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17 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Discovering methylated DNA motifs in bacterial nanopore sequencing data with MIJAMP

Abstract Bacterial DNA methylation is involved in diverse cellular functions, including modulation of gene expression, DNA repair, and restriction–modification systems for defense against viruses and other foreign DNA. Restriction systems hinder efforts to engineer organisms to produce fuels and chemicals from waste and renewable feedstocks by degrading DNA during transformation. Methylome analysis allows identification of motifs within a bacterial chromosome that may be targeted by native restriction enzymes. Further expression of the corresponding methyltransferases in Escherichia coli allows plasmid DNA to be protected from restriction in the target organism, thereby drastically enhancing transformation efficiency. Nanopore sequencing can detect methylated bases, but software is needed to transform modified base coordinates into methylated motifs. Here, we develop MIJAMP (MIJAMP Is Just A MethylBED Parser), a software package that was developed to discover methylated motifs from the output of ONT’s Modkit or other data in the methylBED format. MIJAMP employs a human-driven refinement strategy that empirically validates all motifs against genome-wide methylation data, thus eliminating incorrect motifs. MIJAMP also reports methylation data on specific, user-defined motifs. Using MIJAMP, we determined the methylated motifs both in a control strain (wild-type E. coli) and in Synecococcus sp. strain PCC7002, laying the foundation for improved transformation in this organism. MIJAMP is available at https://code.ornl.gov/alexander-public/mijamp/. One Sentence Summary: Here we describe software written to discover DNA methylation motifs from nanopore sequencing data.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Nanopore Activity Assays for Detection of Biomarker Protease Activity: Design and Testing of Substrates for Both Nanopore Sequencing and PCR-Based Detection Methods

The work performed in this project has demonstrated the ability to construct proteolytic enzyme substrates that are PCR and sequencing-readable reporter molecules. Specifically, the goal was to detect those reporter molecules via PCR and Oxford Nanopore Technologies MinION sequencing methods following exposure to the biomarker protease thrombin. The assay development focused on binding the constructed peptide-oligonucleotide chimera to immobilized streptavidin. The action of thrombin on the peptide portion of the molecule released the oligonucleotide for detection. Detection of protease activity was demonstrated in a concentration-dependent manner using MALDI-MS, RT-PCR and DNA sequencing. Additional steps to remove background release of reporter molecules during the assay was used to improve the difference in detected oligonucleotide reporter following protease activity. Additional steps in assay development will be to (1) test the assay in an appropriate matrix, (2) investigate detection using additional DNA sequencing platforms and (3) demonstrate multiplexed detection of multiple protease markers in a single reaction.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Full-Length ASFV B646L Gene Sequencing by Nanopore Offers a Simple and Rapid Approach for Identifying ASFV Genotypes

African swine fever (ASF) is an acute, highly hemorrhagic viral disease in domestic pigs and wild boars. The disease is caused by African swine fever virus, a double stranded DNA virus of the Asfarviridae family. ASF can be classified into 25 different genotypes, based on a 478 bp fragment corresponding to the C-terminal sequence of the B646L gene, which is highly conserved among strains and encodes the major capsid protein p72. The C-terminal end of p72 has been used as a PCR target for quick diagnosis of ASF, and its characterization remains the first approach for epidemiological tracking and identification of the origin of ASF in outbreak investigations. Recently, a new classification of ASF, based on the complete sequence of p72, reduced the 25 genotypes into only six genotypes; therefore, it is necessary to have the capability to sequence the full-length B646L gene (p72) in a rapid manner for quick genotype characterization. Here, we evaluate the use of an amplicon approach targeting the whole B646L gene, coupled with nanopore sequencing in a multiplex format using Flongle flow cells, as an easy, low cost, and rapid method for the characterization and genotyping of ASF in real-time.

Virology↗
DOE OSTI2024

LevSeq: Rapid Generation of Sequence-Function Data for Directed Evolution and Machine Learning

Sequence-function data provides valuable information about the protein functional landscape but is rarely obtained during directed evolution campaigns. Here, we present Long-read every variant Sequencing (LevSeq), a pipeline that combines a dual barcoding strategy with nanopore sequencing to rapidly generate sequence-function data for entire protein-coding genes. LevSeq integrates into existing protein engineering workflows and comes with open-source software for data analysis and visualization. The pipeline facilitates data-driven protein engineering by consolidating sequence-function data to inform directed evolution and provide the requisite data for machine learning-guided protein engineering (MLPE). LevSeq enables quality control of mutagenesis libraries prior to screening, which reduces time and resource costs. Simulation studies demonstrate LevSeq’s ability to accurately detect variants under various experimental conditions. Lastly, we show LevSeq’s utility in engineering protoglobins for new-to-nature chemistry. Widespread adoption of LevSeq and sharing of the data will enhance our understanding of protein sequence-function landscapes and empower data-driven directed evolution.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Complete genome of Erwinia spp. str . LJJL01 isolated from waste charcoal

We present the complete genome sequence of Erwinia spp. str. LJJL01, isolated from waste charcoal in Colorado, USA, using Oxford Nanopore sequencing. This sequence provides important insights into this bacterium’s metabolic and catabolic robustness to utilize sugars, acids, and aromatics, highlighting its potential as a bio-industrial strain for various feedstocks.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Emerging protein sequencing technologies: proteomics without mass spectrometry?

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been a leading method for proteomics for 30 years. Advantages provided by LC-MS/MS are offset by significant disadvantages, including cost. Recently, several non-mass spectrometric methods have emerged, but little information is available about their capacity to analyze the complex mixtures routine for mass spectrometry. Areas Covered: We review recent non-mass-spectrometric methods for sequencing proteins and peptides, including those using nanopores, sequencing by degradation, reverse translation, and short-epitope mapping, with comments on bioinformatics challenges, fundamental limitations, and areas where new technologies will be more or less competitive with LC-MS/MS. In addition to conventional literature searches, instrument vendor websites, patents, webinars, and preprints were also consulted to give a more up-to-date picture. Expert Opinion: Many new technologies are promising. However, demonstrations that they outperform mass spectrometry in terms of peptides and proteins identified have not yet been published, and astute observers note important disadvantages, especially relating to the dynamic range of single-molecule measurements of complex mixtures. Still, even if the performance of emerging methods proves inferior to LC-MS/MS, their low cost could create a different kind of revolution: a dramatic increase in the number of biology laboratories engaging in new forms of proteomics research.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Decomposing a San Francisco estuary microbiome using long-read metagenomics reveals species- and strain-level dominance from picoeukaryotes to viruses

ABSTRACT Although long-read sequencing has enabled obtaining high-quality and complete genomes from metagenomes, many challenges still remain to completely decompose a metagenome into its constituent prokaryotic and viral genomes. This study focuses on decomposing an estuarine metagenome to obtain a more accurate estimate of microbial diversity. To achieve this, we developed a new bead-based DNA extraction method, a novel bin refinement method, and obtained 150 Gbp of Nanopore sequencing. We estimate that there are ~500 bacterial and archaeal species in our sample and obtained 68 high-quality bins (>90% complete, <5% contamination, ≤5 contigs, contig length of >100 kbp, and all ribosomal and tRNA genes). We also obtained many contigs of picoeukaryotes, environmental DNA of larger eukaryotes such as mammals, and complete mitochondrial and chloroplast genomes and detected ~40,000 viral populations. Our analysis indicates that there are only a few strains that comprise most of the species abundances. IMPORTANCE Ocean and estuarine microbiomes play critical roles in global element cycling and ecosystem function. Despite the importance of these microbial communities, many species still have not been cultured in the lab. Environmental sequencing is the primary way the function and population dynamics of these communities can be studied. Long-read sequencing provides an avenue to overcome limitations of short-read technologies to obtain complete microbial genomes but comes with its own technical challenges, such as needed sequencing depth and obtaining high-quality DNA. We present here new sampling and bioinformatics methods to attempt decomposing an estuarine microbiome into its constituent genomes. Our results suggest there are only a few strains that comprise most of the species abundances from viruses to picoeukaryotes, and to fully decompose a metagenome of this diversity requires 1 Tbp of long-read sequencing. We anticipate that as long-read sequencing technologies continue to improve, less sequencing will be needed.

Lui, Lauren M.↗
DOE OSTI2024

Borg extrachromosomal elements of methane-oxidizing archaea have conserved and expressed genetic repertoires

Borgs are huge extrachromosomal elements (ECE) of anaerobic methane-consuming “Candidatus Methanoperedens” archaea. Here, we used nanopore sequencing to validate published complete genomes curated from short reads and to reconstruct new genomes. 13 complete and four near-complete linear genomes share 40 genes that define a largely syntenous genome backbone. We use these conserved genes to identify new Borgs from peatland soil and to delineate Borg phylogeny, revealing two major clades. Remarkably, Borg genes encoding nanowire-like electron-transferring cytochromes and cell surface proteins are more highly expressed than those of host Methanoperedens, indicating that Borgs augment the Methanoperedens activity in situ. We reconstructed the first complete 4.00 Mbp genome for a Methanoperedens that is inferred to be a Borg host and predicted its methylation motifs, which differ from pervasive TC and CC methylation motifs of the Borgs. Thus, methylation may enable Methanoperedens to distinguish their genomes from those of Borgs. Very high Borg to Methanoperedens ratios and structural predictions suggest that Borgs may be capable of encapsulation. The findings clearly define Borgs as a distinct class of ECE with shared genomic signatures, establish their diversification from a common ancestor with genetic inheritance, and raise the possibility of periodic existence outside of host cells.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Molecular and structural characterization of a Bacillus cereus strain producing an anthrax-like capsule

Bacillus cereus is a ubiquitous Gram-positive, spore-forming, rod-shaped saprophytic bacterium, occasionally reported to cause food-borne illnesses. However, instances of B. cereus strains harboring anthrax toxin and capsule genes have elevated certain strains as formidable pathogens and biothreats. This study focuses on the genomic analysis and the structural characterization of capsular material produced by the virulent B. cereus PATH2418 strain, isolated from the wound of a traumatic open fracture patient. The genome was sequenced using Nanopore MinION sequencing, revealing a chromosome of 5,270,283 bp and three plasmids. One plasmid, pATH1, was found to encode an operon for the biosynthesis of a bacterial capsule. This operon had sequence homology to the Bacillus anthracis capBCADE operon, which encodes the poly-γ-D-glutamate (PDGA) capsule. The capsule production in B. cereus PATH2418 was influenced by temperature and CO 2 levels. Structural analysis of the capsular material using a combined approach of nuclear magnetic resonance (NMR) and high-performance liquid chromatography (HPLC) techniques confirmed the presence of a high-molecular-weight poly-γ-glutamate capsule, with an enantiomeric composition of approximately 67% D-glutamic acid and 33% L-glutamic acid, matching that of B. anthracis.

Bacillus cereus↗
DOE OSTI2024

Multiplex detection and identification of viral, bacterial, and protozoan pathogens in human blood and plasma using an expanded high-density resequencing microarray platform

Introduction: Nucleic acid tests for blood donor screening have improved the safety of the blood supply; however, increasing numbers of emerging pathogen tests are burdensome. Multiplex testing platforms are a potential solution. Methods: The Blood Borne Pathogen Resequencing Microarray Expanded (BBP-RMAv.2) can perform multiplex detection and identification of 80 viruses, bacteria and parasites. This study evaluated pathogen detection in human blood or plasma. Samples spiked with selected pathogens, each with one of 6 viruses, 2 bacteria and 5 protozoans were tested on this platform. The nucleic acids were extracted, amplified using multiplexed sets of primers, and hybridized to a microarray. The reported sequences were aligned to a database to identify the pathogen. To directly compare the microarray to an emerging molecular approach, the amplified nucleic acids were also submitted to nanopore next generation sequencing (NGS). Results: The BBP-RMAv.2 detected viral pathogens at a concentration as low as 100 copies/ml and a range of concentrations from 1,000 to 100,000 copies/ml for all the spiked pathogens. Coded specimens were identified correctly demonstrating the effectiveness of the platform. The nanopore sequencing correctly identified most samples and the results of the two platforms were compared. Discussion: These results indicated that the BBP-RMAv.2 could be employed for multiplex detection with potential for use in blood safety or disease diagnosis. The NGS was nearly as effective at identifying pathogens in blood and performed better than BBP-RMAv.2 at identifying pathogen-negative samples.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Complete genome sequence of Luteolibacter sp. strain Populi, a member of phylum Verrucomicrobiota isolated from the Populus trichocarpa rhizosphere

Luteolibacter sp. strain Populi is a bacterium from the phylum Verrucomicrobiota, isolated from the rhizosphere of a black cottonwood tree, Populus trichocarpa, from the Cascade mountains in Washington. Its 6.6-Mb chromosome was completely sequenced using Oxford Nanopore long-read sequencing and is predicted to encode 5,301 proteins and 60 RNAs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Full ribosomal operon sequencing of anaerobic gut fungi (phylum Neocallimastigomycota ): insights on its markers and phylogenetic resolution

The phylogenetic affiliations of anaerobic gut fungi (Neocallimastigomycota) are typically evaluated using single-gene markers. However, this approach often fails to resolve relationships between closely related lineages. To address this issue and identify alternative markers, we created a curated database comprising the complete ribosomal operon sequences of 156 isolates, representing 20 of the 22 recognized genera and two new genus-level clades. Using long-read sequencing, we obtained ~9 kbp operon sequences and developed a robust analysis pipeline. Incorporating both coding genes and non-coding regions (excluding IGS1) improved phylogenetic resolution. This phylogenetic approach successfully resolved the Cyllamyces and Caecomyces clades (hard-to-distinguish genetically), as well as seven analysed Piromyces species. We also scanned the operon for markers that are suitable for short-read sequencing platforms, with the aim of enhancing biodiversity and phylogenetic studies. Notably, the ETS1 genetic region also enabled the distinction between these lineages, indicating its phylogenetic value within the ribosomal operon. The resulting database is a valuable resource for expanding and strengthening phylogenetic frameworks.

High-throughput sequencing↗
DOE OSTI2024

Luteolibacter sp. strain Populi

Luteolibacter sp. strain Populi is bacterium from the phylum Verrucomicrobiota, isolated from the rhizosphere of a black cottonwood tree, Populus trichocarpa, from the Cascade mountains in Washington. Its 6.6 Mb chromosome was completely sequenced using Oxford Nanopore long-reads and is predicted to encode 5301 proteins and 60 RNAs. The bacteria was isolated from the rhizosphere of a mature Populus trichocarpa from the Tieton riverwatershed of Washington state, USA (Lat: 46°42’9” N, Lon: 120°25 39’36” W). A rhizosphere sample (fine roots and adhering soil) was used to obtain a microbial fraction by centrifugation on Histodenz (12) and stained with 5µM Syto59 (Thermo Fisher Scientific Inc). A Cytopeia Influx cell sorter (BD, Franklin Lakes, NJ) was used to sort and array single cells (100 per plate) based on forward-side scatter and fluorescence intensity on asparagine-glucose nutrient agar (ATCC medium 184). The Luteolibacter sp. Populi genome sequence has been deposited in GenBank under the accession number CP161812. A draft genome annotated with Prokka and DRAM is available in this Narrative as Luteolibacter_sp_Prokka.240711.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Nanopore Readable Activity Probes for Ribosomal Inactivating Protein (RIP) Toxins

Ribosome inactivating proteins (RIPs) such as ricin and abrin depurinate an adenine base in the sarcin/ricin loop in the large ribosomal subunit, leading to inhibtion of protein synthesis and cell death. Here, we demonstrate that RIP toxin activity can be detected via nanopore-based DNA sequencing using synthetic oligonucleotide substrates. This is achieved by monitoring the mismatch proportion at the canonical target sequences incorporated into the synthetic substrate and determining the sequence length distribution throughout the entire substrate sequence. The mismatch proportion increases and sequence length distribution decreases with increasing toxin concentration for both ricin and abrin in buffer as well as in more complex backgrounds such as saliva and nasal secretions.

Turner, Matthew W [Pacific Northwest National Labo↗
DOE OSTI2024

Pyoverdine binding aptamers and label-free electrochemical detection of pseudomonads

Pyoverdines are iron-chelating siderophores employed by various pseudomonads to promote their growth in iron-limited environments, facilitating both beneficial and detrimental interactions with co-inhabiting microbes or hosts, including plants and animals. The fluorescent pseudomonads produce fluorescent pyoverdines comprised of a conserved central chromophore and a unique strain-specific peptidic side chain produced by non-ribosomal peptide synthetases. Pyoverdine Pf5 (PVD-Pf5) is produced by Pseudomonas protegens Pf-5, a species known for supporting plant growth and its involvement in plant pathogen control. To develop a means of exploring the dynamics of P. protegens activity in soil and in the rhizosphere, we selected DNA aptamers that specifically recognize PVD-Pf5 with high affinities. Two selected aptamers with only 16% identity in sequence were examined for structure and function. We found evidence that both aptamers form structures in their apo-forms and one aptamer has structural features suggesting the presence of a G-quadruplex. Although their tertiary structures are predicted to be different, both aptamers bind the target PVD-Pf5 with similar affinities and do not bind other siderophores, including the related pyoverdine, pseudobactin, produced by Pseudomonas sp. B10. One aptamer binds the pyoverdine peptide component and may also interact with the chromophore. This aptamer was integrated into a nanoporous aluminum oxide biosensor and demonstrated to successfully detect PVD-Pf5 and not to detect other siderophores that do not bind to the aptamer when evaluated in solution. This sensor provides a future opportunity to track the locations of P. protegens around plant roots and to monitor PVD-Pf5 production and movement through the soil.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2026

Computationally designed coiled coil ‘bundlemers’ as model colloidal nanoparticles for solution assembly and materials design (Final Report)

As a collaborative team at the University of Delaware and the University of Pennsylvania, Kloxin, Pochan and Saven designed new biomimetic nanomaterials de novo, leveraging a variety of complementary areas of expertise: computational design of biopolymers (Saven at the University of Pennsylvania), and synthesis and characterization (Kloxin and Pochan at the University of Delaware). Overall activities included: sequence-specific peptide synthesis; covalent crosslinking; noncovalent assembly; site-specific functionalization; and nanostructural characterization using electron microscopy and solution-phase (x-ray and neutron) scattering. Using natural and non-natural amino acids, the team created modular, functional peptide building blocks for elaboration of new nanostructured materials. Ultimately, the development of robust peptide-based, building blocks provides tools for researchers to readily produce complex nanomaterial structures in a wide range of applications. The project had three, interconnecting goals in an effort to provide the broader scientific community with a new peptide-based paradigm for materials design and characterization. First, we further developed the coiled-coil bundle-based toolbox (otherwise known as the ‘bundlemer’ toolbox) via computational design with experimental bundle assembly verification. Second, we developed new uses of covalent interactions, in addition to desired physical (noncovalent) interactions, to assemble bundlemers into 1-D polymer chains with targeted chain rigidity, length, and dispersity. Thirds, we used the above designs to experimentally realize (physical or covalent) polymers to target the creation of liquid crystals or to realize interparticle assembly into nanoporous lattices. The close integration of the three groups was instrumental in success of the biomolecular materials design, formation, and understanding for future designs.

36 MATERIALS SCIENCE↗