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NASA NTRSDate not supplied

Method Development for Multiplex, In-Situ, and Real-Time Detection of Herpesvirus Reactivation in Spaceflight Crews using Nanopore Sequencing

Reactivation of latent herpesviruses in crews onboard the International Space Station (ISS) is a well-established spaceflight-associated phenomenon and has been linked to overall immune stress. Beyond an indicator of an altered immune state, this stress-induced reactivation of viruses such as herpesvirus simplex virus 1 (HSV-1), Epstein-Barr virus (EBV), and Varicella-Zoster virus (VZV) may cause clinical symptoms in crew. There is currently no established protocol for in-flight monitoring, and samples are analyzed post-flight using ground-based assays. A real-time, in-flight method for herpesvirus detection followed by stress-mitigation strategies would be a significant advance. In this study, we developed a real-time assay for the multiplex detection of HSV-1, EBV, and VZV from saliva that could be implemented for in-situ monitoring of ISS crew. This method builds on previously validated spaceflight-compatible portable molecular protocols and platforms, such as the miniPCR thermal cycler and the MinION sequencer. Since a metagenomic approach is not currently permitted for crew samples (NASA policy), we employed multiplexing-ready primers directed toward targeted regions within the HSV-1, EBV, and VZV genomes. We also used primers for the human gene, Statherin (STATH), as an internal control. These primers were validated using spiked-in, positive control HSV-1, EBV, and VZV from virus-negative saliva extracted using the Zymo-Research Quick-DNA/RNA Viral MagBead Kit. The PCR Barcoding Kit was used to prepare DNA libraries that were then sequenced on the MK1C and analyzed against known reference genomes using minimap2. Following validation of this method with spiked saliva samples, suspected herpesvirus-positive clinical saliva samples were successfully tested. Prior to use onboard the ISS, this method will be deployed to an analog environment during overwintering at Palmer Station, Antarctica in 2023. This work represents the successful application of molecular technologies currently onboard the ISS for the real-time monitoring of latent herpesvirus reactivation from saliva samples. This assay, in combination with stress-reduction strategies, holds the potential to manage herpesvirus reactivation in ISS crew, thereby improving health outcomes and overall immunity.

Patrick M. Rydzak↗
NASA NTRSDate not supplied

Nanopore Sequencing-Based Microbial Air Profiling Method for Crewed Spacecraft

Microbial monitoring of the International Space Station (ISS) atmosphere is vital to maintaining the health of the spacecraft and crew. Key to NASA’s microbial risk assessment is the identity of contaminating microorganisms in the environment. Historically, this has been achieved through impaction-based air sampling followed by culture. Identification of the microorganisms present requires sample return to Earth and lab-based analyses. While this culture-based approach has served to provide alerts to anomalies and overall confidence in the controls in place, it is not suitable for exploration missions with no sample return. Recently, significant advancements in molecular-based microbial monitoring via nanopore sequencing have been implemented onboard the ISS. Building on this work, multiple commercially available air samplers, compatible with downstream molecular analysis, were evaluated for use in the spaceflight environment. Through this assessment, the Coriolis Compact (Bertin Technologies), which uses cyclonic technology to collect bioaerosols onto the surface of a sterile cone, was selected for a larger-scale comparison to the current culture-based monitoring method. Using the Coriolis Compact, 1000 L of air was collected from the breakroom of an office building and a fitness center. The buffer used to dissociate the microbial cells from the surface of the cone was split between the NASA Microbiology Laboratory’s standard culture and Sanger sequencing-based method and the culture-independent nanopore sequencing method. The bacteria identified through culture were present in the nanopore data, with Micrococcus, Staphylococcus, and Moraxella being the most common cultured isolates, which is expected based on the media and growth conditions. Not surprisingly, the nanopore data yielded much higher diversity and paralleled that of previous atmospheric microbiome studies of human-occupied built environments. As compared to the culture-based data where the breakroom and fitness center data sets cluster in proximity, the nanopore data depicts the contrast of these atmospheric microbiomes. Moreover, the nanopore data were sufficient to meet NASA’s risk assessment needs and noted the culturable isolates routinely observed. This nanopore-based atmospheric microbial profiling method will enable near real-time environmental monitoring of crewed spacecraft as future missions extend beyond low-Earth orbit.

Brandon Dunbar↗
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 OSTI2023

Topography of respiratory tract and gut microbiota in mice with influenza A virus infection

Introduction Influenza A virus (IAV)-induced dysbiosis may predispose to severe bacterial superinfections. Most studies have focused on the microbiota of single mucosal surfaces; consequently, the relationships between microbiota at different anatomic sites in IAV-infected mice have not been fully studied. Methods We characterized respiratory and gut microbiota using full-length 16S rRNA gene sequencing by Nanopore sequencers and compared the nasopharyngeal, oropharyngeal, lung and gut microbiomes in healthy and IAV-infected mice. Results The oropharyngeal, lung and gut microbiota of healthy mice were dominated by Lactobacillus spp., while nasopharyngeal microbiota were comprised primarily of Streptococcus spp. However, the oropharyngeal, nasopharyngeal, lung, and gut microbiota of IAV-infected mice were dominated by Pseudomonas, Escherichia , Streptococcus , and Muribaculum spp., respectively. Lactobacillus murinus was identified as a biomarker and was reduced at all sites in IAV-infected mice. The microbiota composition of lung was more similar to that of the nasopharynx than the oropharynx in healthy mice. Discussion These findings suggest that the main source of lung microbiota in mice differs from that of adults. Moreover, the similarity between the nasopharyngeal and lung microbiota was increased in IAV-infected mice. We found that IAV infection reduced the similarity between the gut and oropharyngeal microbiota. L. murinus was identified as a biomarker of IAV infection and may be an important target for intervention in post-influenza bacterial superinfections.

Chen, Qichao↗
DOE OSTI2024

Long- and short-read sequencing methods discover distinct circular RNA pools in Lotus japonicus

Circular RNAs (circRNAs) are covalently closed single-stranded RNAs, generated through a back-splicing process that links a downstream 5′ site to an upstream 3′ end. The only distinction in the sequence between circRNA and their linear cognate RNA is the back splice junction. Their low abundance and sequence similarity with their linear origin RNA have made the discovery and identification of circRNA challenging. We have identified almost 6000 novel circRNAs from Lotus japonicus leaf tissue using different enrichment, amplification, and sequencing methods as well as alternative bioinformatics pipelines. The different methodologies identified different pools of circRNA with little overlap. We validated circRNA identified by the different methods using reverse transcription polymerase chain reaction and characterized sequence variations using nanopore sequencing. We compared validated circRNA identified in L. japonicus to other plant species and showed conservation of high-confidence circRNA-expressing genes. This is the first identification of L. japonicus circRNA and provides a resource for further characterization of their function in gene regulation. CircRNAs identified in this study originated from genes involved in all biological functions of eukaryotic cells. The comparison of methodologies and technologies to sequence, identify, analyze, and validate circRNA from plant tissues will enable further research to characterize the function and biogenesis of circRNA in L. japonicus.

59 BASIC BIOLOGICAL SCIENCES↗
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 OSTI2024

Arrayed in vivo barcoding for multiplexed sequence verification of plasmid DNA and demultiplexing of pooled libraries

Sequence verification of plasmid DNA is critical for many cloning and molecular biology workflows. To leverage high-throughput sequencing, several methods have been developed that add a unique DNA barcode to individual samples prior to pooling and sequencing. However, these methods require an individual plasmid extraction and/or in vitro barcoding reaction for each sample processed, limiting throughput and adding cost. Here, we develop an arrayed in vivo plasmid barcoding platform that enables pooled plasmid extraction and library preparation for Oxford Nanopore sequencing. This method has a high accuracy and recovery rate, and greatly increases throughput and reduces cost relative to other plasmid barcoding methods or Sanger sequencing. We use in vivo barcoding to sequence verify >45 000 plasmids and show that the method can be used to transform error-containing dispersed plasmid pools into sequence-perfect arrays or well-balanced pools. In vivo barcoding does not require any specialized equipment beyond a low-overhead Oxford Nanopore sequencer, enabling most labs to flexibly process hundreds to thousands of plasmids in parallel.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRSDate not supplied

Method Development for In Situ Microbiome Profiling of the Water Recovery System’s Wastewater Tank Onboard the International Space Station

A distinctive microbial community has inhabited the wastewater tank within the International Space Station Water Recovery System (WRS) for over 14 years and experienced the stressors associated with the microgravity environment. The WRS generates potable water for the crew from urine distillate, humidity condensate, Sabatier product water, and the occasional off-loading of ground-supplied water. The reservoir for these products, the wastewater tank, does not have a means of microbial control. While samples are occasionally collected for analysis, the time between sample collection and the return to Earth, as well as the lack of preservation, results in a skewed depiction of the microbiome. Routinely observed from these returned samples are high counts (105 – 106 colony forming units per mL) and two prevailing genera, Burkholderia and Ralstonia. The wastewater tank likely contains a more diverse microbiome, as a higher diversity of bacteria and fungus has been noted upstream and downstream of the tank. To characterize the microbial profile of the tank, analysis needs to occur at the time of sample collection. Toward this goal, a method for in situ analysis based on nanopore sequencing was developed. The filter-to-sequencer method evolved from previous work that has been validated onboard the ISS (BEST payload and the BioMole Crew Health Care Systems Facility). The method, including filtration, DNA extraction, purification, amplification, library preparation, and nanopore sequencing will be described. Additionally, data collected with this method from both ISS and terrestrial samples will be detailed. The consumables needed to support in situ analysis of the tank are set to the launch to the ISS in the spring of 2023. This investigation will allow for the first accurate characterization of the microbiome of the tank providing insight for crew health, planetary protection, and has the potential to enable engineering controls for future space station water systems.

Sarah Stahl-Rommel↗
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↗
NASA NTRSDate not supplied

Development of Computational Environmental Microbiome Workflows for the Laboratory and the International Space Station

Identification of microorganisms in the spaceflight environment is critical for crew health risk assessment on the International Space Station (ISS). Since 2017, nanopore sequencing technology has been used to support thein situ identification of microbial species during spaceflight. Beginning in 2018, a culture-independent, swab-to-sequencer method was implemented onboard the ISS to provide a more thorough insight of the ISS microbiome. Eliminating microbial culture enables identification of difficult-to-culture organisms, reduces risks associated with potentially pathogenic cultures, and could significantly reduce the time from sample-to-answer. However, this molecular-based approach generates large metagenomic datasets that require substantial computational resources for analysis. To process nanopore-generated sequencing data, the JSC Microbiology Laboratory established a bioinformatics workflow on Amazon EC2 under the security guidance of the NASA Science Managed Cloud Environment (SMCE).This resource allows for the development, testing, and accessing of computational tools for processing large and complex datasets. The work described here will address the downlinking of data from the ISS, the automated pipeline developed to identify targeted bacterial and fungal organisms, and the time from sampling onboard to microbial identification. The pipelines have been enhanced to address high and low biomass samples using optimization based on sample source (air, water, or surface) and type of collection (filter, colony, or swab).The resulting microbiome data can be assessed beyond microbial identifications to gain understanding toward population changes over time, potential selective environmental pressures, and evaluating correlations with a wide range of additional data sets. Metagenome analysis pipelines in development could allow for simultaneous identification of microbial species, gene function, and gene pathways present in the environment. Beyond the ground processing, the developed analysis pipeline is currently deployed onboard the ISS to allow for near real-time assessments of the ISS microbiome. This study serves as a critical foundation for exploration missions, where rapid microbiome analyses will be required.

G. Marie Sharp↗
DOE OSTI2024

Recommendations for Uniform Variant Calling of SARS-CoV-2 Genome Sequence across Bioinformatic Workflows

Genomic sequencing of clinical samples to identify emerging variants of SARS-CoV-2 has been a key public health tool for curbing the spread of the virus. As a result, an unprecedented number of SARS-CoV-2 genomes were sequenced during the COVID-19 pandemic, which allowed for rapid identification of genetic variants, enabling the timely design and testing of therapies and deployment of new vaccine formulations to combat the new variants. However, despite the technological advances of deep sequencing, the analysis of the raw sequence data generated globally is neither standardized nor consistent, leading to vastly disparate sequences that may impact identification of variants. Here, we show that for both Illumina and Oxford Nanopore sequencing platforms, downstream bioinformatic protocols used by industry, government, and academic groups resulted in different virus sequences from same sample. These bioinformatic workflows produced consensus genomes with differences in single nucleotide polymorphisms, inclusion and exclusion of insertions, and/or deletions, despite using the same raw sequence as input datasets. Here, we compared and characterized such discrepancies and propose a specific suite of parameters and protocols that should be adopted across the field. Consistent results from bioinformatic workflows are fundamental to SARS-CoV-2 and future pathogen surveillance efforts, including pandemic preparation, to allow for a data-driven and timely public health response.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Genome sequence, phylogenetic analysis, and structure-based annotation reveal metabolic potential of Chlorella sp. SLA-04

Algae are a broad class of photosynthetic eukaryotes that are phylogenetically and physiologically diverse. Most of the phylogenetic diversity has been inferred from 18S rDNA sequencing since there are only a few complete genomes available in public databases. Here we use ultra-long-read Nanopore sequencing to determine a gapless, telomere-to-telomere complete genome sequence of Chlorella sp. SLA-04, previously described as Chlorella sorokiniana SLA-04. Chlorella sp. SLA-04 is a green alga that grows to high cell density in a wide variety of environments - high and neutral pH, high and low alkalinity, and high and low salinity. SLA-04's ability to grow in high pH and high alkalinity media without external CO 2 supply is favorable for large-scale algal biomass production. Phylogenetic analysis performed using ribosomal DNA and conserved protein sequences consistently reveal that Chlorella sp. SLA-04 forms a distinct lineage from other strains of Chlorella sorokiniana. We complement traditional genome annotation methods with high throughput structural predictions and demonstrate that this approach expands functional prediction of the SLA-04 proteome. Genomic analysis of the SLA-04 genome identifies the genes capable of utilizing TCA cycle intermediates to replenish cytosolic acetyl-CoA pools for lipid production. We also identify a complete metabolic pathway for sphingolipid anabolism that may allow SLA-04 to readily adapt to changing environmental conditions and facilitate robust cultivation in mass production systems. Altogether, this work clarifies the phylogeny of Chlorella sp. SLA-04 within Trebouxiophyceae and demonstrates how structural predictions can be used to improve annotation beyond sequencebased methods.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Complete genome sequence of Cellulomonas sp., strain ES6, a chromate-reducing bacterium isolated from chromium-contaminated subsurface sediment

ABSTRACT Cellulomonas sp. strain ES6 is a chromate-reducing bacterium isolated from chromium contaminated subsurface sediment. Illumina MiSeq and Oxford Nanopore sequencing were used to assemble the genome sequence which consisted of a single circular chromosome of 4.13 Mb, contained 3,960 protein encoding genes and with an overall G + C content 75.38%.

Perea, Katheryn R.↗
NASA NTRSDate not supplied

Congruence of Clusters Defined By Whole Genome Sequencing and MALDI-TOF for Bacteria Isolated From Cleanrooms

Introduction: Oligotrophic conditions can render cleanrooms inhospitable to microbes. Despite these constraints, fungi and bacteria are frequently isolated from surfaces in astromaterials cleanrooms at the Johnson Space Center. Bacillus species are of particular concern because endospores belonging to this genus are resilient and can affect astromaterials. Current monitoring programs rely on 16S rRNA sequencing and the VITEK2 Compact system. These methods have limited power to resolve Bacillus species. Matrix-assisted laser desorption - time of flight mass spectrometry (MALDI-TOF MS), provides a rapid, low cost, method of identifying bacterial isolates and has a higher resolution than 16S rRNA sequencing, particularly for Bacillus species; however, few studies have compared this method to the industry gold standard, whole genome sequencing (WGS). Methods: Based on 16S rRNA classification, we selected 14 isolates for analysis with MALDI-TOF and WGS. Mass spectra were generated with MALDI-TOF MS and processed with custom scripts to identify clusters of closely related isolates and calculate a matrix of pairwise cosine similarity scores. Hybrid Illumina and Nanopore sequencing were used to generate draft genomes. Pairwise similarity scores were calculated from these genomes based on the average amino acid identity (AAI) predicted from single copy core genes. Congruence of clustering between these methods, was assessed by calculating adjusted Rand and Wallace coefficients. Results: Clusters of species generated from MALDI-TOF MS showed good agreement of phylotypes generated with WGS. Pairs of strains that were > 94% similar to each other in terms of predicted amino acid sequences consistently showed cosine similarities of mass spectra > 0.65 and, of the 9 clusters identified with WGS, 8 were identical with MALDI-TOF. This corresponds to an adjusted Rand index of 0.95 and a 95% confidence interval of 0.80 – 1.00 for adjusted Wallace coefficients. The only discordance was for a pair of isolates that were classified as Paenibacillus species. This pair showed relatively high similarity (0.84) in terms of MALDI-TOF MS but only 85% similarity in terms of AAI. Conclusion: This study shows that MALDI-TOF and WGS exhibit a similar ability to delineate Bacillus species isolated from cleanrooms and taxonomic units described by these two methods are consistent with one another. Since MALDI-TOF MS is low in cost and high in throughput, this approach appears to be an ideal option for routine microbial monitoring and identifying Bacillus species.

Farnaz Mazhari↗