cell wall polysaccharides. • The data show that barley papillae induced during infection with Bgh contain, in addition to callose, significant concentrations of cellulose and arabinoxylan. Higher concentrations of callose, arabinoxylan and cellulose are found in effective papillae, compared with ineffective papillae. The papillae have a layered structure, with the inner core consisting of callose and arabinoxylan and the outer layer containing arabinoxylan and cellulose. • The association of arabinoxylan and cellulose with penetration resistance suggests new targets for the improvement of papilla composition and enhanced disease resistance.Jamil Chowdhury, Marilyn Henderson, Patrick Schweizer, Rachel A. Burton, Geoffrey B. Fincher and Alan Littl