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At least 757 records · Page 42

All sources NASA NTRS DOE OSTI

DOE OSTI2016

Whole Genome Sequence Analysis Using JSpecies Tool Establishes Clonal Relationships between Listeria monocytogenes Strains from Epidemiologically Unrelated Listeriosis Outbreaks

In an effort to build a comprehensive genomic approach to food safety challenges, the FDA has implemented a whole genome sequencing effort, GenomeTrakr, which involves the sequencing and analysis of genomes of foodborne pathogens. As a part of this effort, we routinely sequence whole genomes of Listeria monocytogenes (Lm) isolates associated with human listeriosis outbreaks, as well as those isolated through other sources. To rapidly establish genetic relatedness of these genomes, we evaluated tetranucleotide frequency analysis via the JSpecies program to provide a cursory analysis of strain relatedness. The JSpecies tetranucleotide (tetra) analysis plots standardized (z-score) tetramer word frequencies of two strains against each other and uses linear regression analysis to determine similarity (r 2 ). This tool was able to validate the close relationships between outbreak related strains from four different outbreaks. Included in this study was the analysis of Lm strains isolated during the recent caramel apple outbreak and stone fruit incident in 2014. We identified that many of the isolates from these two outbreaks shared a common 4b variant (4bV) serotype, also designated as IVb-v1, using a qPCR protocol developed in our laboratory. The 4bV serotype is characterized by the presence of a 6.3 Kb DNA segment normally found in serotype 1/2a, 3a, 1/2c and 3c strains but not in serotype 4b or 1/2b strains. We decided to compare these strains at a genomic level using the JSpecies Tetra tool. Specifically, we compared several 4bV and 4b isolates and identified a high level of similarity between the stone fruit and apple 4bV strains, but not the 4b strains co-identified in the caramel apple outbreak or other 4b or 4bV strains in our collection. This finding was further substantiated by a SNP-based analysis. Additionally, we were able to identify close relatedness between isolates from clinical cases from 1993–1994 and a single case from 2011 as well as links between two isolates from over 30 years ago. The identification of these potential links shows that JSpecies Tetra analysis can be a useful tool in rapidly assessing genetic relatedness of Lm isolates during outbreak investigations and for comparing historical isolates. In conclusion, our analyses led to the identification of a highly related clonal group involved in two separate outbreaks, stone fruit and caramel apple, and suggests the possibility of a new genotype that may be better adapted for certain foods and/or environment.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2015

Genome-Wide Association Study Based on Multiple Imputation with Low-Depth Sequencing Data: Application to Biofuel Traits in Reed Canarygrass

Genotyping by sequencing allows for large-scale genetic analyses in plant species with no reference genome, but sets the challenge of sound inference in presence of uncertain genotypes. We report an imputation-based genome-wide association study (GWAS) in reed canarygrass (Phalaris arundinacea L., Phalaris caesia Nees), a cool-season grass species with potential as a biofuel crop. Our study involved two linkage populations and an association panel of 590 reed canarygrass genotypes. Plants were assayed for up to 5228 single nucleotide polymorphism markers and 35 traits. The genotypic markers were derived from lowdepth sequencing with 78% missing data on average. To soundly infer marker-trait associations, multiple imputation (MI) was used: several imputes of the marker data were generated to reflect imputation uncertainty and association tests were performed on marker effects across imputes. A total of nine significant markers were identified, three of which showed significant homology with the Brachypodium dystachion genome. Because no physical map of the reed canarygrass genome was available, imputation was conducted using classification trees. In general, MI showed good consistency with the complete-case analysis and adequate control over imputation uncertainty. A gain in significance of marker effects was achieved through MI, but only for rare cases when missing data were ,45%. In addition to providing insight into the genetic basis of important traits in reed canarygrass, this study presents one of the first applications of MI to genome-wide analyses and provides useful guidelines for conducting GWAS based on genotyping-by-sequencing data.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2015

Shared Genomic Regions Between Derivatives of a Large Segregating Population of Maize Identified Using Bulked Segregant Analysis Sequencing and Traditional Linkage Analysis

Delayed transition from the vegetative stage to the reproductive stage of development and increased plant height have been shown to increase biomass productivity in grasses. The goal of this project was to detect quantitative trait loci using extremes from a large synthetic population, as well as a related recombinant inbred line mapping population for these two traits. Ten thousand individuals from a B73 × Mo17 noninbred population intermated for 14 generations (IBM Syn14) were grown at a density of approximately 16,500 plants ha -1 . Flowering time and plant height were measured within this population. DNA was pooled from the 46 most extreme individuals from each distributional tail for each of the traits measured and used in bulk segregant analysis (BSA) sequencing. Allelic divergence at each of the ~1.1 million SNP loci was estimated as the difference in allele frequencies between the selected extremes. Additionally, 224 intermated B73 × Mo17 recombinant inbred lines were concomitantly grown at a similar density adjacent to the large synthetic population and were assessed for flowering time and plant height. Using the BSA sequencing method, 14 and 13 genomic regions were identified for flowering time and plant height, respectively. Linkage mapping with the RIL population identified eight and three regions for flowering time and plant height, respectively. Of the regions identified, three colocalized between the two populations for flowering time and two colocalized for plant height. This study demonstrates the utility of using BSA sequencing for the dissection of complex quantitative traits important for production of lignocellulosic ethanol.

Genetics & Heredity↗

DOE OSTI2017

Diversity and Structure of Diazotrophic Communities in Mangrove Rhizosphere, Revealed by High-Throughput Sequencing

Diazotrophic communities make an essential contribution to the productivity through providing new nitrogen. However, knowledge of the roles that both mangrove tree species and geochemical parameters play in shaping mangove rhizosphere diazotrophic communities is still elusive. Here, a comprehensive examination of the diversity and structure of microbial communities in the rhizospheres of three mangrove species, Rhizophora apiculata , Avicennia marina , and Ceriops tagal , was undertaken using high-throughput sequencing of the 16S rRNA and nifH genes. Our results revealed a great diversity of both the total microbial composition and the diazotrophic composition specifically in the mangrove rhizosphere. Deltaproteobacteria and Gammaproteobacteria were both ubiquitous and dominant, comprising an average of 45.87 and 86.66% of total microbial and diazotrophic communities, respectively. Sulfate-reducing bacteria belonging to the Desulfobacteraceae and Desulfovibrionaceae were the dominant diazotrophs. Community statistical analyses suggested that both mangrove tree species and additional environmental variables played important roles in shaping total microbial and potential diazotroph communities in mangrove rhizospheres. In contrast to the total microbial community investigated by analysis of 16S rRNA gene sequences, most of the dominant diazotrophic groups identified by nifH gene sequences were significantly different among mangrove species. The dominant diazotrophs of the family Desulfobacteraceae were positively correlated with total phosphorus, but negatively correlated with the nitrogen to phosphorus ratio. Finally, the Pseudomonadaceae were positively correlated with the concentration of available potassium, suggesting that diazotrophs potentially play an important role in biogeochemical cycles, such as those of nitrogen, phosphorus, sulfur, and potassium, in the mangrove ecosystem.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

A Two-Step PCR Protocol Enabling Flexible Primer Choice and High Sequencing Yield for Illumina MiSeq Meta-Barcoding

High-throughput amplicon sequencing that primarily targets the 16S ribosomal DNA (rDNA) (for bacteria and archaea) and the Internal Transcribed Spacer rDNA (for fungi) have facilitated microbial community discovery across diverse environments. A three-step PCR that utilizes flexible primer choices to construct the library for Illumina amplicon sequencing has been applied to several studies in forest and agricultural systems. The three-step PCR protocol, while producing high-quality reads, often yields a large number (up to 46%) of reads that are unable to be assigned to a specific sample according to its barcode. Here, we improve this technique through an optimized two-step PCR protocol. We tested and compared the improved two-step PCR meta-barcoding protocol against the three-step PCR protocol using four different primer pairs (fungal ITS: ITS1F-ITS2 and ITS1F-ITS4, and bacterial 16S: 515F-806R and 341F-806R). We demonstrate that the sequence quantity and recovery rate were significantly improved with the two-step PCR approach (fourfold more read counts per sample; determined reads ≈90% per run) while retaining high read quality (Q30 > 80%). Given that synthetic barcodes are incorporated independently from any specific primers, this two-step PCR protocol can be broadly adapted to different genomic regions and organisms of scientific interest.

16S rDNA↗

DOE OSTI2019

Genome Sequencing of Cladobotryum protrusum Provides Insights into the Evolution and Pathogenic Mechanisms of the Cobweb Disease Pathogen on Cultivated Mushroom

Cladobotryum protrusum is one of the mycoparasites that cause cobweb disease on cultivated edible mushrooms. However, the molecular mechanisms of evolution and pathogenesis of C. protrusum on mushrooms are largely unknown. Here, we report a high-quality genome sequence of C. protrusum using the single-molecule, real-time sequencing platform of PacBio and perform a comparative analysis with closely related fungi in the family Hypocreaceae. The C. protrusum genome, the first complete genome to be sequenced in the genus Cladobotryum, is 39.09 Mb long, with an N50 of 4.97 Mb, encoding 11,003 proteins. The phylogenomic analysis confirmed its inclusion in Hypocreaceae, with its evolutionary divergence time estimated to be ~170.1 million years ago. The genome encodes a large and diverse set of genes involved in secreted peptidases, carbohydrate-active enzymes, cytochrome P450 enzymes, pathogen–host interactions, mycotoxins, and pigments. Moreover, C. protrusum harbors arrays of genes with the potential to produce bioactive secondary metabolites and stress response-related proteins that are significant for adaptation to hostile environments. Knowledge of the genome will foster a better understanding of the biology of C. protrusum and mycoparasitism in general, as well as help with the development of effective disease control strategies to minimize economic losses from cobweb disease in cultivated edible mushrooms.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2016

POST-MAIN SEQUENCE EVOLUTION OF ICY MINOR PLANETS: IMPLICATIONS FOR WATER RETENTION AND WHITE DWARF POLLUTION

Most observations of polluted white dwarf atmospheres are consistent with accretion of water-depleted planetary material. Among tens of known cases, merely two involve accretion of objects that contain a considerable mass fraction of water. The purpose of this study is to investigate the relative scarcity of these detections. Based on a new and highly detailed model, we evaluate the retention of water inside icy minor planets during the high-luminosity stellar evolution that follows the main sequence. Our model fully considers the thermal, physical, and chemical evolution of icy bodies, following their internal differentiation as well as water depletion, from the moment of their birth and through all stellar evolution phases preceding the formation of the white dwarf. We also account for different initial compositions and formation times. Our results differ from previous studies, which have either underestimated or overestimated water retention. We show that water can survive in a variety of circumstances and in great quantities, and therefore other possibilities are discussed in order to explain the infrequency of water detection. We predict that the sequence of accretion is such that water accretes earlier, and more rapidly, than the rest of the silicate disk, considerably reducing the chance of its detection in H-dominated atmospheres. In He-dominated atmospheres, the scarcity of water detections could be observationally biased. It implies that the accreted material is typically intrinsically dry, which may be the result of the inside-out depopulation sequence of minor planets.

79 ASTRONOMY AND ASTROPHYSICS↗

DOE OSTI2020

Bayesian Characterization of Main-sequence Binaries in the Old Open Cluster NGC 188

The binary fractions of open and globular clusters yield powerful constraints on their dynamical state and evolutionary history. We apply publicly available Bayesian analysis tools to a UBV RIJHK {sub S} photometric catalog of the open cluster NGC 188 to detect and characterize photometric binaries along the cluster main sequence. This technique has the advantage of self-consistently handling photometric errors, missing data in various bandpasses, and star-by-star prior constraints on cluster membership. Simulations are used to verify uncertainties and quantify selection biases in our analysis, illustrating that among binaries with mass ratios >0.5, we recover the binary fraction to better than 7% in the mean, with no significant dependence on binary fraction and a mild dependence on assumed mass-ratio distribution. Using our photometric catalog, we recover the majority (65% ± 11%) of spectroscopically identified main-sequence binaries, including eight of the nine with spectroscopically measured mass ratios. Accounting for incompleteness and systematics, we derive a mass-ratio distribution that rises toward lower mass ratios (within our q > 0.5 analysis domain). We observe a raw binary fraction for solar-type main-sequence stars with mass ratios q > 0.5 of 42% ± 4%, independent of the assumed mass-ratio distribution to within its uncertainties, consistent with literature values for old open clusters but significantly higher than the field solar-type binary fraction. We confirm that the binaries identified by our method are more concentrated than single stars, in agreement with previous studies, and we demonstrate that the binary nature of those candidates that remain unidentified spectroscopically is strongly supported by photometry from Gaia DR2.

79 ASTRONOMY AND ASTROPHYSICS↗

DOE OSTI2013

Complete genome sequence of Enterobacter sp. IIT-BT 08: A potential microbial strain for high rate hydrogen production

Enterobacter sp. IIT-BT 08 belongs to Phylum: Proteobacteria, Class: Gammaproteobacteria, Order: Enterobacteriales, Family: Enterobacteriaceae. The organism was isolated from the leaves of a local plant near the Kharagpur railway station, Kharagpur, West Bengal, India. It has been extensively studied for fermentative hydrogen production because of its high hydrogen yield. For further enhancement of hydrogen production by strain development, complete genome sequence analysis was carried out. Sequence analysis revealed that the genome was linear, 4.67 Mbp long and had a GC content of 56.01%. The genome properties encode 4,393 protein-coding and 179 RNA genes. Additionally, a putative pathway of hydrogen production was suggested based on the presence of formate hydrogen lyase complex and other related genes identified in the genome. Thus, in the present study we describe the specific properties of the organism and the generation, annotation and analysis of its genome sequence as well as discuss the putative pathway of hydrogen production by this organism.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1998

A 5.8S nuclear ribosomal RNA gene sequence database: applications to ecology and evolution

We complied a 5.8S nuclear ribosomal gene sequence database for animals, plants, and fungi using both newly generated and GenBank sequences. We demonstrate the utility of this database as an internal check to determine whether the target organism and not a contaminant has been sequenced, as a diagnostic tool for ecologists and evolutionary biologists to determine the placement of asexual fungi within larger taxonomic groups, and as a tool to help identify fungi that form ectomycorrhizae.

Databases, Factual↗

NASA NTRSDate not supplied

Elevating the Quality of Space Omics Sequencing Data: Innovations and Methodologies from NASA GeneLab Sample Processing Laboratory

NASA’s GeneLab, part of the NASA Open Science Data Repository, is a space-related database that hosts a diverse range of transcriptomics, proteomics, epigenomics and genomics data. The NASA GeneLab Sample Processing Laboratory (SPL) generates omics data from biological experiments conducted aboard the International Space Station, Space Shuttle and space related ground experiments, this omics data then hosted on the GeneLab repository. Samples generated such experiments pose numerous technical challenges such as small experimental sample size, variance in dissection times, limited tissue preservation methods, prolonged storage time, and more. GeneLab SPL team had developed specialized expertise in nucleic acid extraction, library preparation and sequencing of such biological samples via extensive training and years of experience. In order to ensure data accuracy and consistency across experiments, SPL has developed standardized protocols for each species and tissue type. These protocols in conjunction with quality control metrics and data standards are crucial in generating of high-quality data. SPL protocols and standards have been developed in collaboration with the scientific community and had been made publicly available on the GeneLab portal, guaranteeing comparability of datasets across spaceflight experiments. To ensure reliability of data generation, SPL leverages cutting-edge innovations in laboratory automation for sample processing. By leveraging these state-of-the-art platforms, SPL achieves high levels of data reproducibility while significantly minimizing sources of bias and variability, especially across experiments with large numbers of samples. Over the past few years, the space biology investigator community has accessed SPL-generated data from the Open Science Data Repository for a myriad of data re-analysis and re-use studies. We observe a trend that in-house SPL-generated data consistently outperforms outsourced sequencing data in terms of technical standards, quality control metrics, timeliness of data delivery, and sequencing and reagent efficiency. Superior data generation has and will continue to enable discoveries in disease, diagnostic tools, and the biological effects of long duration spaceflight.

GeneLab↗

NASA NTRS1995

Structurally complex and highly active RNA ligases derived from random RNA sequences

Seven families of RNA ligases, previously isolated from random RNA sequences, fall into three classes on the basis of secondary structure and regiospecificity of ligation. Two of the three classes of ribozymes have been engineered to act as true enzymes, catalyzing the multiple-turnover transformation of substrates into products. The most complex of these ribozymes has a minimal catalytic domain of 93 nucleotides. An optimized version of this ribozyme has a kcat exceeding one per second, a value far greater than that of most natural RNA catalysts and approaching that of comparable protein enzymes. The fact that such a large and complex ligase emerged from a very limited sampling of sequence space implies the existence of a large number of distinct RNA structures of equivalent complexity and activity.

Non-NASA Center↗

NASA NTRS1991

Phylogenetic diversity in the genus Bacillus as seen by 16S rRNA sequencing studies

Comparative sequence analysis of 16S ribosomal (r)RNAs or DNAs of Bacillus alvei, B. laterosporus, B. macerans, B. macquariensis, B. polymyxa and B. stearothermophilus revealed the phylogenetic diversity of the genus Bacillus. Based on the presently available data set of 16S rRNA sequences from bacilli and relatives at least four major "Bacillus clusters" can be defined: a "Bacillus subtilis cluster" including B. stearothermophilus, a "B. brevis cluster" including B. laterosporus, a "B. alvei cluster" including B. macerans, B. maquariensis and B. polymyxa and a "B. cycloheptanicus branch".

NASA Discipline Number 52-30↗

NASA NTRS1992

Purification and sequence analysis of two rat tissue inhibitors of metalloproteinases

Two protein inhibitors of metalloproteinases (TIMP) were isolated from medium conditioned by the clonal rat osteosarcoma line UMR 106-01. Initial purification of both a 30-kDa inhibitor and a 20-kDa inhibitor was accomplished using heparin-Sepharose chromatography with dextran sulfate elution followed by DEAE-Sepharose and CM-Sepharose chromatography. Purification of the 20-kDa inhibitor to homogeneity was completed with reverse-phase high-performance liquid chromatography. The 20-kDa inhibitor was identified as rat TIMP-2. The 30-kDa inhibitor, although not purified to homogeneity, was identified as rat TIMP-1. Amino terminal amino acid sequence analysis of the 30-kDa inhibitor demonstrated 86% identity to human TIMP-1 for the first 22 amino acids while the sequence of the 20-kDa inhibitor was identical to that of human TIMP-2 for the first 22 residues. Treatment with peptide:N-glycosidase F indicated that the 30-kDa rat inhibitor is glycosylated while the 20-kDa inhibitor is apparently unglycosylated. Inhibition of both rat and human interstitial collagenase by rat TIMP-2 was stoichiometric, with a 1:1 molar ratio required for complete inhibition. Exposure of UMR 106-01 cells to 10(-7) M parathyroid hormone resulted in approximately a 40% increase in total inhibitor production over basal levels.

NASA Discipline Musculoskeletal↗

DOE OSTI2016

Triazine‐Based Sequence‐Defined Polymers with Side‐Chain Diversity and Backbone–Backbone Interaction Motifs

Abstract Sequence control in polymers, well‐known in nature, encodes structure and functionality. Here we introduce a new architecture, based on the nucleophilic aromatic substitution chemistry of cyanuric chloride, that creates a new class of sequence‐defined polymers dubbed TZPs. Proof of concept is demonstrated with two synthesized hexamers, having neutral and ionizable side chains. Molecular dynamics simulations show backbone–backbone interactions, including H‐bonding motifs and pi–pi interactions. This architecture is arguably biomimetic while differing from sequence‐defined polymers having peptide bonds. The synthetic methodology supports the structural diversity of side chains known in peptides, as well as backbone–backbone hydrogen‐bonding motifs, and will thus enable new macromolecules and materials with useful functions.

Grate, Jay W.↗

DOE OSTI2024

Hierarchical Self‐Assembly of Multidimensional Functional Materials from Sequence‐Defined Peptoids

Abstract Hierarchical self‐assembly represents a powerful strategy for the fabrication of functional materials across various length scales. However, achieving precise formation of functional hierarchical assemblies remains a significant challenge and requires a profound understanding of molecular assembly interactions. In this study, we present a molecular‐level understanding of the hierarchical assembly of sequence‐defined peptoids into multidimensional functional materials, including twisted nanotube bundles serving as a highly efficient artificial light harvesting system. By employing synchrotron‐based powder X‐ray diffraction and analyzing single crystal structures of model compounds, we elucidated the molecular packing and mechanisms underlying the assembly of peptoids into multidimensional nanostructures. Our findings demonstrate that incorporating aromatic functional groups, such as tetraphenyl ethylene (TPE), at the termini of assembling peptoid sequences promotes the formation of twisted bundles of nanotubes and nanosheets, thus enabling the creation of a highly efficient artificial light harvesting system. This research exemplifies the potential of leveraging sequence‐defined synthetic polymers to translate microscopic molecular structures into macroscopic assemblies. It holds promise for the development of functional materials with precisely controlled hierarchical structures and designed functions.

Shao, Li↗

DOE OSTI2016

Triazine‐Based Sequence‐Defined Polymers with Side‐Chain Diversity and Backbone–Backbone Interaction Motifs

36 MATERIALS SCIENCE↗

DOE OSTI2024

Hierarchical Self‐Assembly of Multidimensional Functional Materials from Sequence‐Defined Peptoids

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗