We sequenced sarbecovirus from Rhinolophus spp. bats in Bandarban District, Bangladesh, in a genomic surveillance campaign during 2022-2023. Sequences shared identity with SARS-CoV-1 Tor2, which caused an outbreak of human illnesses in 2003. Describing the genetic diversity and zoonotic potential of reservoir pathogens can aid in identifying sources of future spillovers.

In this work we identify changes in high-resolution zones across the globe linked by environmental similarity that have implications for agriculture, bioenergy, and zoonosis. We refine exhaustive vector comparison methods with improved similarity metrics as well as provide multiple methods of amalgamation across 744 months of climatic data. The results of the vector comparison are captured as networks which are analyzed using static and longitudinal comparison methods to reveal locations around the globe experiencing dramatic changes in abiotic stress. Specifically we (i) incorporate updated similarity scores and provide a comparison between similarity metrics, (ii) implement a new feature for resource optimization, (iii) compare an agglomerative view to a longitudinal view, (iv) compare across 2-way and 3-way vector comparisons, (v) implement a new form of analysis, and (vi) demonstrate biological applications and discuss implications across a diverse set of species distributions by detecting changes that affect their habitats. Species of interest are related to agriculture (e.g., coffee, wine, chocolate), bioenergy (e.g., poplar, switchgrass, pennycress), as well as those living in zones of concern for zoonotic spillover that may lead to pandemics (e.g., eucalyptus, flying foxes).

Current methods for defining SARS-CoV-2 lineages ignore the vast majority of the SARS-CoV-2 genome. We develop and apply an exhaustive vector comparison method that directly compares all known SARS-CoV-2 genome sequences to produce novel lineage classifications. We utilize data-driven models that (i) accurately capture the complex interactions across the set of all known SARS-CoV-2 genomes, (ii) scale to leadership-class computing systems, and (iii) enable tracking how such strains evolve geospatially over time. We show that during the height of the original Omicron surge, countries across Europe, Asia, and the Americas had a spatially asynchronous distribution of Omicron sub-strains. Moreover, neighboring countries were often dominated by either different clusters of the same variant or different variants altogether throughout the pandemic. Analyses of this kind may suggest a different pattern of epidemiological risk than was understood from conventional data, as well as produce actionable insights and transform our ability to prepare for and respond to current and future biological threats.

A major challenge in the field of metagenomics is the selection of the correct combination of sequencing platform and downstream metagenomic analysis algorithm, or ‘classifier’. Here, we present the Metagenomic Evaluation Tool Analyzer (META), which produces simulated data and facilitates platform and algorithm selection for any given metagenomic use case. META-generated in silico read data are modular, scalable, and reflect user-defined community profiles, while the downstream analysis is done using a variety of metagenomic classifiers. Reported results include information on resource utilization, time-to-answer, and performance. Real-world data can also be analyzed using selected classifiers and results benchmarked against simulations. To test the utility of the META software, simulated data was compared to real-world viral and bacterial metagenomic samples run on four different sequencers and analyzed using 12 metagenomic classifiers. Lastly, we introduce ‘META Score’: a unified, quantitative value which rates an analytic classifier’s ability to both identify and count taxa in a representative sample.

In situ sampling missions to detect biosignatures on ocean worlds requires thorough sample preparation to manage the expected chemical complexity of such environments. Proposed instruments must be capable of automatic liquid sample handling to ensure sensitive and accurate detections of biosignatures, regardless of the initial chemical composition. Herein, we outline the design, build, and test of the integrated Biosignature Preparation for Ocean Worlds (BioPOW) system capable of purifying amino acids from icy samples. This four step modular instrument 1) melts ice samples, 2) purifies amino acids via cation exchange chromatography, 3) concentrates via vacuum drying, and 4) derivatizes amino acids to volatilize and enable detection with downstream analytical instruments. Initial experiments validated the thermal performance of the system by melting ice in the sample cup (1 mL sample, 3°C–28°C, <5 min, 1.4 kJ) and heating the derivatization tank past the concentration temperature (20°C–60°C, 12 min, 3.6 kJ) to the derivatization temperature (60°C–90°C, 25 min, 7.5 kJ). Later experiments investigated important factors for automatic cation exchange using a design of experiments approach, and found that initial salt concentration, sample and eluate flow rates, and water wash volumes all play significant roles in reducing conductivity (1.1 x–6.7 x) while maintaining phenylalanine yields between 31% and 94%. The modules were then integrated into a 12 cm × 20 cm × 20 cm fieldable platform for analysis, and the maturation of this design for future spaceflight is discussed.

Increasing interest in the detection of biogenic signatures, such as amino acids, on icy moons and bodies within our solar system has led to the development of compact in situ instruments. Given the expected dilute bio-signatures and high salinities of these extreme environments, purification of icy samples before analysis enables increased detection sensitivity. Herein, we outline a novel compact cation exchange method to desalinate proteinogenic amino acids in solution, independent of the type and concentration of salts in the sample. Using a modular microfluidic device, initial experiments explored operational limits of binding capacity with phenyl-alanine and three model cations, Na+, Mg2+, and Ca2+. Phenylalanine recovery (94-17%) with reduced conductivity (30-200 times) was seen at high salt-to-amino-acid ratios between 25:1 and 500:1. Later experiments tested competition between mixtures of 17 amino acids and other chemistries present in a terrestrial ocean sample. Recoveries ranged from 11% to 85% depending on side chain chemistry and cation competition, with concentration shown for select high affinity amino acids. This work outlines a nondestructive amino acid purification device capable of coupling to multiple downstream analytical techniques for improved characterization of icy samples at remote ocean worlds.

Enceladus’s long-lived plume of ice grains and water vapor makes accessing oceanic material readily achievable from orbit (around Saturn or Enceladus) and from the moon’s surface. In preparation for the National Academies of Sciences, Engineering and Medicine 2023–2032 Planetary Science and Astrobiology Decadal Survey, we investigated four architectures capable of collecting and analyzing plume material from orbit and/or on the surface to address the most pressing questions at Enceladus: Is the subsurface ocean inhabited? Why, or why not? Trades specific to these four architectures were studied to allow an evaluation of the science return with respect to investment. The team found that Orbilander, a mission concept that would first orbit and then land on Enceladus, represented the best balance. Orbilander was thus studied at a higher fidelity, including a more detailed science operations plan during both orbital and landed phases, landing site characterization and selection analyses, and landing procedures. The Orbilander mission concept demonstrates that scientifically compelling but resource-conscious Flagship-class missions can be executed in the next decade to search for life at Enceladus.

Reference genome fidelity is critically important for genome wide association studies, yet most vary widely from the study population. A typical whole genome sequencing approach implies short-read technologies resulting in fragmented assemblies with regions of ambiguity. Further information is lost by economic necessity when genotyping populations, as lower resolution technologies such as genotyping arrays are commonly used. Here, we present a phased reference genome for Canis lupus familiaris using high molecular weight DNA-sequencing technologies. We tested wet laboratory and bioinformatic approaches to demonstrate a minimum workflow to generate the 2.4 gigabase genome for a Labrador Retriever. The de novo assembly required eight Oxford Nanopore R9.4 flowcells (∼23X depth) and running a 10X Genomics library on the equivalent of one lane of an Illumina NovaSeq S1 flowcell (∼88X depth), bringing the cost of generating a nearly complete reference genome to less than $10K (USD). Mapping of short-read data from 10 Labrador Retrievers against this reference resulted in 1% more aligned reads versus the current reference (CanFam3.1, P < 0.001), and a 15% reduction of variant calls, increasing the chance of identifying true, low-effect size variants in a genome-wide association studies. We believe that by incorporating the cost to produce a full genome assembly into any large-scale genotyping project, an investigator can improve study power, decrease costs, and optimize the overall scientific value of their study.

ABSTRACTBackgroundMonoclonal antibodies (mAbs) against SARS-CoV-2 are a promising treatment for limiting the progression of COVID-19 and decreasing strain on hospitals. Their use, however, remains limited, particularly in disadvantaged populations.MethodsElectronic health records were reviewed from SARS-CoV-2 patients at a single medical center in the United States that initiated mAb infusions in January 2021 with the support of the U.S. Department of Health and Human Services’ National Disaster Medical System. Patients who received mAbs were compared to untreated patients from the time period before mAb availability who met eligibility criteria for mAb treatment. We used logistic regression to measure the effect of mAb treatment on the risk of hospitalization or emergency department (E.D.) visit within 30 days of laboratory-confirmed COVID-19.ResultsOf 598 COVID-19 patients, 270 (45%) received bamlanivimab and 328 (55%) were untreated. Two hundred and thirty-one patients (39%) were Hispanic. Among treated patients, 5/270 (1.9%) presented to the E.D. or required hospitalization within 30 days of a positive SARS-CoV-2 test, compared to 39/328 (12%) untreated patients (p<0.001). After adjusting for age, gender, and comorbidities, the risk of E.D. visit or hospitalization was 82% lower in mAb-treated patients compared to untreated patients (95% confidence interval [CI]: 66%-94%).ConclusionsIn this diverse, real-world COVID-19 patient population, mAb treatment significantly decreased the risk of subsequent E.D. visit or hospitalization. Broader treatment with mAbs, including in disadvantaged patient populations, can decrease the burden on hospitals and should be facilitated in all populations in the United States to ensure health equity.SummaryIn a diverse, real-world COVID-19 patient population, treatment with monoclonal antibodies significantly decreased the risk of subsequent emergency department visit or hospitalization within 30 days of a positive SARS-CoV-2 viral test.

Astrobiology is an exciting field of science focused on understanding the origins, evolution, distribution, and future of life in the universe. NASA focuses much of its research and technology developments on astrobiology, and the Johns Hopkins University Applied Physics Laboratory (APL) is a major contributor through research, technology, and missions. Astrobiology efforts at APL range from constraining when life first emerged on Earth and researching biosignature (i.e., signals of past or present life) preservation, to developing instruments and missions aiming to detect biosignatures and characterize the capability of an extreme planetary environment to harbor and support life. Beginning with APL's Compact Reconnaissance Imaging Spectrometer for Mars (CRISM) on the Mars Reconnaissance Orbiter (MRO), which searches for past wet and potentially habitable regions on Mars, APL has continued to develop cutting-edge techniques and instruments to search for biosignatures, remotely and in situ. Additionally, APL is leading and serving as a key partner in several exciting NASA missions that will occur in the coming decades with habitability and biosignature detection goals. In this article, we summarize current efforts and look forward, over the coming 25 years, to the potential astrobiology exploration and discoveries that await.

Abstract Background The state-of-the-art in nucleic acid based biodetection continues to be polymerase chain reaction (PCR), and many real-time PCR assays targeting biodefense pathogens for biosurveillance are in widespread use. These assays are predominantly singleplex; i.e. one assay tests for the presence of one target, found in a single organism, one sample at a time. Due to the intrinsic limitations of such tests, there exists a critical need for high-throughput multiplex assays to reduce the time and cost incurred when screening multiple targets, in multiple pathogens, and in multiple samples. Such assays allow users to make an actionable call while maximizing the utility of the small volumes of test samples. Unfortunately, current multiplex real-time PCR assays are limited in the number of targets that can be probed simultaneously due to the availability of fluorescence channels in real-time PCR instruments. Results To address this gap, we developed a pipeline in which the amplicons produced by a 14-plex end-point PCR assay using spiked samples were subsequently sequenced using Nanopore technology. We used bar codes to sequence multiple samples simultaneously, leading to the generation and subsequent analysis of sequence data resulting from a short sequencing run time (< 10 min). We compared the limits of detection (LoD) of real-time PCR assays to Oxford Nanopore Technologies (ONT)-based amplicon sequencing and estimated the sample-to-answer time needed for this approach. Overall, LoDs determined from the first 10 min of sequencing data were at least one to two orders of magnitude lower than real-time PCR. Given enough time, the amplicon sequencing approach is approximately 100 times more sensitive than real-time PCR, with detection of amplicon specific reads even at the lowest tested spiking concentration (around 2.5–50 Colony Forming Units (CFU)/ml). Conclusions Based on these results, we propose amplicon sequencing assay as a viable alternative to replace the current real-time PCR based singleplex assays for higher throughput biodefense applications. We note, however, that targeted amplicon specific reads were not detectable even at the highest tested spike concentrations (2.5 X 104–5.0 X105 CFU/ml) without an initial amplification step, indicating that PCR is still necessary when utilizing this protocol.

Precision medicine shares many goals with environmental health medicine. Genetics and environment contribute to risks for most chronic diseases, and many genome-wide association studies (GWAS) have been done to establish their genomic components. As these fields converge, there is opportunity to combine quantitative risks and leverage phenotypical measurements to better define both genomic and environmental components. Importantly, the improving technology of genomic assessment tools means that older GWAS could be improved and new variants and DNA-based biomarkers could be discovered. The military maintains resources for tracking exposures and large-scale populations that can be used for better genotype, to phenotype, to environmental source discovery and characterization. The clinical translational framework being developed by national precision medicine efforts, and the ethical policy considerations for military service members that have been established in recent years, provide a roadmap to combine clinical useful aspects of these two different fields.

ABSTRACT Reference genome fidelity is critically important for genome wide association studies (GWAS), yet many are incomplete or too dissimilar from the study population. A typical whole genome sequencing approach implies short-read technologies resulting in fragmented assemblies with regions of ambiguity low complexity. Further information is lost by economic necessity when genotyping populations, as lower resolution technologies such as genotyping arrays are commonly utilized. Here we present a phased reference genome for Canis lupus familiaris utilizing high molecular weight sequencing technologies. We tested wet lab and bioinformatic approaches to demonstrate a minimum workflow to generate the 2.4 gigabase genome for a Labrador Retriever. The resulting de novo assembly required eight Oxford Nanopore R9.4 flowcells (~23X depth) and running a 10X Genomics library on the equivalent of one lane of an Illumina NovaSeq S1 flowcell (~88X depth), bringing the cost of generating a nearly complete reference genome to less than $10K. Mapping of publicly available short-read data from ten Labrador Retrievers against this breed-specific reference resulted in an average of approximately 1% more aligned reads compared to mapping against the current gold standard reference (CanFam3.1, p<0.001), indicating a more complete breed-specific reference. An average 15% reduction of variant calls was observed from the same mapped data, which increases the chance of identifying low effect size variants in a GWAS. We believe that by incorporating the cost to produce a full genome assembly into any large-scale canine genotyping study, an investigator can make an informed cost/benefit analysis regarding genotyping technology.

Introduction: Many challenges exist for in situ techniques searching for signatures of life in planetary environments. Whether exploring the ocean worlds Europa and Enceladus or the dunes of Mars, collected samples are likely to contain high salinity and are expected to have very low biomass, if present. These environmental factors would act as significant challenges to the instruments/techniques currently used to analyze in situ samples for biosignatures (e.g. amino acids and their chirality, or DNA/RNA) and may include mass spectrometers, fluorescent-based optics, and nanopore sequencers [1]. To surmount these challenges, we are developing a robust sample preparation method to desalinate proteinogenic amino acids, independent of type and concentration of salt in the sample on a microfluidic chip. This technique can be used as a single purification tool for multiple downstream biosignature detection analyses. Though similar methods have been used to desalinate meteorite samples on Earth [2], this has yet to be developed as an in situ space flight instrument. Methods: Here we describe the separation achieved using a modular microfluidic chip (Figure 1) that allows for non-destructive sample preparation to remove ionic interference, minimize loss of amino acids, and increase downstream detection sensitivity. Salts and amino acids were chosen as planetary analogs for environments of icy ocean worlds such as Europa and Enceladus [3, 4] and the amino acids used by terrestrial biology [5]. For the experiments, we introduced solutions of one salt (NaCl, CaCl2, or MgSO4) and an aromatic amino acid (phenylalanine).