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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 127 records · Page 7
All sourcesNASA NTRSDOE OSTI
DOE OSTI2026

Spatio-temporal dynamics of Hendra virus in Australia reveal stable maintenance of diverse viral clades among Pteropus bats

Hendra virus (HeV) was discovered in 1994 in Australia. Limited genomic data have hindered comprehensive understanding of HeV’s evolutionary dynamics. Here, in this work, we recovered 48 HeV genomes from bats and 9 from horses from Australia between 2016 and 2020, revealing four distinct clades. Each clade was distributed over a large spatial area with multiple clades co-circulating within a single bat roost on the same day and over consecutive years. The diversity and temporal stability of co-circulating clades suggest that viral dynamics are driven by episodic shedding of existing lineages maintained at the population level, rather than immune-driven strain-replacement dynamics. HeV isolates of different clades displayed variation in phenotypic properties but minimal antigenic differences. We provide an overview of evolutionary dynamics, phenotypic properties and assessment of countermeasures for HeV, and provide insights into the processes that maintain virus diversity in bats and influence the potential for viral emergence.

genetic variation↗
DOE OSTI2021

Conservation of endo -glucanase 16 (EG16) activity across highly divergent plant lineages

Plant cell walls are highly dynamic structures that are composed predominately of polysaccharides. As such, endogenous carbohydrate active enzymes (CAZymes) are central to the synthesis and subsequent modification of plant cells during morphogenesis. The endo-glucanase 16 (EG16) members constitute a distinct group of plant CAZymes, angiosperm orthologs of which were recently shown to have dual β-glucan/xyloglucan hydrolase activity. Molecular phylogeny indicates that EG16 members comprise a sister clade with a deep evolutionary relationship to the widely studied apoplastic xyloglucan endo-transglycosylases/hydrolases (XTH). A cross-genome survey indicated that EG16 members occur as a single ortholog across species and are widespread in early diverging plants, including the non-vascular bryophytes, for which functional data were previously lacking. Remarkably, enzymological characterization of an EG16 ortholog from the model moss Physcomitrella patens (PpEG16) revealed that EG16 activity and sequence/structure are highly conserved across 500 million years of plant evolution, vis-à-vis orthologs from grapevine and poplar. Ex vivo biomechanical assays demonstrated that the application of EG16 gene products caused abrupt breakage of etiolated hypocotyls rather than slow extension, thereby indicating a mode-of-action distinct from endogenous expansins and microbial endo-glucanases. As a result, the biochemical data presented here will inform future genomic, genetic, and physiological studies of EG16 enzymes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Representing plant diversity in land models: An evolutionary approach to make “Functional Types” more functional

Plants are critical mediators of terrestrial mass and energy fluxes, and their structural and functional traits have profound impacts on local and global climate, biogeochemistry, biodiversity, and hydrology. Yet, Earth System Models (ESMs), our most powerful tools for predicting the effects of humans on the coupled biosphere-atmosphere system, simplify the incredible diversity of land plants into a handful of coarse categories of "Plant Functional Types" (PFTs) that often fail to capture ecological dynamics such as biome distributions. The inclusion of more realistic functional diversity is a recognized goal for ESMs, yet there is currently no consistent, widely accepted way to add diversity to models, that is, to determine what new PFTs to add and with what data to constrain their parameters. Here we review approaches to representing plant diversity in ESMs and draw on recent ecological and evolutionary findings to present an evolution-based functional type approach for further disaggregating functional diversity. Specifically, the prevalence of niche conservatism, or the tendency of closely related taxa to retain similar ecological and functional attributes through evolutionary time, reveals that evolutionary relatedness is a powerful framework for summarizing functional similarities and differences among plant types. We advocate that Plant Functional Types based on dominant evolutionary lineages ("Lineage Functional Types") will provide an ecologically defensible, tractable, and scalable framework for representing plant diversity in next-generation ESMs, with the potential to improve parameterization, process representation, and model benchmarking. We highlight how the importance of evolutionary history for plant function can unify the work of disparate fields to improve predictive modeling of the Earth system.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Inferring Viral Transmission Time from Phylogenies for Known Transmission Pairs

When the time of an HIV transmission event is unknown, methods to identify it from virus genetic data can reveal the circumstances that enable transmission. We developed a single-parameter Markov model to infer transmission time from an HIV phylogeny constructed of multiple virus sequences from people in a transmission pair. Our method finds the statistical support for transmission occurring in different possible time slices. We compared our time-slice model results to previously described methods: a tree-based logical transmission interval, a simple parsimony-like rules-based method, and a more complex coalescent model. Across simulations with multiple transmitted lineages, different transmission times relative to the source’s infection, and different sampling times relative to transmission, we found that overall our time-slice model provided accurate and narrower estimates of the time of transmission. We also identified situations when transmission time or direction was difficult to estimate by any method, particularly when transmission occurred long after the source was infected and when sampling occurred long after transmission. Applying our model to real HIV transmission pairs showed some agreement with facts known from the case investigations. We also found, however, that uncertainty on the inferred transmission time was driven more by uncertainty from time calibration of the phylogeny than from the model inference itself. Encouragingly, comparable performance of the Markov time-slice model and the coalescent model—which make use of different information within a tree—suggests that a new method remains to be described that will make full use of the topology and node times for improved transmission time inference.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

GENESPACE R Package (GENESPACE) v1.0

In short, the GENESPACE pipeline conducts analysis of orthology networks, constrained within syntenic regions. Since analyses are limited to local tests conducted within syntenic blocks, GENESPACE is agnostic to ploidy, duplicated regions, inversions or other whole-genome chromosomal complexities that are common across many evolutionary lineages. This advantage allows for evolutionary tests in polyploids (e.g. switchgrass, manuscript in review), species with ancient, but retained whole-genome duplications (e.g. pecan, manuscript in prep), high levels of tandem array proliferation (e.g. eukalypts, manuscript in review) and many other factors that can confound comparative genomic analyses. The major advances of GENESPACE are three-fold: First, this is the first R package to integrate visualization and analysis of large-scale comparative genomics. R, which offers a high-level environment for graphical and statistical exploration of data, is often speed- and memory-limited and not used for computationally intensive tasks such as comparative genomics. The highly efficient C++ scripts used in GENESPACE (via data.table) permit a much faster and computationally lightweight implementation of comparative genomics than is currently available. Second, the pipeline itself is novel. To the best of our knowledge, no other program accomplishes synteny-constrained and ploidy-agnostic comparative genomics. Since nearly all plants and many animals have a history of whole-genome duplications, this is a major and necessary advance to the field. Third, GENESPACE offers high-level and intuitive multi-genome graphical outputs. The dotplots and 'riparian' plots produced herein, which are produced entirely through original R code, are publication-ready and easily customizable.

Schmutz, Jeremy↗
DOE OSTI2022

Predominance of Methanomicrobiales and diverse hydrocarbon–degrading taxa in the Appalachian coalbed biosphere revealed through metagenomics and genome–resolved metabolisms

Coalbed deposits are a unique subsurface environment and represent an underutilized resource for methane generation. Microbial communities extant in coalbed deposits are responsible for key subsurface biogeochemical cycling and could be utilized to enhance methane production in areas where existing gas wells have depleted methane stores, or in coalbeds that are unmined, or conversely be utilized for mitigation of methane release. Here we utilize metagenomics and metagenome-assembled genomes to identify extant microbial lineages and genome-resolved microbial metabolisms of coalbed produced water, which has not yet been explored in the Appalachian Basin. Our analyses resulted in the recovery of over 40 metagenome-assembled genomes (MAGs) from eight coalbed methane wells. The most commonly identified taxa among samples were hydrogenotrophic methanogens from the order Methanomicrobiales and these dominant MAGs were highly similar to one another. Conversely, low-abundance coalbed bacterial populations were taxonomically and functionally diverse, mostly belonging to a variety of Proteobacteria classes, and encoding various hydrocarbon solubilization and degradation pathways. Further, the data presented herein provides novel insights into Appalachian Basin coalbed microbial ecology, and our findings provide new perspectives on underrepresented Methanocalculus species and low-relative abundance bacterial assemblages in coalbed environments, and their potential roles in stimulation or mitigation of methane release.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

An evaluation of methodology to determine algal genome completeness

The advancement of sequencing technologies has resulted in a rapid expansion of genome sequencing. One challenge in processing and analyzing this abundance of genomics data is development and application of tools to accurately assess the quality of novel genomes. The quality of a given genome assembly is commonly assessed using the presence of conserved orthologs. However, the applicability and accuracy of identifying conserved orthologs applied to algal genome assemblies, which are uniquely diverse, is unclear. Here, in this study, we analyze the utility of a genome content assessment tool, Benchmarking Universal Single-Copy Orthologs (BUSCO), to evaluate algal genome assembly completeness. We find that Chlorophyta and Stramenopile BUSCO databases are effective tools to analyze genome completeness of these lineages. For other lineages, the Eukaryota database should be utilized until additional lineage-specific databases are developed. Based on these results, suggested best practices for current usage and necessary future improvements are provided.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Phylogenomics and the first higher taxonomy of Placozoa, an ancient and enigmatic animal phylum

Placozoa is an ancient phylum of extraordinarily unusual animals: miniscule, ameboid creatures that lack most fundamental animal features. Despite high genetic diversity, only recently have the second and third species been named. While prior genomic studies suffer from incomplete placozoan taxon sampling, we more than double the count with protein sequences from seven key genomes and produce the first nuclear phylogenomic reconstruction of all major placozoan lineages. This leads us to the first complete Linnaean taxonomic classification of Placozoa, over a century after its discovery: This may be the only time in the 21st century when an entire higher taxonomy for a whole animal phylum is formalized. Our classification establishes 2 new classes, 4 new orders, 3 new families, 1 new genus, and 1 new species, namely classes Polyplacotomia and Uniplacotomia; orders Polyplacotomea, Trichoplacea, Cladhexea, and Hoilungea; families Polyplacotomidae, Cladtertiidae, and Hoilungidae; and genus Cladtertia with species Cladtertia collaboinventa, nov. Our likelihood and gene content tree topologies refine the relationships determined in previous studies. Adding morphological data into our phylogenomic matrices suggests sponges (Porifera) as the sister to other animals, indicating that modest data addition shifts this node away from comb jellies (Ctenophora). Furthermore, by adding the first genomic protein data of the exceptionally distinct and branching Polyplacotoma mediterranea , we solidify its position as sister to all other placozoans; a divergence we estimate to be over 400 million years old. Yet even this deep split sits on a long branch to other animals, suggesting a bottleneck event followed by diversification. Ancestral state reconstructions indicate large shifts in gene content within Placozoa, with Hoilungia hongkongensis and its closest relatives having the most unique genetics.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2022

Phylogenomic analysis of the hemp family (Cannabaceae) reveals deep cyto‐nuclear discordance and provides new insights into generic relationships

Abstract Cannabaceae are a relatively small family of angiosperms, but they include several species of huge economic and cultural significance: marijuana or hemp ( Cannabis sativa ) and hops ( Humulus lupulus ). Previous phylogenetic studies have clarified the most deep relationships in Cannabaceae, but relationships remain ambiguous among several major lineages. Here, we sampled 82 species representing all genera of Cannabaceae and utilized a new dataset of 90 nuclear genes and 82 chloroplast loci from Hyb‐Seq to investigate the phylogenomics of Cannabaceae. Nuclear phylogenetic analyses revealed a robust and consistent backbone for Cannabaceae. We observed nuclear gene‐tree conflict at several deep nodes in inferred species trees, also cyto‐nuclear discordance concerning the relationship between Gironniera and Lozanella and the relationships among Trema s.l. (including Parasponia ), Cannabis + Humulus , and Chaetachme + Pteroceltis . Coalescent simulations and network analyses suggest that observed deep cyto‐nuclear discordances were most likely to stem from incomplete lineage sorting (ILS); nuclear gene‐tree conflict might be caused by both ILS and gene flow between species. All genera of Cannabaceae were recovered as monophyletic, except for Celtis , which consisted of two distinct clades: Celtis I (including most Celtis species) and Celtis II (including Celtis gomphophylla and Celtis schippii ). We suggest that Celtis II should be recognized as the independent genus Sparrea based on both molecular and morphological evidence. Our work provides the most comprehensive and reliable phylogeny to date for Cannabaceae, enabling further exploration of evolutionary patterns across this family and highlighting the necessity of comparing nuclear with chloroplast data to examine the evolutionary history of plant groups.

Plant Sciences↗
DOE OSTI2024

Detectability of Varied Hybridization Scenarios Using Genome-Scale Hybrid Detection Methods

Hybridization events complicate the accurate reconstruction of phylogenies, as they lead to patterns of genetic heritability that are unexpected under traditional, bifurcating models of species trees. This phenomenon has led to the development of methods to infer these varied hybridization events, both methods that reconstruct networks directly, as well as summary methods that predict individual hybridization events from a subset of taxa. However, a lack of empirical comparisons between methods – especially those pertaining to large networks with varied hybridization scenarios – hinders their practical use. Here, we provide a comprehensive review of popular summary methods: TICR, MSCquartets, HyDe, Patterson’s D-Statistic (ABBA-BABA), D3, and Dp. TICR and MSCquartets are based on quartet concordance factors gathered from gene tree topologies and HyDe, Patterson’s D-Statistic, D3, and Dp use site pattern frequencies to identify hybridization events between sets of three taxa. We then use simulated data to address questions of method accuracy and ideal use scenarios by testing methods against complex networks which depict gene flow events that differ in depth (timing), quantity (single vs. multiple, overlapping hybridizations), and rate of gene flow (γ). We find that deeper or multiple hybridization events may introduce noise and weaken the signal of hybridization, leading to higher relative false negative rates across all methods. Despite some forms of hybridization eluding quartet-based detection methods, MSCquartets displays high precision in most scenarios. While HyDe results in high false negative rates when tested on hybridizations involving extinct or unsampled ghost lineages, HyDe is the only method able to identify the direction of hybridization, distinguishing the source parental lineages from recipient hybrid lineages. Lastly, we test the methods on a dataset of ultraconserved elements from the bee subfamily Nomiinae, finding possible hybridization events between clades which correspond to regions of poor support in the species tree estimated in a previous study.

Bjorner, Marianne B.↗
DOE OSTI2022

Single-nuclei transcriptome analysis of the shoot apex vascular system differentiation in Populus

ABSTRACT Differentiation of stem cells in the plant apex gives rise to aerial tissues and organs. Presently, we lack a lineage map of the shoot apex cells in woody perennials – a crucial gap considering their role in determining primary and secondary growth. Here, we used single-nuclei RNA-sequencing to determine cell type-specific transcriptomes of the Populus vegetative shoot apex. We identified highly heterogeneous cell populations clustered into seven broad groups represented by 18 transcriptionally distinct cell clusters. Next, we established the developmental trajectories of the epidermis, leaf mesophyll and vascular tissue. Motivated by the high similarities between Populus and Arabidopsis cell population in the vegetative apex, we applied a pipeline for interspecific single-cell gene expression data integration. We contrasted the developmental trajectories of primary phloem and xylem formation in both species, establishing the first comparison of vascular development between a model annual herbaceous and a woody perennial plant species. Our results offer a valuable resource for investigating the principles underlying cell division and differentiation conserved between herbaceous and perennial species while also allowing us to examine species-specific differences at single-cell resolution.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Genome-scale phylogenetic analyses confirm Olpidium as the closest living zoosporic fungus to the non-flagellated, terrestrial fungi

Abstract The zoosporic obligate endoparasites, Olpidium, hold a pivotal position to the reconstruction of the flagellum loss in fungi, one of the key morphological transitions associated with the colonization of land by the early fungi. We generated genome and transcriptome data from non-axenic zoospores of Olpidium bornovanus and used a metagenome approach to extract phylogenetically informative fungal markers. Our phylogenetic reconstruction strongly supported Olpidium as the closest zoosporic relative of the non-flagellated terrestrial fungi. Super-alignment analyses resolved Olpidium as sister to the non-flagellated terrestrial fungi, whereas a super-tree approach recovered different placements of Olpidium, but without strong support. Further investigations detected little conflicting signal among the sampled markers but revealed a potential polytomy in early fungal evolution associated with the branching order among Olpidium , Zoopagomycota and Mucoromycota. The branches defining the evolutionary relationships of these lineages were characterized by short branch lengths and low phylogenetic content and received equivocal support for alternative phylogenetic hypotheses from individual markers. These nodes were marked by important morphological innovations, including the transition to hyphal growth and the loss of flagellum, which enabled early fungi to explore new niches and resulted in rapid and temporally concurrent Precambrian diversifications of the ancestors of several phyla of fungi.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Antigenic Characterization and Pandemic Risk Assessment of North American H1 Influenza A Viruses Circulating in Swine

The first pandemic of the 21st century was caused by an H1N1 influenza A virus (IAV) introduced from pigs into humans, highlighting the importance of swine as reservoirs for pandemic viruses. Two major lineages of swine H1 circulate in North America: the 1A classical swine lineage (including that of the 2009 H1N1 pandemic) and the 1B human seasonal-like lineage. Here, we investigated the evolution of these H1 IAV lineages in North American swine and their potential pandemic risk. We assessed the antigenic distance between the HA of representative swine H1 and human seasonal vaccine strains (1978 to 2015) in hemagglutination inhibition (HI) assays using a panel of monovalent antisera raised in pigs. Antigenic cross-reactivity varied by strain but was associated with genetic distance. Generally, the swine 1A lineage viruses that seeded the 2009 H1 pandemic were antigenically most similar to the H1 pandemic vaccine strains, with the exception of viruses in the genetic clade 1A.1.1.3, which had a two-amino acid deletion mutation near the receptor-binding site, which dramatically reduced antibody recognition. The swine 1B lineage strains, which arose from previously circulating (pre-2009 pandemic) human seasonal viruses, were more antigenically similar to pre-2009 human seasonal H1 vaccine viruses than post-2009 strains. Human population immunity was measured by cross-reactivity in HI assays to representative swine H1 strains. There was a broad range of titers against each swine strain that was not associated with age, sex, or location. However, there was almost no cross-reactivity in human sera to the 1A.1.1.3 and 1B.2.1 genetic clades of swine viruses, and the 1A.1.1.3 and 1B.2.1 clades were also the most antigenically distant to the human vaccine strains. Our data demonstrate that the antigenic distances of representative swine strains from human vaccine strains represent an important part of the rational assessment of swine IAV for zoonotic risk research and pandemic preparedness prioritization.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Plant microbiomes harbor potential to promote nutrient turnover in impoverished substrates of a Brazilian biodiversity hotspot

The substrates of the Brazilian campos rupestres, a grassland ecosystem, have extremely low concentrations of phosphorus and nitrogen, imposing restrictions to plant growth. Despite that, this ecosystem harbors almost 15% of the Brazilian plant diversity, raising the question of how plants acquire nutrients in such a harsh environment. Here, we set out to uncover the taxonomic profile, the compositional and functional differences and similarities, and the nutrient turnover potential of microbial communities associated with two plant species of the campos rupestres-dominant family Velloziaceae that grow over distinct substrates (soil and rock). Using amplicon sequencing data, we show that, despite the pronounced composition differentiation, the plant-associated soil and rock communities share a core of highly efficient colonizers that tend to be highly abundant and is enriched in 21 bacterial families. Functional investigation of metagenomes and 522 metagenome-assembled genomes revealed that the microorganisms found associated to plant roots are enriched in genes involved in organic compound intake, and phosphorus and nitrogen turnover. We show that potential for phosphorus transport, mineralization, and solubilization are mostly found within bacterial families of the shared microbiome, such as Xanthobacteraceae and Bryobacteraceae. We also detected the full repertoire of nitrogen cycle-related genes and discovered a lineage of Isosphaeraceae that acquired nitrogen-fixing potential via horizontal gene transfer and might be also involved in nitrification via a metabolic handoff association with Binataceae. We highlight that plant-associated microbial populations in the campos rupestres harbor a genetic repertoire with potential to increase nutrient availability and that the microbiomes of biodiversity hotspots can reveal novel mechanisms of nutrient turnover.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Integrating Intermediate Traits in Phylogenetic Genotype-to-Phenotype Studies

A major goal of research in evolution and genetics is linking genotype to phenotype. This work could be direct, such as determining the genetic basis of a phenotype by leveraging genetic variation or divergence in a developmental, physiological, or behavioral trait. The work could also involve studying the evolutionary phenomena (e.g., reproductive isolation, adaptation, sexual dimorphism, behavior) that reveal an indirect link between genotype and a trait of interest. When the phenotype diverges across evolutionarily distinct lineages, this genotype-to-phenotype problem can be addressed using phylogenetic genotype-to-phenotype (PhyloG2P) mapping, which uses genetic signatures and convergent phenotypes on a phylogeny to infer the genetic bases of traits. The PhyloG2P approach has proven powerful in revealing key genetic changes associated with diverse traits, including the mammalian transition to marine environments and transitions between major mechanisms of photosynthesis. However, there are several intermediate traits layered in between genotype and the phenotype of interest, including but not limited to transcriptional profiles, chromatin states, protein abundances, structures, modifications, metabolites, and physiological parameters. Each intermediate trait is interesting and informative in its own right, but synthesis across data types has great promise for providing a deep, integrated, and predictive understanding of how genotypes drive phenotypic differences and convergence. We argue that an expanded PhyloG2P framework (the PhyloG2P matrix) that explicitly considers intermediate traits, and imputes those that are prohibitive to obtain, will allow a better mechanistic understanding of any trait of interest. Furthermore, this approach provides a proxy for functional validation and mechanistic understanding in organisms where laboratory manipulation is impractical.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Shed Light in the DaRk LineagES of the Fungal Tree of Life—STRES

The polyphyletic group of black fungi within the Ascomycota (Arthoniomycetes, Dothideomycetes, and Eurotiomycetes) is ubiquitous in natural and anthropogenic habitats. Partly because of their dark, melanin-based pigmentation, black fungi are resistant to stresses including UV- and ionizing-radiation, heat and desiccation, toxic metals, and organic pollutants. Consequently, they are amongst the most stunning extremophiles and poly-extreme-tolerant organisms on Earth. Even though ca. 60 black fungal genomes have been sequenced to date, [mostly in the family Herpotrichiellaceae (Eurotiomycetes)], the class Dothideomycetes that hosts the largest majority of extremophiles has only been sparsely sampled. By sequencing up to 92 species that will become reference genomes, the “Shed light in The daRk lineagES of the fungal tree of life” (STRES) project will cover a broad collection of black fungal diversity spread throughout the Fungal Tree of Life. Interestingly, the STRES project will focus on mostly unsampled genera that display different ecologies and life-styles (e.g., ant- and lichen-associated fungi, rock-inhabiting fungi, etc.). With a resequencing strategy of 10- to 15-fold depth coverage of up to ~550 strains, numerous new reference genomes will be established. To identify metabolites and functional processes, these new genomic resources will be enriched with metabolomics analyses coupled with transcriptomics experiments on selected species under various stress conditions (salinity, dryness, UV radiation, oligotrophy). The data acquired will serve as a reference and foundation for establishing an encyclopedic database for fungal metagenomics as well as the biology, evolution, and ecology of the fungi in extreme environments.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Peripheral blood hematology, plasma biochemistry, and the optimization of an in vitro immune-based assay in the brown watersnake ( Nerodia taxispilota )

Reptiles represent a phylogenetic lineage that provides a unique link between ectothermic anamniotes and endothermic amniotes. Compared to mammalian and avian species, our understanding of the reptilian immune system is greatly lacking. This gap in knowledge is largely due to an absence of established immune-based assays or specific reagents for these species. In the present study, brown watersnakes (Nerodia taxispilota) were live-captured in the wild, sexed, weighed, measured, bled via the caudal vein and released. At 24 hr post-collection, peripheral blood leukocytes were enriched and evaluated with an established mammalian in-vitro lymphocyte proliferation assay. Snake peripheral blood leukocyte enrichment yielded > 90% lymphocytes with viabilities averaging 81.5%. Baseline physiologic data for N. taxispilota, including hematology and total solids, leukocyte differentials, cell recovery and plasma biochemistry, were also collected. Cells cultured with Concanavalin A exhibited significantly increased proliferation at both 72 and 96 hr. Furthermore, these preliminary results show that enriched peripheral blood from wild-caught N. taxispilota provides a sufficient yield of leukocytes that can be cultured and functionally evaluated using a standard mammalian in-vitro immune-based assay.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Performance of methods for SARS-CoV-2 variant detection and abundance estimation within mixed population samples

The accurate identification of SARS-CoV-2 (SC2) variants and estimation of their abundance in mixed population samples (e.g., air or wastewater) is imperative for successful surveillance of community level trends. Assessing the performance of SC2 variant composition estimators (VCEs) should improve our confidence in public health decision making. Here, we introduce a linear regression based VCE and compare its performance to four other VCEs: two re-purposed DNA sequence read classifiers (Kallisto and Kraken2), a maximum-likelihood based method (Lineage deComposition for Sars-Cov-2 pooled samples (LCS)), and a regression based method (Freyja). We simulated DNA sequence datasets of known variant composition from both Illumina and Oxford Nanopore Technologies (ONT) platforms and assessed the performance of each VCE. We also evaluated VCEs performance using publicly available empirical wastewater samples collected for SC2 surveillance efforts. Bioinformatic analyses were performed with a custom NextFlow workflow (C-WAP, CFSAN Wastewater Analysis Pipeline). Relative root mean squared error (RRMSE) was used as a measure of performance with respect to the known abundance and concordance correlation coefficient (CCC) was used to measure agreement between pairs of estimators. Based on our results from simulated data, Kallisto was the most accurate estimator as it had the lowest RRMSE, followed by Freyja. Kallisto and Freyja had the most similar predictions, reflected by the highest CCC metrics. We also found that accuracy was platform and amplicon panel dependent. For example, the accuracy of Freyja was significantly higher with Illumina data compared to ONT data; performance of Kallisto was best with ARTICv4. However, when analyzing empirical data there was poor agreement among methods and variations in the number of variants detected (e.g., Freyja ARTICv4 had a mean of 2.2 variants while Kallisto ARTICv4 had a mean of 10.1 variants). This work provides an understanding of the differences in performance of a number of VCEs and how accurate they are in capturing the relative abundance of SC2 variants within a mixed sample (e.g., wastewater). Such information should help officials gauge the confidence they can have in such data for informing public health decisions.

60 APPLIED LIFE SCIENCES↗