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All sourcesNASA NTRSDOE OSTI
DOE OSTI2017

Front-End Electron Transfer Dissociation Coupled to a 21 Tesla FT-ICR Mass Spectrometer for Intact Protein Sequence Analysis

High resolution mass spectrometry is a key technology for in-depth protein characterization. High-field Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) enables high-level interrogation of intact proteins in the most detail to date. However, an appropriate complement of fragmentation technologies must be paired with FTMS to provide comprehensive sequence coverage, as well as characterization of sequence variants, and post-translational modifications. Here we describe the integration of front-end electron transfer dissociation (FETD) with a custom-built 21 tesla FT-ICR mass spectrometer, which yields unprecedented sequence coverage for proteins ranging from 2.8 to 29 kDa, without the need for extensive spectral averaging (e.g., ~60% sequence coverage for apo-myoglobin with four averaged acquisitions). The system is equipped with a multipole storage device separate from the ETD reaction device, which allows accumulation of multiple ETD fragment ion fills. Consequently, an optimally large product ion population is accumulated prior to transfer to the ICR cell for mass analysis, which improves mass spectral signal-to-noise ratio, dynamic range, and scan rate. We find a linear relationship between protein molecular weight and minimum number of ETD reaction fills to achieve optimum sequence coverage, thereby enabling more efficient use of instrument data acquisition time. Finally, real-time scaling of the number of ETD reactions fills during method-based acquisition is shown, and the implications for LC-MS/MS top-down analysis are discussed. .

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗
DOE OSTI2015

Targeted Switchgrass BAC Library Screening and Sequence Analysis Identifies Predicted Biomass and Stress Response-Related Genes

To identify switchgrass homologs of rice genes, known/predicted to control biomass and stress response-related traits, we screened 96,000 clones from two switchgrass bacterial artificial chromosome (BAC) libraries. Full-length sequencing of 311 BAC clones revealed sequence for ~3.2 % (51.7 Mb) of the switchgrass genome, coding for 3948 genes. A comparison with Arabidopsis and five grass genomes revealed that switchgrass genes share the highest number of homologs with rice (95.5 %) followed by foxtail millet (91.7 %) and Sorghum (91.5 %). One hundred eighteen of the annotated genes are unique to switchgrass. Gene annotation and ontology analysis revealed 695 genes belonging to gene families targeted in the screening. These include 350 kinase, 203 glycosyltransferase (GT), 109 glycoside hydrolase (GH), and 33 ethylene responsive transcription factor (ERF) family genes. Rice homologs of 65 genes, identified here, have demonstrated roles in bioenergy-relevant traits. These include 14 GT2 family genes involved in the synthesis of cellulose and hemicelluloses. Finally, comparative expression analysis in six switchgrass organs revealed a conserved expression pattern for three cellulose synthase (CesA1, CesA2, and CesA9) and five cellulose-synthase-like genes (CslA2, CslA11, CslC1, CslD4, and CslE6). CslF genes that encode mixed linkage glucans are expressed in wider range of tissues in switchgrass compared with rice.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Application of prophage sequence analysis to investigate a disease outbreak involving Salmonella Adjame, a rare serovar and implications for the population structure

Introduction Outbreak investigation of foodborne salmonellosis is hindered when the food source is contaminated by multiple strains of Salmonella , creating difficulties matching an incriminated organism recovered from patients with the specific strain in the suspect food. An outbreak of the rare Salmonella Adjame was caused by multiple strains of the organism as revealed by single-nucleotide polymorphism (SNP) variation. The use of highly discriminatory prophage analysis to characterize strains of Salmonella should enable a more precise strain characterization and aid the investigation of foodborne salmonellosis. Methods We have carried out genomic analysis of S. Adjame strains recovered during the course of a recent outbreak and compared them with other strains of the organism ( n = 38 strains), using SNPs to evaluate strain differences present in the core genome, and prophage sequence typing (PST) to evaluate the accessory genome. Phylogenetic analyses were performed using both total prophage content and conserved prophages. Results The PST analysis of the S. Adjame isolates showed a high degree of strain heterogeneity. We observed small clusters made up of 2-6 isolates ( n = 27) and singletons ( n = 11) in stark contrast with the three clusters observed by SNP analysis. In total, we detected 24 prophages of which only four were highly prevalent, namely: Entero_p88 (36/38 strains), Salmon_SEN34 (35/38 strains), Burkho_phiE255 (33/38 strains) and Edward_GF (28/38 strains). Despite the marked strain diversity seen with prophage analysis, the distribution of the four most common prophages matched the clustering observed using core genome. Discussion Mutations in the core and accessory genomes of S. Adjame have shed light on the evolutionary relationships among the Adjame strains and demonstrated a convergence of the variations observed in both fractions of the genome. We conclude that core and accessory genomes analyses should be adopted in foodborne bacteria outbreak investigations to provide a more accurate strain description and facilitate reliable matching of isolates from patients and incriminated food sources. The outcomes should translate to a better understanding of the microbial population structure and an 46 improved source attribution in foodborne illnesses.

Gao, Ruimin↗
DOE OSTI2020

KENO V.a Primer: Performing Calculations using SCALE’s Criticality Safety Analysis Sequence (CSAS5) with Fulcrum

The SCALE code system developed at Oak Ridge National Laboratory is widely used and accepted around the world for criticality safety analyses. The well-known KENO V.a three-dimensional Monte Carlo criticality computer code is one of the primary criticality safety analysis tools in SCALE. The KENO V.a primer is designed to help a new user understand and use the SCALE/KENO V.a Monte Carlo code for nuclear criticality safety analyses. It assumes that the user has a college education in a technical field. There is no assumption of familiarity with Monte Carlo codes in general or with SCALE/KENO V.a in particular. The primer is designed to teach by example, with each example illustrating two or three features of SCALE/KENO V.a that are useful in criticality analyses. The primer is based on SCALE 6.2 and 6.3, which includes the Fulcrum graphical user interface (GUI). Each example uses Fulcrum to provide the framework for preparing input data and viewing output results. Starting with a Quickstart section, the primer gives an overview of the basic requirements for SCALE/KENO V.a input and allows the user to quickly run a simple criticality problem with SCALE/KENO V.a. The sections that follow Quickstart include a list of basic objectives at the beginning that identifies the goal of the section and the individual SCALE/KENO V.a features that are covered in detail in the sample problems in that section. Upon completion of the primer, a new user should be comfortable using Fulcrum to set up criticality problems in SCALE/KENO V.a. The primer provides a starting point for the criticality safety analyst who uses SCALE/KENO V.a. Complete descriptions are provided in the SCALE/KENO V.a manual. Although the primer is self-contained, it is intended as a companion volume to the SCALE/KENO V.a training and documentation. The SCALE manual and training schedule are available at https://scale.ornl.gov. The primer provides specific examples of using SCALE/KENO V.a for criticality analyses; the SCALE/KENO V.a manual provides information on the use of SCALE/KENO V.a and all its modules. The primer also contains an appendix with sample input files. In addition, this primer, its errata and sample inputs are also available at https://code.ornl.gov/scale/primers/kenova.

98 NUCLEAR DISARMAMENT, SAFEGUARDS, AND PHYSICAL P↗
DOE OSTI2022

DNA Sequence Analysis of an Inversion Hot Spot in Lobeliaceae Plastomes

The evolution of plastid genomes (plastomes) in land plants is typically conservative, with extensive structural rearrangements present in only a few groups. Early Southern blot analysis identified two Lobelia species that minimally required deletion of the plastid gene accD and five inversions to account for their plastome arrangement relative to the ancestral organization. Sixty alternative 5-step inversion scenarios could account for the observed arrangement, but only one scenario was consistent with the criterion of ‘common cause’ attributable to a putative rearrangement hot spot at the accD deletion-site. Plastome sequencing demonstrated that this previously hypothesized inversion order is historically accurate. Detailed reconstructions of the ancestral plastome organization before and after each inversion are presented herein. Stem-loop and disruption-rescue models were evaluated for each inversion. One inversion has an obvious stem-loop basis, but the other four inversions were primarily caused by serial insertion of foreign (extra-plastid) DNA bearing large open-reading frames that disrupted plastome organization at the accD deletion-site, and complete plastomes were rescued by seemingly arbitrary ligation or fortuitous recombination at the other inversion endpoint. Transposed copies of DNA segments from elsewhere in the plastome are frequently inserted at inversion junctions, and four junctions are consistent with the stem-loop ligation model.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Comparative Sequence Analysis of Multidrug-Resistant IncA/C Plasmids from Salmonella enterica

Determinants of multidrug resistance (MDR) are often encoded on mobile elements, such as plasmids, transposons, and integrons, which have the potential to transfer among foodborne pathogens, as well as to other virulent pathogens, increasing the threats these traits pose to human and veterinary health. Our understanding of MDR among Salmonella has been limited by the lack of closed plasmid genomes for comparisons across resistance phenotypes, due to difficulties in effectively separating the DNA of these high-molecular weight, low-copy-number plasmids from chromosomal DNA. To resolve this problem, we demonstrate an efficient protocol for isolating, sequencing and closing IncA/C plasmids from Salmonella sp. using single molecule real-time sequencing on a Pacific Biosciences (Pacbio) RS II Sequencer. We obtained six Salmonella enterica isolates from poultry, representing six different serovars, each exhibiting the MDR-Ampc resistance profile. Salmonella plasmids were obtained using a modified mini preparation and transformed with Escherichia coli DH10Br. A Qiagen Large-Construct kitTM was used to recover highly concentrated and purified plasmid DNA that was sequenced using PacBio technology. These six closed IncA/C plasmids ranged in size from 104 to 191 kb and shared a stable, conserved backbone containing 98 core genes, with only six differences among those core genes. The plasmids encoded a number of antimicrobial resistance genes, including those for quaternary ammonium compounds and mercury. We then compared our six IncA/C plasmid sequences: first with 14 IncA/C plasmids derived from S. enterica available at the National Center for Biotechnology Information (NCBI), and then with an additional 38 IncA/C plasmids derived from different taxa. These comparisons allowed us to build an evolutionary picture of how antimicrobial resistance may be mediated by this common plasmid backbone. Our project provides detailed genetic information about resistance genes in plasmids, advances in plasmid sequencing, and phylogenetic analyses, and important insights about how MDR evolution occurs across diverse serotypes from different animal sources, particularly in agricultural settings where antimicrobial drug use practices vary.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2013

Comparative sequence analysis of nitrogen fixation-related genes in six legumes

Legumes play an important role as food and forage crops in international agriculture especially in developing countries. Legumes have a unique biological process called nitrogen fixation (NF) by which they convert atmospheric nitrogen to ammonia. Although legume genomes have undergone polyploidization, duplication and divergence, NF-related genes, because of their essential functional role for legumes, might have remained conserved. To understand the relationship of divergence and evolutionary processes in legumes, this study analyzes orthologs and paralogs for selected 20 NF-related genes by using comparative genomic approaches in six legumes i.e., Medicago truncatula (Mt), Cicer arietinum, Lotus japonicus, Cajanus cajan (Cc), Phaseolus vulgaris (Pv), and Glycine max (Gm). Subsequently, sequence distances, numbers of synonymous substitutions per synonymous site (Ks) and non-synonymous substitutions per non-synonymous site (Ka) between orthologs and paralogs were calculated and compared across legumes. These analyses suggest the closest relationship between Gm and Cc and the highest distance between Mt and Pv in six legumes. Ks proportional plots clearly showed ancient genome duplication in all legumes, whole genome duplication event in Gm and also speciation pattern in different legumes. This study also reports some interesting observations e.g., no peak at Ks 0.4 in Gm-Gm, location of two independent genes next to each other in Mt and low Ks values for outparalogs for three genes as compared to other 12 genes. In summary, this study underlines the importance of NF-related genes and provides important insights in genome organization and evolutionary aspects of six legume species analyzed.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Sequence Analysis of 20,453 Severe Acute Respiratory Syndrome Coronavirus 2 Genomes from the Houston Metropolitan Area Identifies the Emergence and Widespread Distribution of Multiple Isolates of All Major Variants of Concern

Since the beginning of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, there has been international concern about the emergence of virus variants with mutations that increase transmissibility, enhance escape from the human immune response, or otherwise alter biologically important phenotypes. In late 2020, several variants of concern emerged globally, including the UK variant (B.1.1.7), the South Africa variant (B.1.351), Brazil variants (P.1 and P.2), and two related California variants of interest (B.1.429 and B.1.427). These variants are believed to have enhanced transmissibility. For the South Africa and Brazil variants, there is evidence that mutations in spike protein permit it to escape from some vaccines and therapeutic monoclonal antibodies. On the basis of our extensive genome sequencing program involving 20,453 coronavirus disease 2019 patient samples collected from March 2020 to February 2021, we report identification of all six of these SARS-CoV-2 variants among Houston Methodist Hospital (Houston, TX) patients residing in the greater metropolitan area. Although these variants are currently at relatively low frequency (aggregate of 1.1%) in the population, they are geographically widespread. Houston is the first city in the United States in which active circulation of all six current variants of concern has been documented by genome sequencing. Finally, as vaccine deployment accelerates, increased genomic surveillance of SARS-CoV-2 is essential to understanding the presence, frequency, and medical impact of consequential variants and their patterns and trajectory of dissemination.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Diazotroph Community Characterization via a High-Throughput nifH Amplicon Sequencing and Analysis Pipeline

The dinitrogenase reductase gene ( nifH ) is the most widely established molecular marker for the study of nitrogen-fixing prokaryotes in nature. A large number of PCR primer sets have been developed for nifH amplification, and the effective deployment of these approaches should be guided by a rapid, easy-to-use analysis protocol. Bioinformatic analysis of marker gene sequences also requires considerable expertise. In this study, we advance the state of the art for nifH analysis by evaluating nifH primer set performance, developing an improved amplicon sequencing workflow, and implementing a user-friendly bioinformatics pipeline. Furthermore, the developed amplicon sequencing workflow is a three-stage PCR-based approach that uses established technologies for incorporating sample-specific barcode sequences and sequencing adapters. Based on our primer evaluation, we recommend the Ando primer set be used with a modified annealing temperature of 58°C, as this approach captured the largest diversity of nifH templates, including paralog cluster IV/V sequences. To improve nifH sequence analysis, we developed a computational pipeline which infers taxonomy and optionally filters out paralog sequences. In addition, we employed an empirical model to derive optimal operational taxonomic unit (OTU) cutoffs for the nifH gene at the species, genus, and family levels. A comprehensive workflow script named TaxADivA (TAXonomy Assignment and DIVersity Assessment) is provided to ease processing and analysis of nifH amplicons. Our approach is then validated through characterization of diazotroph communities across environmental gradients in beach sands impacted by the Deepwater Horizon oil spill in the Gulf of Mexico, in a peat moss-dominated wetland, and in various plant compartments of a sugarcane field. IMPORTANCE: Nitrogen availability often limits ecosystem productivity, and nitrogen fixation, exclusive to prokaryotes, comprises a major source of nitrogen input that sustains food webs. The nifH gene, which codes for the iron protein of the nitrogenase enzyme, is the most widely established molecular marker for the study of nitrogen-fixing microorganisms (diazotrophs) in nature. In this study, a flexible sequencing/analysis pipeline, named TaxADivA, was developed for nifH amplicons produced by Illumina paired-end sequencing, and it enables an inference of taxonomy, performs clustering, and produces output in formats that may be used by programs that facilitate data exploration and analysis. Diazotroph diversity and community composition are linked to ecosystem functioning, and our results advance the phylogenetic characterization of diazotroph communities by providing empirically derived nifH similarity cutoffs for species, genus, and family levels. Thus, the utility of our pipeline is validated for diazotroph communities in a variety of ecosystems, including contaminated beach sands, peatland ecosystems, living plant tissues, and rhizosphere soil.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

High quality draft genome sequence and analysis of Pontibacter roseus type strain SRC-1T (DSM 17521T) isolated from muddy waters of a drainage system in Chandigarh, India

Pontibacter roseus is a member of genus Pontibacter family Cytophagaceae, class Cytophagia. While the type species of the genus Pontibacter actiniarum was isolated in 2005 from a marine environment, subsequent species of the same genus have been found in different types of habitats ranging from seawater, sediment, desert soil, rhizosphere, contaminated sites, solar saltern and muddy water. Here we describe the features of Pontibacter roseus strain SRC-1 T along with its complete genome sequence and annotation from a culture of DSM 17521 T . In conclusion, the 4,581,480 bp long draft genome consists of 12 scaffolds with 4,003 protein-coding and 50 RNA genes and is a part of Genomic Encyclopedia of Type Strains: KMG-I project.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Sequencing and analysis of 131 SARS-CoV-2 isolates in previously sampled and unsampled regions of Jordan from 2020 to 2023

The Hashemite Kingdom of Jordan remains an understudied country for next generation sequencing analysis of SARS-CoV-2 genomes collected during the 2019 pandemic. Here we provide 131 additional reference genomes collected between 2020–2023 from SARS-CoV-2-positive patients across Jordan. Phylogenetic analysis supports existing pandemic narratives of changing clade dominance over time and adds genomes in novel Jordanian locations and timepoints to make Jordan SARS-CoV-2 databases more comprehensive. Samples from the less-sequenced cities of Ajloun, Jaresh, Karak, and Madaba identified previously unreported lineages while Amman, Irbid, and Zarqa have existing sequencing efforts bolstered. Despite many incomplete patient records and a relatively small sample size, we observe interesting symptom patterns that support existing global and Jordanian pandemic narratives. We note how in-country COVID-19 pandemic genomic studies showcase Jordan’s efforts to expand next generation sequencing capabilities, especially through the leveraging of EDGE COVID-19, a bioinformatics platform for performing rapid, batched analysis of SARS-CoV-2 sequencing that streamlines sample processing prepared from a network of hospital locations.

60 APPLIED LIFE SCIENCES↗
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↗
DOE OSTI2014

D-Tailor: automated analysis and design of DNA sequences

Motivation: Current advances in DNA synthesis, cloning and sequencing technologies afford high-throughput implementation of artificial sequences into living cells. However, flexible computational tools for multi-objective sequence design are lacking, limiting the potential of these technologies. Results: We developed DNA-Tailor (D-Tailor), a fully extendable software framework, for property-based design of synthetic DNA sequences. D-Tailor permits the seamless integration of multiple sequence analysis tools into a generic Monte Carlo simulation that evolves sequences toward any combination of rationally defined properties. As proof of principle, we show that D-Tailor is capable of designing sequence libraries comprising all possible combinations among three different sequence properties influencing translation efficiency in Escherichia coli. The capacity to design artificial sequences that systematically sample any given parameter space should support the implementation of more rigorous experimental designs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Classification of nucleotide sequences by latent semantic analysis

DNA sequences are analyzed using latent semantic analysis. A set of nucleotide sequences is received in which the set has a first number of sequences. A set of basis vectors is determined, in which the set has a second number of basis vectors, the second number being smaller than the first number. Each basis vector represents a specific combination of predetermined nucleotide segments. For each of the nucleotide sequences, an approximate representation of the nucleotide sequence is determined based on a combination of the basis vectors. For each pair of nucleotide sequences, a distance between the pair of nucleotide sequences is determined according the distance between the approximate representation of the pair of nucleotide sequences. The set of nucleotide sequences are classified based on the distances between the pairs of nucleotide sequences.

Sayood, Khalid↗