Icarust, a real-time simulator for Oxford Nanopore adaptive sampling.

MOTIVATION: Oxford Nanopore Technologies (ONT) sequencers enable real-time generation of sequence data, which allows for concurrent analysis during a run. Adaptive sampling leverages this real-time capability in extremis, rejecting or accepting reads for sequencing based on assessment of the sequence from the start of each read. This functionality is provided by ONT's software, MinKNOW (Oxford Nanopore Technologies). Designing and developing software to take advantage of adaptive sampling can be costly in terms of sequencing consumables, using precious samples and preparing sequencing libraries. MinKNOW addresses this in part by allowing the replay of previously sequenced runs for testing. However, as we show, the sequencing output only partially changes in response to adaptive sampling instructions. Here we present Icarust, a tool enabling more accurate approximations of sequencing runs. Icarust recreates all the required endpoints of MinKNOW to perform adaptive sampling and writes output compatible with current base-callers and analysis pipelines. Icarust serves nanopore signal simulating a MinION or PromethION flow cell experiment from any reference genome using either R9 or R10 pore models. We show that simulating sequencing runs with Icarust provides a realistic testing and development environment for software exploiting the real-time nature of Nanopore sequencing. AVAILABILITY AND IMPLEMENTATION: All code is open source and freely available here-https://github.com/LooseLab/Icarust. Icarust is implemented in Rust, with a docker container also available. The data underlying this article will be shared on reasonable request to the corresponding author.

Socioeconomic deprivation and illness trajectory in the Scottish population after COVID-19 hospitalization.

BACKGROUND: The associations between deprivation and illness trajectory after hospitalisation for coronavirus disease-19 (COVID-19) are uncertain. METHODS: A prospective, multicentre cohort study was conducted on post-COVID-19 patients, enrolled either in-hospital or shortly post-discharge. Two evaluations were carried out: an initial assessment and a follow-up at 28-60 days post-discharge. The study encompassed research blood tests, patient-reported outcome measures, and multisystem imaging (including chest computed tomography (CT) with pulmonary and coronary angiography, cardiovascular and renal magnetic resonance imaging). Primary and secondary outcomes were analysed in relation to socioeconomic status, using the Scottish Index of Multiple Deprivation (SIMD). The EQ-5D-5L, Brief Illness Perception Questionnaire (BIPQ), Patient Health Questionnaire-4 (PHQ-4) for Anxiety and Depression, and the Duke Activity Status Index (DASI) were used to assess health status. RESULTS: Of the 252 enrolled patients (mean age 55.0 ± 12.0 years; 40% female; 23% with diabetes), deprivation status was linked with increased BMI and diabetes prevalence. 186 (74%) returned for the follow-up. Within this group, findings indicated associations between deprivation and lung abnormalities (p = 0.0085), coronary artery disease (p = 0.0128), and renal inflammation (p = 0.0421). Furthermore, patients with higher deprivation exhibited worse scores in health-related quality of life (EQ-5D-5L, p = 0.0084), illness perception (BIPQ, p = 0.0004), anxiety and depression levels (PHQ-4, p = 0.0038), and diminished physical activity (DASI, p = 0.002). At the 3-month mark, those with greater deprivation showed a higher frequency of referrals to secondary care due to ongoing COVID-19 symptoms (p = 0.0438). However, clinical outcomes were not influenced by deprivation. CONCLUSIONS: In a post-hospital COVID-19 population, socioeconomic deprivation was associated with impaired health status and secondary care episodes. Deprivation influences illness trajectory after COVID-19.

In our study, we aimed to understand how socioeconomic factors impact recovery from COVID-19 following hospitalisation. We followed 252 patients, collecting health data and utilising advanced imaging techniques. We discovered that individuals from deprived areas experienced more severe health complications, reported worse quality of life, and required more specialist care. However, their clinical outcomes were not significantly different. This underscores that socioeconomic deprivation affects health recovery, underlining the need for tailored care for these individuals. Our findings emphasise the importance of considering socioeconomic factors in recovery plans post-COVID-19, potentially improving healthcare for those in deprived areas.

Decoupling speciation and extinction reveals both abiotic and biotic drivers shaped 250 million years of diversity in crocodile-line archosaurs.

Whereas living representatives of Pseudosuchia, crocodylians, number fewer than 30 species, more than 700 pseudosuchian species are known from their 250-million-year fossil record, displaying far greater ecomorphological diversity than their extant counterparts. With a new time-calibrated tree of >500 species, we use a phylogenetic framework to reveal that pseudosuchian evolutionary history and diversification dynamics were directly shaped by the interplay of abiotic and biotic processes over hundreds of millions of years, supported by information theory analyses. Speciation, but not extinction, is correlated with higher temperatures in terrestrial and marine lineages, with high sea level associated with heightened extinction in non-marine taxa. Low lineage diversity and increased speciation in non-marine species is consistent with opportunities for niche-filling, whereas increased competition may have led to elevated extinction rates. In marine lineages, competition via increased lineage diversity appears to have driven both speciation and extinction. Decoupling speciation and extinction, in combination with ecological partitioning, reveals a more complex picture of pseudosuchian evolution than previously understood. As the number of species threatened with extinction by anthropogenic climate change continues to rise, the fossil record provides a unique window into the drivers that led to clade success and those that may ultimately lead to extinction.

Utility of wastewater genomic surveillance compared to clinical surveillance to track the spread of the SARS-CoV-2 Omicron variant across England.

The world has moved into a new stage of managing the SARS-CoV-2 pandemic with minimal restrictions and reduced testing in the population, leading to reduced genomic surveillance of virus variants in individuals. Wastewater-based epidemiology (WBE) can provide an alternative means of tracking virus variants in the population but decision-makers require confidence that it can be applied to a national scale and is comparable to individual testing data. We analysed 19,911 samples from 524 wastewater sites across England at least twice a week between November 2021 and February 2022, capturing sewage from >70% of the English population. We used amplicon-based sequencing and the phylogeny based de-mixing tool Freyja to estimate SARS-CoV-2 variant frequencies and compared these to the variant dynamics observed in individual testing data from clinical and community settings. We show that wastewater data can reconstruct the spread of the Omicron variant across England since November 2021 in close detail and aligns closely with epidemiological estimates from individual testing data. We also show the temporal and spatial spread of Omicron within London. Our wastewater data further reliably track the transition between Omicron subvariants BA1 and BA2 in February 2022 at regional and national levels. Our demonstration that WBE can track the fast-paced dynamics of SARS-CoV-2 variant frequencies at a national scale and closely match individual testing data in time shows that WBE can reliably fill the monitoring gap left by reduced individual testing in a more affordable way.

NANOG is required to establish the competence for germ-layer differentiation in the basal tetrapod axolotl.

Pluripotency defines the unlimited potential of individual cells of vertebrate embryos, from which all adult somatic cells and germ cells are derived. Understanding how the programming of pluripotency evolved has been obscured in part by a lack of data from lower vertebrates; in model systems such as frogs and zebrafish, the function of the pluripotency genes NANOG and POU5F1 have diverged. Here, we investigated how the axolotl ortholog of NANOG programs pluripotency during development. Axolotl NANOG is absolutely required for gastrulation and germ-layer commitment. We show that in axolotl primitive ectoderm (animal caps; ACs) NANOG and NODAL activity, as well as the epigenetic modifying enzyme DPY30, are required for the mass deposition of H3K4me3 in pluripotent chromatin. We also demonstrate that all 3 protein activities are required for ACs to establish the competency to differentiate toward mesoderm. Our results suggest the ancient function of NANOG may be establishing the competence for lineage differentiation in early cells. These observations provide insights into embryonic development in the tetrapod ancestor from which terrestrial vertebrates evolved.

A framework for real-time monitoring, analysis and adaptive sampling of viral amplicon nanopore sequencing.

The ongoing SARS-CoV-2 pandemic demonstrates the utility of real-time sequence analysis in monitoring and surveillance of pathogens. However, cost-effective sequencing requires that samples be PCR amplified and multiplexed via barcoding onto a single flow cell, resulting in challenges with maximising and balancing coverage for each sample. To address this, we developed a real-time analysis pipeline to maximise flow cell performance and optimise sequencing time and costs for any amplicon based sequencing. We extended our nanopore analysis platform MinoTour to incorporate ARTIC network bioinformatics analysis pipelines. MinoTour predicts which samples will reach sufficient coverage for downstream analysis and runs the ARTIC networks Medaka pipeline once sufficient coverage has been reached. We show that stopping a viral sequencing run earlier, at the point that sufficient data has become available, has no negative effect on subsequent down-stream analysis. A separate tool, SwordFish, is used to automate adaptive sampling on Nanopore sequencers during the sequencing run. This enables normalisation of coverage both within (amplicons) and between samples (barcodes) on barcoded sequencing runs. We show that this process enriches under-represented samples and amplicons in a library as well as reducing the time taken to obtain complete genomes without affecting the consensus sequence.

Evaluation of variant calling algorithms for wastewater-based epidemiology using mixed populations of SARS-CoV-2 variants in synthetic and wastewater samples.

Wastewater-based epidemiology has been used extensively throughout the COVID-19 (coronavirus disease 19) pandemic to detect and monitor the spread and prevalence of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) and its variants. It has proven an excellent, complementary tool to clinical sequencing, supporting the insights gained and helping to make informed public-health decisions. Consequently, many groups globally have developed bioinformatics pipelines to analyse sequencing data from wastewater. Accurate calling of mutations is critical in this process and in the assignment of circulating variants; yet, to date, the performance of variant-calling algorithms in wastewater samples has not been investigated. To address this, we compared the performance of six variant callers (VarScan, iVar, GATK, FreeBayes, LoFreq and BCFtools), used widely in bioinformatics pipelines, on 19 synthetic samples with known ratios of three different SARS-CoV-2 variants of concern (VOCs) (Alpha, Beta and Delta), as well as 13 wastewater samples collected in London between the 15th and 18th December 2021. We used the fundamental parameters of recall (sensitivity) and precision (specificity) to confirm the presence of mutational profiles defining specific variants across the six variant callers. Our results show that BCFtools, FreeBayes and VarScan found the expected variants with higher precision and recall than GATK or iVar, although the latter identified more expected defining mutations than other callers. LoFreq gave the least reliable results due to the high number of false-positive mutations detected, resulting in lower precision. Similar results were obtained for both the synthetic and wastewater samples.

Adjudicated myocarditis and multisystem illness trajectory in healthcare workers post-COVID-19.

BACKGROUND: We investigated the associations of healthcare worker status with multisystem illness trajectory in hospitalised post-COVID-19 individuals. METHODS AND RESULTS: One hundred and sixty-eight patients were evaluated 28-60 days after the last episode of hospital care. Thirty-six (21%) were healthcare workers. Compared with non-healthcare workers, healthcare workers were of similar age (51.3 (8.7) years vs 55.0 (12.4) years; p=0.09) more often women (26 (72%) vs 48 (38%); p<0.01) and had lower 10-year cardiovascular risk (%) (8.1 (7.9) vs 15.0 (11.5); p<0.01) and Coronavirus Clinical Characterisation Consortium in-hospital mortality risk (7.3 (10.2) vs 12.7 (9.8); p<0.01). Healthcare worker status associated with less acute inflammation (peak C reactive protein 48 mg/L (IQR: 14-165) vs 112 mg/L (52-181)), milder illness reflected by WHO clinical severity score distribution (p=0.04) and shorter duration of admission (4 days (IQR: 2-6) vs 6 days (3-12)).In adjusted multivariate logistic regression analysis, healthcare worker status associated with a binary classification (probable/very likely vs not present/unlikely) of adjudicated myocarditis (OR: 2.99; 95% CI (1.01 to 8.89) by 28-60 days postdischarge).After a mean (SD, range) duration of follow-up after hospital discharge of 450 (88) days (range 290, 627 days), fewer healthcare workers died or were rehospitalised (1 (3%) vs 22 (17%); p=0.038) and secondary care referrals for post-COVID-19 syndrome were common (42%) and similar to non-healthcare workers (38%; p=0.934). CONCLUSION: Healthcare worker status was independently associated with the likelihood of adjudicated myocarditis, despite better antecedent health. Two in five healthcare workers had a secondary care referral for post-COVID-19 syndrome. TRIAL REGISTRATION NUMBER: NCT04403607.

Ecological Transitions and the Shape of the Decapod Tree of Life.

Understanding the processes that shaped the distribution of species richness across the Tree of Life is a central macroevolutionary research agenda. Major ecological innovations, including transitions between habitats, may help to explain the striking asymmetries of diversity that are often observed between sister clades. Here, we test the impact of such transitions on speciation rates across decapod crustaceans, modeling diversification dynamics within a phylogenetic framework. Our results show that, while terrestrial lineages have higher speciation rates than either marine or freshwater lineages, there is no difference between mean speciation rates in marine and freshwater lineages across Decapoda. Partitioning our data by infraorder reveals that those clades with habitat heterogeneity have higher speciation rates in freshwater and terrestrial lineages, with freshwater rates up to 1.5 times faster than marine rates, and terrestrial rates approximately four times faster. This averaging out of marine and freshwater speciation rates results from the varying contributions of different clades to average speciation rates. However, with the exception of Caridea, we find no evidence for any causal relationship between habitat and speciation rate. Our results demonstrate that while statistical generalizations about ecological traits and evolutionary rates are valuable, there are many exceptions. Hence, while freshwater and terrestrial lineages typically speciate faster than their marine relatives, there are many atypically slow freshwater lineages and fast marine lineages across Decapoda. Future work on diversification patterns will benefit from the inclusion of fossil data, as well as additional ecological factors.

A multisystem, cardio-renal investigation of post-COVID-19 illness.

The pathophysiology and trajectory of post-Coronavirus Disease 2019 (COVID-19) syndrome is uncertain. To clarify multisystem involvement, we undertook a prospective cohort study including patients who had been hospitalized with COVID-19 (ClinicalTrials.gov ID NCT04403607 ). Serial blood biomarkers, digital electrocardiography and patient-reported outcome measures were obtained in-hospital and at 28-60 days post-discharge when multisystem imaging using chest computed tomography with pulmonary and coronary angiography and cardio-renal magnetic resonance imaging was also obtained. Longer-term clinical outcomes were assessed using electronic health records. Compared to controls (n = 29), at 28-60 days post-discharge, people with COVID-19 (n = 159; mean age, 55 years; 43% female) had persisting evidence of cardio-renal involvement and hemostasis pathway activation. The adjudicated likelihood of myocarditis was 'very likely' in 21 (13%) patients, 'probable' in 65 (41%) patients, 'unlikely' in 56 (35%) patients and 'not present' in 17 (11%) patients. At 28-60 days post-discharge, COVID-19 was associated with worse health-related quality of life (EQ-5D-5L score 0.77 (0.23) versus 0.87 (0.20)), anxiety and depression (PHQ-4 total score 3.59 (3.71) versus 1.28 (2.67)) and aerobic exercise capacity reflected by predicted maximal oxygen utilization (20.0 (7.6) versus 29.5 (8.0) ml/kg/min) (all P < 0.01). During follow-up (mean, 450 days), 24 (15%) patients and two (7%) controls died or were rehospitalized, and 108 (68%) patients and seven (26%) controls received outpatient secondary care (P = 0.017). The illness trajectory of patients after hospitalization with COVID-19 includes persisting multisystem abnormalities and health impairments that could lead to substantial demand on healthcare services in the future.

minoTour, real-time monitoring and analysis for nanopore sequencers.

SUMMARY: minoTour offers a Laboratory Informations Management System (LIMS) system for Oxford Nanopore Technology sequencers, with real-time metrics and analysis available permanently for review. Integration of unique real-time automated analysis can reduce the time required to answer biological questions, including mapping and classification of sequence while a run is in progress. Real-time sequence data require new methods of analysis which do not wait for the completion of a run and minoTour provides a framework to allow users to exploit these features. AVAILABILITY AND IMPLEMENTATION: Source code and documentation are available at https://github.com/LooseLab/minotourcli and https://github.com/LooseLab/minotourapp. Docker images are available from https://hub.docker.com/r/adoni5/, and can be installed using a preconfigured docker-compose script at https://github.com/LooseLab/minotour-docker. An example server is available at http://137.44.59.170. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Designed, highly expressing, thermostable dengue virus 2 envelope protein dimers elicit quaternary epitope antibodies.

Dengue virus (DENV) is a worldwide health burden, and a safe vaccine is needed. Neutralizing antibodies bind to quaternary epitopes on DENV envelope (E) protein homodimers. However, recombinantly expressed soluble E proteins are monomers under vaccination conditions and do not present these quaternary epitopes, partly explaining their limited success as vaccine antigens. Using molecular modeling, we found DENV2 E protein mutations that induce dimerization at low concentrations (<100 pM) and enhance production yield by more than 50-fold. Cross-dimer epitope antibodies bind to the stabilized dimers, and a crystal structure resembles the wild-type (WT) E protein bound to a dimer epitope antibody. Mice immunized with the stabilized dimers developed antibodies that bind to E dimers and not monomers and elicited higher levels of DENV2-neutralizing antibodies compared to mice immunized with WT E antigen. Our findings demonstrate the feasibility of using structure-based design to produce subunit vaccines for dengue and other flaviviruses.

Unusual mode of dimerization of retinitis pigmentosa-associated F220C rhodopsin.

Mutations in the G protein-coupled receptor (GPCR) rhodopsin are a common cause of autosomal dominant retinitis pigmentosa, a blinding disease. Rhodopsin self-associates in the membrane, and the purified monomeric apo-protein opsin dimerizes in vitro as it transitions from detergent micelles to reconstitute into a lipid bilayer. We previously reported that the retinitis pigmentosa-linked F220C opsin mutant fails to dimerize in vitro, reconstituting as a monomer. Using fluorescence-based assays and molecular dynamics simulations we now report that whereas wild-type and F220C opsin display distinct dimerization propensities in vitro as previously shown, they both dimerize in the plasma membrane of HEK293 cells. Unexpectedly, molecular dynamics simulations show that F220C opsin forms an energetically favored dimer in the membrane when compared with the wild-type protein. The conformation of the F220C dimer is unique, with transmembrane helices 5 and 6 splayed apart, promoting widening of the intracellular vestibule of each protomer and influx of water into the protein interior. FRET experiments with SNAP-tagged wild-type and F220C opsin expressed in HEK293 cells are consistent with this conformational difference. We speculate that the unusual mode of dimerization of F220C opsin in the membrane may have physiological consequences.

Readfish enables targeted nanopore sequencing of gigabase-sized genomes.

Nanopore sequencers can be used to selectively sequence certain DNA molecules in a pool by reversing the voltage across individual nanopores to reject specific sequences, enabling enrichment and depletion to address biological questions. Previously, we achieved this using dynamic time warping to map the signal to a reference genome, but the method required substantial computational resources and did not scale to gigabase-sized references. Here we overcome this limitation by using graphical processing unit (GPU) base-calling. We show enrichment of specific chromosomes from the human genome and of low-abundance organisms in mixed populations without a priori knowledge of sample composition. Finally, we enrich targeted panels comprising 25,600 exons from 10,000 human genes and 717 genes implicated in cancer, identifying PML-RARA fusions in the NB4 cell line in <15 h sequencing. These methods can be used to efficiently screen any target panel of genes without specialized sample preparation using any computer and a suitable GPU. Our toolkit, readfish, is available at https://www.github.com/looselab/readfish .

Incidence and Management of Hypertriglyceridemia-Associated Acute Pancreatitis: A Prospective Case Series in a Single Australian Tertiary Centre.

BACKGROUND: Hypertriglyceridemia-associated acute pancreatitis (HTGAP) has been linked with increased severity and morbidity. In this study, triglyceride levels were measured in all patients admitted with acute pancreatitis (AP) to establish the incidence of HTGAP in an Australian center. METHODS: A prospective cohort with AP was collated over an 18-month period in a single tertiary referral hospital. HTGAP was defined as AP with triglycerides ≥ 11.2 mmol/L (1000 mg/dL). Incidence, clinical co-morbidities, severity and management strategies were recorded. RESULTS: Of the 292 episodes of AP, 248 (85%) had triglycerides measured and were included. HTGAP was diagnosed in 10 of 248 (4%) AP cases. Type 2 diabetes, obesity, alcohol misuse and gallstones were common cofactors. The HTGAP group demonstrated severe hypertriglyceridemia compared to the non-HTGAP group (median 51 mmol/L vs. 1.3 mmol/L). Intensive care unit (ICU) admissions were significantly increased (odds ratio (OR) 16; 95% CI 4-62) in the HTGAP group (5/10 vs. 14/238 admissions, p < 0.001) and constituted 26% (5/19) of total ICU admissions for AP. Four patients received intravenous insulin with fasting and had a rapid reduction in triglyceride levels by 65-77% within 24 h; one patient had mild hypoglycemia secondary to therapy. CONCLUSION: HTGAP occurred in 4% of AP cases and was associated with higher risk of ICU admission. Intravenous insulin and fasting appear safe and efficacious for acutely lowering triglyceride levels in HTGAP.

Making the invisible enemy visible.

During the COVID-19 pandemic, structural biologists rushed to solve the structures of the 28 proteins encoded by the SARS-CoV-2 genome in order to understand the viral life cycle and enable structure-based drug design. In addition to the 204 previously solved structures from SARS-CoV-1, 548 structures covering 16 of the SARS-CoV-2 viral proteins have been released in a span of only 6 months. These structural models serve as the basis for research to understand how the virus hijacks human cells, for structure-based drug design, and to aid in the development of vaccines. However, errors often occur in even the most careful structure determination - and may be even more common among these structures, which were solved quickly and under immense pressure. The Coronavirus Structural Task Force has responded to this challenge by rapidly categorizing, evaluating and reviewing all of these experimental protein structures in order to help downstream users and original authors. In addition, the Task Force provided improved models for key structures online, which have been used by Folding@Home, OpenPandemics, the EU JEDI COVID-19 challenge and others.

Author Correction: Nanopore native RNA sequencing of a human poly(A) transcriptome.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.