Metagenomic Methods for Addressing NASA's Planetary Protection Policy Requirements on Future Missions: A Workshop Report.

Molecular biology methods and technologies have advanced substantially over the past decade. These new molecular methods should be incorporated among the standard tools of planetary protection (PP) and could be validated for incorporation by 2026. To address the feasibility of applying modern molecular techniques to such an application, NASA conducted a technology workshop with private industry partners, academics, and government agency stakeholders, along with NASA staff and contractors. The technical discussions and presentations of the Multi-Mission Metagenomics Technology Development Workshop focused on modernizing and supplementing the current PP assays. The goals of the workshop were to assess the state of metagenomics and other advanced molecular techniques in the context of providing a validated framework to supplement the bacterial endospore-based NASA Standard Assay and to identify knowledge and technology gaps. In particular, workshop participants were tasked with discussing metagenomics as a stand-alone technology to provide rapid and comprehensive analysis of total nucleic acids and viable microorganisms on spacecraft surfaces, thereby allowing for the development of tailored and cost-effective microbial reduction plans for each hardware item on a spacecraft. Workshop participants recommended metagenomics approaches as the only data source that can adequately feed into quantitative microbial risk assessment models for evaluating the risk of forward (exploring extraterrestrial planet) and back (Earth harmful biological) contamination. Participants were unanimous that a metagenomics workflow, in tandem with rapid targeted quantitative (digital) PCR, represents a revolutionary advance over existing methods for the assessment of microbial bioburden on spacecraft surfaces. The workshop highlighted low biomass sampling, reagent contamination, and inconsistent bioinformatics data analysis as key areas for technology development. Finally, it was concluded that implementing metagenomics as an additional workflow for addressing concerns of NASA's robotic mission will represent a dramatic improvement in technology advancement for PP and will benefit future missions where mission success is affected by backward and forward contamination.

Microbiome and metagenomic analysis of Lake Hillier Australia reveals pigment-rich polyextremophiles and wide-ranging metabolic adaptations.

Lake Hillier is a hypersaline lake known for its distinctive bright pink color. The cause of this phenomenon in other hypersaline sites has been attributed to halophiles, Dunaliella, and Salinibacter, however, a systematic analysis of the microbial communities, their functional features, and the prevalence of pigment-producing-metabolisms has not been previously studied. Through metagenomic sequencing and culture-based approaches, our results evidence that Lake Hillier is composed of a diverse set of microorganisms including archaea, bacteria, algae, and viruses. Our data indicate that the microbiome in Lake Hillier is composed of multiple pigment-producer microbes, including Dunaliella, Salinibacter, Halobacillus, Psychroflexus, Halorubrum, many of which are cataloged as polyextremophiles. Additionally, we estimated the diversity of metabolic pathways in the lake and determined that many of these are related to pigment production. We reconstructed complete or partial genomes for 21 discrete bacteria (N = 14) and archaea (N = 7), only 2 of which could be taxonomically annotated to previously observed species. Our findings provide the first metagenomic study to decipher the source of the pink color of Australia's Lake Hillier. The study of this pink hypersaline environment is evidence of a microbial consortium of pigment producers, a repertoire of polyextremophiles, a core microbiome and potentially novel species.

Complete Genome Sequence of Klebsiella quasipneumoniae subsp. similipneumoniae Strain IF3SW-P1, Isolated from the International Space Station.

The 5.2-Mb circular genome of Klebsiella quasipneumoniae subsp. similipneumoniae strain IF3SW-P1, isolated from the International Space Station, was sequenced using Oxford Nanopore Technologies. The genome lacks a megaplasmid typical of hypervirulent and multidrug-resistant Klebsiella strains but does contain a chromosomally encoded OqxAB efflux pump associated with carbapenem resistance.

Performance assessment of DNA sequencing platforms in the ABRF Next-Generation Sequencing Study.

Assessing the reproducibility, accuracy and utility of massively parallel DNA sequencing platforms remains an ongoing challenge. Here the Association of Biomolecular Resource Facilities (ABRF) Next-Generation Sequencing Study benchmarks the performance of a set of sequencing instruments (HiSeq/NovaSeq/paired-end 2 × 250-bp chemistry, Ion S5/Proton, PacBio circular consensus sequencing (CCS), Oxford Nanopore Technologies PromethION/MinION, BGISEQ-500/MGISEQ-2000 and GS111) on human and bacterial reference DNA samples. Among short-read instruments, HiSeq 4000 and X10 provided the most consistent, highest genome coverage, while BGI/MGISEQ provided the lowest sequencing error rates. The long-read instrument PacBio CCS had the highest reference-based mapping rate and lowest non-mapping rate. The two long-read platforms PacBio CCS and PromethION/MinION showed the best sequence mapping in repeat-rich areas and across homopolymers. NovaSeq 6000 using 2 × 250-bp read chemistry was the most robust instrument for capturing known insertion/deletion events. This study serves as a benchmark for current genomics technologies, as well as a resource to inform experimental design and next-generation sequencing variant calling.

Immunotranscriptomic profiling the acute and clearance phases of a human challenge dengue virus serotype 2 infection model.

About 20-25% of dengue virus (DENV) infections become symptomatic ranging from self-limiting fever to shock. Immune gene expression changes during progression to severe dengue have been documented in hospitalized patients; however, baseline or kinetic information is difficult to standardize in natural infection. Here we profile the host immunotranscriptome response in humans before, during, and after infection with a partially attenuated rDEN2Δ30 challenge virus (ClinicalTrials.gov NCT02021968). Inflammatory genes including type I interferon and viral restriction pathways are induced during DENV2 viremia and return to baseline after viral clearance, while others including myeloid, migratory, humoral, and growth factor immune regulation factors pathways are found at non-baseline levels post-viremia. Furthermore, pre-infection baseline gene expression is useful to predict rDEN2Δ30-induced immune responses and the development of rash. Our results suggest a distinct immunological profile for mild rDEN2Δ30 infection and offer new potential biomarkers for characterizing primary DENV infection.

Loop-Mediated Isothermal Amplification Detection of SARS-CoV-2 and Myriad Other Applications.

As the second year of the COVID-19 pandemic begins, it remains clear that a massive increase in the ability to test for SARS-CoV-2 infections in a myriad of settings is critical to controlling the pandemic and to preparing for future outbreaks. The current gold standard for molecular diagnostics is the polymerase chain reaction (PCR), but the extraordinary and unmet demand for testing in a variety of environments means that both complementary and supplementary testing solutions are still needed. This review highlights the role that loop-mediated isothermal amplification (LAMP) has had in filling this global testing need, providing a faster and easier means of testing, and what it can do for future applications, pathogens, and the preparation for future outbreaks. This review describes the current state of the art for research of LAMP-based SARS-CoV-2 testing, as well as its implications for other pathogens and testing. The authors represent the global LAMP (gLAMP) Consortium, an international research collective, which has regularly met to share their experiences on LAMP deployment and best practices; sections are devoted to all aspects of LAMP testing, including preanalytic sample processing, target amplification, and amplicon detection, then the hardware and software required for deployment are discussed, and finally, a summary of the current regulatory landscape is provided. Included as well are a series of first-person accounts of LAMP method development and deployment. The final discussion section provides the reader with a distillation of the most validated testing methods and their paths to implementation. This review also aims to provide practical information and insight for a range of audiences: for a research audience, to help accelerate research through sharing of best practices; for an implementation audience, to help get testing up and running quickly; and for a public health, clinical, and policy audience, to help convey the breadth of the effect that LAMP methods have to offer.

Factors influencing recovery of SARS-CoV-2 RNA in raw sewage and wastewater sludge using polyethylene glycol-based concentration method.

Wastewater surveillance for monitoring severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important epidemiologic tool for the assessment of population-wide coronavirus disease 2019 (COVID-19). This tool can be successfully implemented only if SARS-CoV-2 RNA in wastewater can be accurately recovered and quantified. The lack of standardized procedure for wastewater virus analysis has resulted in varying SARS-CoV-2 concentrations for the same sample. This study reports the effect of 4 key factors-sample volume, percentage polyethylene glycol (PEG)-NaCl, incubation period, and storage duration at 4°C-on the recovery of spiked noninfectious SARS-CoV-2 RNA in raw sewage and sludge samples. N1 and N2 genes of SARS-CoV-2 were quantified using the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and digital droplet PCR (RT-ddPCR) techniques. Results indicate that 1) for raw sewage, 50-ml sample volume, 30% PEG-NaCl addition, 6-h incubation, and sample analysis within 24 h of collection can result in much better RNA recovery (RT-qPCR: 72% for N1 and 82% for N2; RT-ddPCR: 55% for N1 and 85% for N2) when compared with commonly used PEG-based method; 2) for sludge, the sample analysis using raw sewage protocol and all other variations of each factor mostly resulted in false negatives for both N1 and N2. The absence of N1 and N2 suggests that sludge samples probably need a pretreatment step that releases RNA entrapped in sludge solids back into bulk solution. In conclusion, our modified PEG-based concentration method can cut down the analysis time at least by half, which in turn helps to implement early detection system for SARS-CoV-2 in wastewater.

Molecular Characterization of Increased Amplicon Lengths in SARS-CoV-2 Reverse Transcription Loop-Mediated Isothermal Amplification Assays.

Loop-mediated isothermal amplification (LAMP) is a power tool for the amplification of specific RNA and DNA targets. Much like PCR, LAMP requires primers that surround a target amplification region and generates exponential product through a unique highly specific daisy-chain single-temperature amplification reaction. However, until recently, attempts to amplify targets of greater than 200 base pairs (bp) have been mostly unsuccessful and limited to short amplicon targets of less than 150 bp. Although short amplicons have the benefit of a rapid detection (<40 min), they do not allow for the prediction of RNA integrity based on RNA length and possible intactness. In this study, 8 primer sets were developed using 2 LAMP primer-specific software packages against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid gene with insert lengths ranging from 262 to 945 bp in order to amplify and infer the integrity of viral RNA. Because these amplification lengths are greater than the current methods that use an insert length of 130 or less, they require a longer incubation, modified primer and temperature strategies, and the addition of specific adjuncts to prevent nonspecific amplification. This proof of concept study resulted in successful reverse transcription LAMP reactions for amplicon targets of 262, 687, 693, and 945 bp using a clinical nasopharyngeal NP sample, purified SARS-CoV-2 RNA, and crude lysate containing inactivated virus.

Advanced biofilm analysis in streams receiving organic deicer runoff.

Prolific heterotrophic biofilm growth is a common occurrence in airport receiving streams containing deicers and anti-icers, which are composed of low-molecular weight organic compounds. This study investigated biofilm spatiotemporal patterns and responses to concurrent and antecedent (i.e., preceding biofilm sampling) environmental conditions at stream sites upstream and downstream from Milwaukee Mitchell International Airport in Milwaukee, Wisconsin, during two deicing seasons (2009-2010; 2010-2011). Biofilm abundance and community composition were investigated along spatial and temporal gradients using field surveys and microarray analyses, respectively. Given the recognized role of Sphaerotilus in organically enriched environments, additional analyses were pursued to specifically characterize its abundance: a consensus sthA sequence was determined via comparison of whole metagenome sequences with a previously identified sthA sequence, the primers developed for this gene were used to characterize relative Sphaerotilus abundance using quantitative real-time PCR, and a Sphaerotilus strain was isolated to validate the determined sthA sequence. Results indicated that biofilm abundance was stimulated by elevated antecedent chemical oxygen demand concentrations, a surrogate for deicer concentrations, with minimal biofilm volumes observed when antecedent chemical oxygen demand concentrations remained below 48 mg/L. Biofilms were composed of diverse communities (including sheathed bacterium Thiothrix) whose composition appeared to shift in relation to antecedent temperature and chemical oxygen demand. The relative abundance of sthA correlated most strongly with heterotrophic biofilm volume (positive) and dissolved oxygen (negative), indicating that Sphaerotilus was likely a consistent biofilm member and thrived under low oxygen conditions. Additional investigations identified the isolate as a new strain of Sphaerotilus montanus (strain KMKE) able to use deicer components as carbon sources and found that stream dissolved oxygen concentrations related inversely to biofilm volume as well as to antecedent temperature and chemical oxygen demand. The airport setting provides insight into potential consequences of widescale adoption of organic deicers for roadway deicing.

Life and its traces in Antarctica's McMurdo Dry Valley paleolakes: a survey of preservation.

The extremely cold and arid conditions of Antarctica make it uniquely positioned to investigate fundamental questions regarding the persistence of life in extreme environments. Within the McMurdo Dry Valleys and surrounding mountain ranges are multiple ancient relict lakes, paleolakes, with lacustrine deposits spanning from thousands to millions of years in age. Here we present data from light microscopy, scanning electron microscopy, electron dispersive x-ray spectroscopy, and radiocarbon dating to catalog the remarkable range of life preserved within these deposits. This includes intact microbes and nanobacteria-sized cocci, CaCO3 precipitations consistent with biogenic calcium, previously undescribed net-like structures, possible dormant spores, and long-extinct yet exquisitely preserved non-vascular plants. These images provide an important reference for further microbiome investigations of Antarctic paleolake samples. In addition, these findings may provide a visual reference for the use of subsurface groundwater microbial communities as an analog for paleolake subsurface water on planets such as Mars.

Characterizing species interactions that contribute to biofilm formation in a multispecies model of a potable water bacterial community.

Microbial biofilms are ubiquitous in drinking water systems, yet our understanding of drinking water biofilms lags behind our understanding of those in other environments. Here, a six-member model bacterial community was used to identify the interactions and individual contributions of each species to community biofilm formation. These bacteria were isolated from the International Space Station potable water system and include Cupriavidus metallidurans, Chryseobacterium gleum, Ralstonia insidiosa, Ralstonia pickettii, Methylorubrum (Methylobacterium) populi and Sphingomonas paucimobilis, but all six species are common members of terrestrial potable water systems. Using reconstituted assemblages, from pairs to all 6 members, community biofilm formation was observed to be robust to the absence of any single species and only removal of the C. gleum/S. paucimobilis pair, out of all 15 possible 2-species subtractions, led to loss of community biofilm formation. In conjunction with these findings, dual-species biofilm formation assays supported the view that the contribution of C. gleum to community biofilm formation was dependent on synergistic biofilm formation with either R. insidiosa or C. metallidurans. These data support a model of multiple, partially redundant species interactions to generate robustness in biofilm formation. A bacteriophage and multiple predatory bacteria were used to test the resilience of the community to the removal of individual members in situ, but the combination of precise and substantial depletion of a single target species was not achievable. We propose that this assemblage can be used as a tractable model to understand the molecular bases of the interactions described here and to decipher other functions of drinking water biofilms.

Real-Time DNA Sequencing in the Antarctic Dry Valleys Using the Oxford Nanopore Sequencer.

The ability to sequence DNA outside of the laboratory setting has enabled novel research questions to be addressed in the field in diverse areas, ranging from environmental microbiology to viral epidemics. Here, we demonstrate the application of offline DNA sequencing of environmental samples using a hand-held nanopore sequencer in a remote field location: the McMurdo Dry Valleys, Antarctica. Sequencing was performed using a MK1B MinION sequencer from Oxford Nanopore Technologies (ONT; Oxford, United Kingdom) that was equipped with software to operate without internet connectivity. One-direction (1D) genomic libraries were prepared using portable field techniques on DNA isolated from desiccated microbial mats. By adequately insulating the sequencer and laptop, it was possible to run the sequencing protocol for up to 2½ h under arduous conditions.

Acetate supplementation as a means of inducing glioblastoma stem-like cell growth arrest.

Glioblastoma (GBM), the most common primary adult malignant brain tumor, is associated with a poor prognosis due, in part, to tumor recurrence mediated by chemotherapy and radiation resistant glioma stem-like cells (GSCs). The metabolic and epigenetic state of GSCs differs from their non-GSC counterparts, with GSCs exhibiting greater glycolytic metabolism and global hypoacetylation. However, little attention has been focused on the potential use of acetate supplementation as a therapeutic approach. N-acetyl-l-aspartate (NAA), the primary storage form of brain acetate, and aspartoacylase (ASPA), the enzyme responsible for NAA catalysis, are significantly reduced in GBM tumors. We recently demonstrated that NAA supplementation is not an appropriate therapeutic approach since it increases GSC proliferation and pursued an alternative acetate source. The FDA approved food additive Triacetin (glyceryl triacetate, GTA) has been safely used for acetate supplementation therapy in Canavan disease, a leukodystrophy due to ASPA mutation. This study characterized the effects of GTA on the proliferation and differentiation of six primary GBM-derived GSCs relative to established U87 and U251 GBM cell lines, normal human cerebral cortical astrocytes, and murine neural stem cells. GTA reduced proliferation of GSCs greater than established GBM lines. Moreover, GTA reduced growth of the more aggressive mesenchymal GSCs greater than proneural GSCs. Although sodium acetate induced a dose-dependent reduction of GSC growth, it also reduced cell viability. GTA-mediated growth inhibition was not associated with differentiation, but increased protein acetylation. These data suggest that GTA-mediated acetate supplementation is a novel therapeutic strategy to inhibit GSC growth.

Multi-platform assessment of transcriptome profiling using RNA-seq in the ABRF next-generation sequencing study.

High-throughput RNA sequencing (RNA-seq) greatly expands the potential for genomics discoveries, but the wide variety of platforms, protocols and performance capabilitites has created the need for comprehensive reference data. Here we describe the Association of Biomolecular Resource Facilities next-generation sequencing (ABRF-NGS) study on RNA-seq. We carried out replicate experiments across 15 laboratory sites using reference RNA standards to test four protocols (poly-A-selected, ribo-depleted, size-selected and degraded) on five sequencing platforms (Illumina HiSeq, Life Technologies PGM and Proton, Pacific Biosciences RS and Roche 454). The results show high intraplatform (Spearman rank R > 0.86) and inter-platform (R > 0.83) concordance for expression measures across the deep-count platforms, but highly variable efficiency and cost for splice junction and variant detection between all platforms. For intact RNA, gene expression profiles from rRNA-depletion and poly-A enrichment are similar. In addition, rRNA depletion enables effective analysis of degraded RNA samples. This study provides a broad foundation for cross-platform standardization, evaluation and improvement of RNA-seq.

Acetate supplementation induces growth arrest of NG2/PDGFRα-positive oligodendroglioma-derived tumor-initiating cells.

Cancer is associated with globally hypoacetylated chromatin and considerable attention has recently been focused on epigenetic therapies. N-acetyl-L-aspartate (NAA), the primary storage form of acetate in the brain, and aspartoacylase (ASPA), the enzyme responsible for NAA catalysis to generate acetate and ultimately acetyl-Coenzyme A for histone acetylation, are reduced in oligodendroglioma. The short chain triglyceride glyceryl triacetate (GTA), which increases histone acetylation and inhibits histone deacetylase expression, has been safely used for acetate supplementation in Canavan disease, a leukodystrophy due to ASPA mutation. We demonstrate that GTA induces cytostatic G0 growth arrest of oligodendroglioma-derived cells in vitro, without affecting normal cells. Sodium acetate, at doses comparable to that generated by complete GTA catalysis, but not glycerol also promoted growth arrest, whereas long chain triglycerides promoted cell growth. To begin to elucidate its mechanism of action, the effects of GTA on ASPA and acetyl-CoA synthetase protein levels and differentiation of established human oligodendroglioma cells (HOG and Hs683) and primary tumor-derived oligodendroglioma cells that exhibit some features of cancer stem cells (grade II OG33 and grade III OG35) relative to an oligodendrocyte progenitor line (Oli-Neu) were examined. The nuclear localization of ASPA and acetyl-CoA synthetase-1 in untreated cells was regulated during the cell cycle. GTA-mediated growth arrest was not associated with apoptosis or differentiation, but increased expression of acetylated proteins. Thus, GTA-mediated acetate supplementation may provide a safe, novel epigenetic therapy to reduce the growth of oligodendroglioma cells without affecting normal neural stem or oligodendrocyte progenitor cell proliferation or differentiation.