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DOE OSTI2013

A Microfluidic DNA Library Preparation Platform for Next-Generation Sequencing

Next-generation sequencing (NGS) is emerging as a powerful tool for elucidating genetic information for a wide range of applications. Unfortunately, the surging popularity of NGS has not yet been accompanied by an improvement in automated techniques for preparing formatted sequencing libraries. To address this challenge, we have developed a prototype microfluidic system for preparing sequencer-ready DNA libraries for analysis by Illumina sequencing. Our system combines droplet-based digital microfluidic (DMF) sample handling with peripheral modules to create a fully-integrated, sample-in library-out platform. In this report, we use our automated system to prepare NGS libraries from samples of human and bacterial genomic DNA. E. coli libraries prepared on-device from 5 ng of total DNA yielded excellent sequence coverage over the entire bacterial genome, with .99% alignment to the reference genome, even genome coverage, and good quality scores. Furthermore, we produced a de novo assembly on a previously unsequenced multi-drug resistant Klebsiella pneumoniae strain BAA-2146 (KpnNDM). The new method described here is fast, robust, scalable, and automated. Our device for library preparation will assist in the integration of NGS technology into a wide variety of laboratories, including small research laboratories and clinical laboratories.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Design and calculations for HWR Strongback

The PIP-II Superconductive Linac is a linear accelerator that provides the first stage of beam acceleration. The velocity increase is achieved through a sequence of different cryomodules, where all the cavities are aligned to form a continuous string. The cryomodule team is currently developing a spare for the HWR cryomodule. This spare will not be an exact replica of the existing one, but rather will be based on the SSR f650 cryomodule design created for PIP-II . Each cavity , magnet , and coupler form a unit, with a total of eight units per string. These components, together with additional equipment (such as piping , the thermal shield , and beamline interconnections ), are supported by the Strongback . The aim of this project is to develop a brand-new Strongback design for HWR cavities, and to perform the related analyses and calculations in order to meet the required specifications.

43 PARTICLE ACCELERATORS↗

DOE OSTI2026

GenomeDepot: data management system for microbial comparative genomics

Summary GenomeDepot is an open-source web-based platform for annotation, management, and comparative analysis of microbial genomic sequences and associated data including ortholog families, protein domains, operons, regulatory interactions, strain taxonomy, and sample metadata. GenomeDepot supports rapid creation of websites for user-defined genome collections that include bioinformatic tools for interactive genome browsing, Basic Local Alignment Search Tool (BLAST) search, annotation search, comparative genomic neighborhood visualization, and sequence download. Gene function annotations are generated by a customizable annotation pipeline. The pipeline runs annotation tools in Conda environments and can be easily extended with additional user-specified tools. Availability and implementation GenomeDepot is open source and distributed under the GNU General Public License via GitHub (https://github.com/aekazakov/genome-depot). GenomeDepot is implemented in Python and was tested in Ubuntu Linux. Full installation instructions and documentation are available at https://aekazakov.github.io/genome-depot/. GenomeDepot demo server is freely accessible at https://iseq.lbl.gov/demogd/.

Kazakov, Alexey [Lawrence Berkeley National Labora↗

DOE OSTI2025

Spatially Aligned Binary Single-Site Catalyst on Defective SiO 2 for Cascading Reactions

Capitalizing on the success of single-atom catalysts (SACs), dual-atom catalysts (DACs) have emerged as a new frontier in heterogeneous catalysis. However, most SACs and DACs studies seek to uniformly distribute the catalytic sites on the support material, which can hinder their effectiveness in intricate multistep cascading reactions. Particularly, it is a grand challenge to precisely control the spatial distribution of two different single sites forming binary sites so that reactants and intermediates contact the catalytic sites in the exact sequence required by the reaction steps. Here, in this work, we report a new type of binary single-site catalyst, Cu 1 –Zr 1 @SiO 2 , with Cu 1 and Zr 1 sites spatially aligned with the reaction sequence of the cascade reactions. The catalyst is synthesized by a modified reverse microemulsion approach, with single Cu sites anchored by nonbridging oxygen hole centers, which were induced by doping single Zr sites into SiO 2 . Low-energy ion scattering spectroscopy (LEIS) reveals that the outermost surface of the catalyst contains only Cu single sites, while the Zr sites are dispersed in the bulk. The catalytic performance is demonstrated in ethanol conversion to butenes, a model cascade reaction which includes ethanol dehydrogenation and aldol condensation steps. The precisely spatially controlled binary sites enable ethanol to first undergo dehydrogenation to acetaldehyde on Cu sites, followed by aldol condensation of acetaldehyde on Zr sites. As a result, C 3+ olefins selectivity as high as 77.0% (56.0% selectivity of butenes) is achieved by suppressing ethylene formation.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

NASA NTRS2012

Real-Time X-ray Radiography Diagnostics of Components in Solid Rocket Motors

Solid rocket motors (SRMs) typically use nozzle materials which are required to maintain their shape as well as insulate the underlying support structure during the motor operation. In addition, SRMs need internal insulation materials to protect the motor case from the harsh environment resulting from the combustion of solid propellant. In the nozzle, typical materials consist of high density graphite, carbon-carbon composites and carbon phenolic composites. Internal insulation of the motor cases is typically a composite material with carbon, asbestos, Kevlar, or silica fibers in an ablative matrix such as EPDM or NBR. For both nozzle and internal insulation materials, the charring process occurs when the hot combustion products heat the material intensely. The pyrolysis of the matrix material takes away a portion of the thermal energy near the wall surface and leaves behind a char layer. The fiber reinforcement retains the porous char layer which provides continued thermal protection from the hot combustion products. It is of great interest to characterize both the total erosion rates of the material and the char layer thickness. By better understanding of the erosion process for a particular ablative material in a specific flow environment, the required insulation material thickness can be properly selected. The recession rates of internal insulation and nozzle materials of SRMs are typically determined by testing in some sort of simulated environment; either arc-jet testing, flame torch testing, or subscale SRMs of different size. Material recession rates are deduced by comparison of pre- and post-test measurements and then averaging over the duration of the test. However, these averaging techniques cannot be used to determine the instantaneous recession rates of the material. Knowledge of the variation in recession rates in response to the instantaneous flow conditions during the motor operation is of great importance. For example, in many SRM configurations the recession of the solid propellant grain can drastically alter the flow-field and effect the recession of internal insulation and nozzle materials. Simultaneous measurement of the overall erosion rate, the development of the char layer, and the recession of the char-virgin interface during the motor operation can be rather difficult. While invasive techniques have been used with limited success, they have serious drawbacks. Break wires or make wire sensors can be installed into a sufficient number of locations in the charring material from which a time history of the charring surface can be deduced. These sensors fundamentally alter the local structure of the material in which they are imbedded. Also, the location of these sensors within the material is not known precisely without the use of an X-ray. To determine instantaneous recession rates, real-time X-ray radiography (X-ray RTR) has been utilized in several SRM experiments at PSU. The X-ray RTR system discussed in this paper consists of an X-ray source, X-ray image intensifier, and CCD camera connected to a capture computer. The system has been used to examine the ablation process of internal insulation as well as nozzle material erosion in a subscale SRM. The X-ray source is rated to 320 kV at 10 mA and has both a large (5.5 mm) and small (3.0 mm) focal spot. The lead-lined cesium iodide X-ray image intensifier produces an image which is captured by a CCD camera with a 1,000 x 1,000 pixel resolution. To produce accurate imagery of the object of interest, the alignment of the X-ray source to the X-ray image intensifier is crucial. The image sequences captured during the operation of an SRM are then processed to enhance the quality of the images. This procedure allows for computer software to extract data on the total erosion rate and the char layer thickness. Figure 1 Error! Reference source not found.shows a sequence of images captured during the operation the subscale SRM with the X-ray RTR system. The X-rayTR system, alignment procedure, uncertainty determination, and image analysis process will be discussed in detail in the full manuscript.

Cortopassi, A. C.↗

NASA NTRS1982

Planar multijunction high voltage solar cell chip

A new innovative planar multijunction solar cell chip for concentrated sunlight applications is proposed. The chip consists of many voltage-generating regions, called unit cells, which are connected in series within a single silicon wafer, thereby providing a high open-circuit voltage at multiple sun illumination levels. The unit cells are fabricated on 75 micron thick p-type single crystal silicon substrate. Each chip consists of 1.42 x 9.63 mm n(+)/p collecting junctions on the back of the wafer, while the illuminated front surface area is divided into 0.3 micron deep n(+) regions. The fabrication sequence includes standard degreasing and cleaning procedures, double-sided alignment photomasking, introduction of boron and phosphorus impurities, and photolithography. The open circuit voltage of the chip increased rapidly with illumination up to about 4 AM1 suns, and then began to saturate at the sum of the individual unit cell voltages of 3.5 above 4 AM1 suns. A short circuit density per unit cell of 300 mA/sq cm at 20 AM1 suns was observed.

Valco, G. J.↗

DOE OSTI2024

Evaluation of the Impact of Concentration and Extraction Methods on the Targeted Sequencing of Human Viruses from Wastewater

Sequencing human viruses in wastewater is challenging due to their low abundance compared to the total microbial background. This study compared the impact of four virus concentration/extraction methods (Innovaprep, Nanotrap, Promega, and Solids extraction) on probe-capture enrichment for human viruses followed by sequencing. Different concentration/extraction methods yielded distinct virus profiles. Innovaprep ultrafiltration (following solids removal) had the highest sequencing sensitivity and richness, resulting in the successful assembly of several near-complete human virus genomes. However, it was less sensitive in detecting SARS-CoV-2 by digital polymerase chain reaction (dPCR) compared to Promega and Nanotrap. Across all preparation methods, astroviruses and polyomaviruses were the most highly abundant human viruses, and SARS-CoV-2 was rare. These findings suggest that sequencing success can be increased using methods that reduce nontarget nucleic acids in the extract, though the absolute concentration of total extracted nucleic acid, as indicated by Qubit, and targeted viruses, as indicated by dPCR, may not be directly related to targeted sequencing performance. Further, using broadly targeted sequencing panels may capture viral diversity but risks losing signals for specific low-abundance viruses. Overall, this study highlights the importance of aligning wet lab and bioinformatic methods with specific goals when employing probe-capture enrichment for human virus sequencing from wastewater.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2019

PNNL-CompBio/perseq

Per sequence functional classification and Functional annotation in addition to linking those functions to taxonomic groups presents a challenge due to many established tools performing one task and not the other creating disparate downstream data. PerSeq aims to address these challenges by performing local alignments against a reference database annotated for both aspects, function and taxonomy, to annotate sequences individually. Downstream results are merged into count tables facilitating complex analyses based on observed function or functional potential in addition to being able to separate out function by taxonomic level to determine metabolic contributions by present organisms.

Brown, Joe↗

DOE OSTI2024

Profiling expression strategies for a type III polyketide synthase in a lysate-based, cell-free system

Abstract Some of the most metabolically diverse species of bacteria (e.g., Actinobacteria) have higher GC content in their DNA, differ substantially in codon usage, and have distinct protein folding environments compared to tractable expression hosts like Escherichia coli . Consequentially, expressing biosynthetic gene clusters (BGCs) from these bacteria in E. coli often results in a myriad of unpredictable issues with regard to protein expression and folding, delaying the biochemical characterization of new natural products. Current strategies to achieve soluble, active expression of these enzymes in tractable hosts can be a lengthy trial-and-error process. Cell-free expression (CFE) has emerged as a valuable expression platform as a testbed for rapid prototyping expression parameters. Here, we use a type III polyketide synthase from Streptomyces griseus , RppA, which catalyzes the formation of the red pigment flaviolin, as a reporter to investigate BGC refactoring techniques. We applied a library of constructs with different combinations of promoters and rppA coding sequences to investigate the synergies between promoter and codon usage. Subsequently, we assess the utility of cell-free systems for prototyping these refactoring tactics prior to their implementation in cells. Overall, codon harmonization improves natural product synthesis more than traditional codon optimization across cell-free and cellular environments. More importantly, the choice of coding sequences and promoters impact protein expression synergistically, which should be considered for future efforts to use CFE for high-yield protein expression. The promoter strategy when applied to RppA was not completely correlated with that observed with GFP, indicating that different promoter strategies should be applied for different proteins. In vivo experiments suggest that there is correlation, but not complete alignment between expressing in cell free and in vivo. Refactoring promoters and/or coding sequences via CFE can be a valuable strategy to rapidly screen for catalytically functional production of enzymes from BCGs, which advances CFE as a tool for natural product research.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2016

Intrinsic alignments in redMaPPer clusters – I. Central galaxy alignments and angular segregation of satellites

The shapes of cluster central galaxies are not randomly oriented, but rather exhibit coherent alignments with the shapes of their parent clusters as well as with the surrounding large-scale structures. In this work, we aim to identify the galaxy and cluster quantities that most strongly predict the central galaxy alignment phenomenon among a large parameter space with a sample of 8237 clusters and 94 817 members within 0.1 < z < 0.35, based on the red-sequence Matched-filter Probabilistic Percolation cluster catalogue constructed from the Sloan Digital Sky Survey. We first quantify the alignment between the projected central galaxy shapes and the distribution of member satellites, to understand what central galaxy and cluster properties most strongly correlate with these alignments. Next, we investigate the angular segregation of satellites with respect to their central galaxy major axis directions, to identify the satellite properties that most strongly predict their angular segregation. We find that central galaxies are more aligned with their member galaxy distributions in clusters that are more elongated and have higher richness, and for central galaxies with larger physical size, higher luminosity and centring probability, and redder colour. Satellites with redder colour, higher luminosity, located closer to the central galaxy, and with smaller ellipticity show a stronger angular segregation towards their central galaxy major axes. Lastly, we provide physical explanations for some of the identified correlations, and discuss the connection to theories of central galaxy alignments, the impact of primordial alignments with tidal fields, and the importance of anisotropic accretion.

79 ASTRONOMY AND ASTROPHYSICS↗

DOE OSTI2011

Identification and removal of ribosomal RNA sequences from metatranscriptomes

Here, we present riboPicker, a robust framework for the rapid, automated identification and removal of ribosomal RNA sequences from metatranscriptomic datasets. The results can be exported for subsequent analysis, and the databases used for the web-based version are updated on a regular basis. riboPicker categorizes rRNA-like sequences and provides graphical visualizations and tabular outputs of ribosomal coverage, alignment results and taxonomic classifications.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2019

Decoupled and semi-decoupled bands in Hg 197 and Hg 199

Here, the structure of decoupled positive-parity and semidecoupled negative-parity bands up to high spin in 197,199 Hg has been studied using multinucleon transfer reactions with the Gammasphere array. A positive-parity band in 199 Hg with the (vi 13/2 ) 3 decoupled configuration that deexcites to levels in the negative-parity sequences is newly established. The semidecoupled sequences in both isotopes are extended to higher spin. Rotation-aligned angular momenta and crossing frequencies determined from experiment are in fair agreement with those from cranking calculations. Finally, a transition from oblate collective rotation at low spin to triaxial noncollective shapes at high spin is indicated.

73 NUCLEAR PHYSICS AND RADIATION PHYSICS↗

DOE OSTI2026

Automation of Laser Plasma Focused Ion Beam Microscopy for Next-Gen Energy Materials

Automation can revolutionize the use of ultrafast laser ablation and plasma-focused ion beam (PFIB) techniques for high-throughput, reproducible cross-sectioning and various sample preparation in materials characterization. As these methods become essential for analyzing complex energy materials and next-generation devices, efficient, standardized workflows are needed to minimize variability and enhance precision. This work highlights our advancements in developing automated processes for sample preparation that integrates machine learning, workflow optimization, and large-scale data acquisition to improve efficiency and scalability in applications such as electrolyzers, photovoltaic cells, and microelectronics. To streamline cross-sectioning and lamella fabrication, we have implemented fully automated workflows that standardize laser ablation and PFIB milling sequences. These workflows incorporate pre-programmed protocols for material removal, alignment, and thinning, reducing user intervention and ensuring consistency across different sample types. Machine learning algorithms further enhance automation by predicting optimal milling strategies and adapting parameters based on material properties and sectioning requirements. This approach significantly improves throughput while maintaining the structural integrity of prepared samples for high-resolution imaging and analysis, including transmission electron microscopy. Beyond sample preparation, our automation platform enables the acquisition of large, high-resolution datasets through serial sectioning, image alignment, and 3D reconstruction. These automated routines facilitate multi-scale characterization, capturing structural and compositional details from the nanoscale to the device level. By reducing variability and increasing efficiency, our automated approach enhances defect analysis, failure diagnostics, and process optimization, accelerating advancements in materials research and device engineering.

36 MATERIALS SCIENCE↗

NASA NTRS1986

Halogen occultation experiment (HALOE) performance verification test procedure

The Performance Verification Test Procedure is given for the Halogen Occultation Experiment (HALOE) instrument, which is being developed in house at the Langley Research Center for the Upper Atmosphere Research Satellite (UARS). This procedure is used for comprehensive performance testing of the HALOE instrument which occurs before, during, and after flight environmental tests. The radiometric performance tests include noise, drift, linearity, instantaneous field-of-view, cal wheel gas cell characterization, and self thermal emissions. Pointer/tracker performance tests include sun sensor performance, gimbal performance, control system performance, and boresight alignment. In addition, the instrument is tested functionally in simulated orbit sequences and all command operating modes are exercised. The data analysis required for each test is specified and pass/fail criteria are given where applicable. This test will fully demonstrate the HALOE instrument's ability to achieve science mission requirements. The HALOE instrument is a gas correlation radiometer that measures vertical distribution of eight upper atmospheric constituents: O3, HCl, HF, NO, CH4, H2O, NO2, and CO2.

Mauldin, L. E., III↗

NASA NTRS1993

Isolation of new ribozymes from a large pool of random sequences

An iterative in vitro selection procedure was used to isolate a new class of catalytic RNAs (ribozymes) from a large pool of random-sequence RNA molecules. These ribozymes ligate two RNA molecules that are aligned on a template by catalyzing the attack of a 3'-hydroxyl on an adjacent 5'-triphosphate - a reaction similar to that employed by the familiar protein enzymes that synthesize RNA. The corresponding uncatalyzed reaction also yields a 3',5'-phosphodiester bond. In vitro evolution of the population of new ribozymes led to improvement of the average ligation activity and the emergence of ribozymes with reaction rates 7 million times faster than the uncatalyzed reaction rate.

Bartel, David P.↗

DOE OSTI2017

Progress Updates for the Hanford Waste Treatment and Immobilization Plant Project - 17275

12 MANAGEMENT OF RADIOACTIVE AND NON-RADIOACTIVE W↗

DOE OSTI2018

Progress Updates for the Hanford Waste Treatment and Immobilization Plant Project - 18327

The Waste Treatment and Immobilization Plant (WTP) Project is vital to the U.S. Department of Energy's (DOE) mission to cleanup radioactive waste at the Hanford Site, located in southeastern Washington State. The overall WTP Project objective is to design, build, and commission the facilities and systems that will treat and immobilize approximately 56 million gallons of radioactive waste stored in Hanford's 177 underground storage tanks. The combination of technologies in the WTP makes it a first-of-a-kind facility at a scale larger than ever, built for radioactive waste processing. DOE is proceeding with its approach to complete the WTP Project in phases, which includes the following principal actions: - Completing construction of the Low-Activity Waste (LAW) Facility, Balance of Facilities (BOF), and Analytical Laboratory (LAB) (collectively referred to as LBL); - Implementing a direct-feed low-activity waste (DFLAW) process to treat low-activity waste independent of the Pretreatment (PT) Facility; - Resolving the technical issues associated with primarily the PT Facility; - Completing the design and construction of the High-Level Waste (HLW) and PT facilities to enable integrated WTP operations. In March 2016, a federal judge amended the 2010 Consent Decree 1 completion milestones for the Hanford WTP, which generally aligns with DOE's plans for completing the WTP facilities in sequence: - Complete LAW Facility hot commissioning: December 31, 2023; - Complete HLW Facility hot commissioning: December 31, 2033; - Complete PT Facility hot commissioning: December 31, 2033; - Achieve initial plant operations for the WTP: December 31, 2036. Having established milestones for completing the WTP facilities has enabled DOE to formally implement its plans for completing the WTP facilities in sequence, starting with the LAW Facility. In December 2016, DOE executed a contract modification and approved a change to the project's performance baseline to complete the LBL facilities and incorporate modifications needed to allow direct feed of low-activity waste to the LAW Facility. Significant progress has been made toward completing the design, construction, and planning for commissioning of the facilities needed to enable the DFLAW operations to commence as early as January 2022. In 2012, DOE partially suspended production work on the WTP's PT Facility and to a lesser degree the HLW Facility because of unresolved technical issues associated with those facilities. In September 2017, DOE acknowledged the WTP contractor had met all the criteria for full resumption of engineering, procurement, and construction (EPC) activities for the HLW Facility. By December 2017, DOE had completed the actions needed to resolve the six of the eight primary technical issues associated with the PT Facility, with the remaining two issues expected to be resolved in the first quarter of 2018. Although significant progress has been made to prepare for resuming production activities for the HLW and PT facilities, DOE has made the decision to constrain EPC work on those facilities in the near term based on DOE's higher priority of completing the WTP facilities needed to begin DFLAW operations. (authors)

12 MANAGEMENT OF RADIOACTIVE AND NON-RADIOACTIVE W↗

DOE OSTI2024

VIBES: a workflow for annotating and visualizing viral sequences integrated into bacterial genomes

Abstract Bacteriophages are viruses that infect bacteria. Many bacteriophages integrate their genomes into the bacterial chromosome and become prophages. Prophages may substantially burden or benefit host bacteria fitness, acting in some cases as parasites and in others as mutualists. Some prophages have been demonstrated to increase host virulence. The increasing ease of bacterial genome sequencing provides an opportunity to deeply explore prophage prevalence and insertion sites. Here we present VIBES (Viral Integrations in Bacterial genomES), a workflow intended to automate prophage annotation in complete bacterial genome sequences. VIBES provides additional context to prophage annotations by annotating bacterial genes and viral proteins in user-provided bacterial and viral genomes. The VIBES pipeline is implemented as a Nextflow-driven workflow, providing a simple, unified interface for execution on local, cluster and cloud computing environments. For each step of the pipeline, a container including all necessary software dependencies is provided. VIBES produces results in simple tab-separated format and generates intuitive and interactive visualizations for data exploration. Despite VIBES’s primary emphasis on prophage annotation, its generic alignment-based design allows it to be deployed as a general-purpose sequence similarity search manager. We demonstrate the utility of the VIBES prophage annotation workflow by searching for 178 Pf phage genomes across 1072 Pseudomonas spp. genomes.

59 BASIC BIOLOGICAL SCIENCES↗