A novel approach to interrogating the effects of chemical warfare agent exposure using organ-on-a-chip technology and multiomic analysis.

Organ-on-a-chip platforms are utilized in global bioanalytical and toxicological studies as a way to reduce materials and increase throughput as compared to in vivo based experiments. These platforms bridge the infrastructure and regulatory gaps between in vivo animal work and human systems, with models that exemplify active biological pathways. In conjunction with the advent of increased capabilities associated with next generation sequencing and mass spectrometry based '-omic' technologies, organ-on-a-chip platforms provide an excellent opportunity to investigate the global changes at multiple biological levels, including the transcriptome, proteome and metabolome. When investigated concurrently, a complete profile of cellular and regulatory perturbations can be characterized following treatment with specific agonists. In this study, global effects were observed and analyzed following liver chip exposure to the chemical warfare agent, VX. Even though the primary mechanism of action of VX (i.e. acetylcholinesterase inhibition) is well characterized, recent in vivo studies suggest additional protein binding partners that are implicated in metabolism and cellular energetic pathways. In addition, secondary toxicity associated with peripheral organ systems, especially in human tissues, is not well defined. Our results demonstrate the potential of utilizing an organ-on-a-chip platform as a surrogate system to traditional in vivo studies. This is realized by specifically indicating significant dysregulation of several cellular processes in response to VX exposure including but not limited to amino acid synthesis, drug metabolism, and energetics pathways.

A Rapid, Whole Genome Sequencing Assay for Detection and Characterization of Novel Coronavirus (SARS-CoV-2) Clinical Specimens Using Nanopore Sequencing.

A new human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged at the end of 2019 in Wuhan, China that caused a range of disease severities; including fever, shortness of breath, and coughing. This disease, now known as coronavirus disease 2019 (COVID-19), quickly spread throughout the world, and was declared a pandemic by the World Health Organization in March of 2020. As the disease continues to spread, providing rapid characterization has proven crucial to better inform the design and execution of control measures, such as decontamination methods, diagnostic tests, antiviral drugs, and prophylactic vaccines for long-term control. Our work at the United States Army's Combat Capabilities Development Command Chemical Biological Center (DEVCOM CBC) is focused on engineering workflows to efficiently identify, characterize, and evaluate the threat level of any potential biological threat in the field and more remote, lower resource settings, such as forward operating bases. While we have successfully established untargeted sequencing approaches for detection of pathogens for rapid identification, our current work entails a more in-depth sequencing analysis for use in evolutionary monitoring. We are developing and validating a SARS-CoV-2 nanopore sequencing assay, based on the ARTIC protocol. The standard ARTIC, Illumina, and nanopore sequencing protocols for SARS-CoV-2 are elaborate and time consuming. The new protocol integrates Oxford Nanopore Technology's Rapid Sequencing Kit following targeted RT-PCR of RNA extracted from human clinical specimens. This approach decreases sample manipulations and preparation times. Our current bioinformatics pipeline utilizes Centrifuge as the classifier for quick identification of SARS-CoV-2 and RAMPART software for verification and mapping of reads to the full SARS-CoV-2 genome. ARTIC rapid sequencing results, of previous RT-PCR confirmed patient samples, showed that the modified protocol produces high quality data, with up to 98.9% genome coverage at >1,000x depth for samples with presumably higher viral loads. Furthermore, whole genome assembly and subsequent mutational analysis of six of these sequences identified existing and unique mutations to this cluster, including three in the Spike protein: V308L, P521R, and D614G. This work suggests that an accessible, portable, and relatively fast sample-to-sequence process to characterize viral outbreaks is feasible and effective.

Putative Phenotypically Neutral Genomic Insertion Points in Prokaryotes.

The barriers to effective genome editing in diverse prokaryotic organisms have been falling at an accelerated rate. As editing becomes easier in more organisms, quickly identifying genomic locations to insert new genetic functions without disrupting organism fitness becomes increasingly useful. When the insertion is noncoding DNA for applications such as information storage or barcoding, a neutral insertion point can be especially important. Here we describe an approach to identify putatively neutral insertion sites in prokaryotes. An algorithm (targetFinder) finds convergently transcribed genes with gap sizes within a specified range, and looks for annotations within the gaps. We report putative editing targets for 10 common synthetic biology chassis organisms, including coverage of available RNA-seq data, and provide software to apply to others. We further experimentally evaluate the neutrality of six identified targets in Escherichia coli through insertion of a DNA barcode. We anticipate this information and the accompanying tool will prove useful for synthetic biologists seeking neutral insertion points for genome editing.

A ribosomal operon database and MegaBLAST settings for strain-level resolution of microbiomes.

Current methods to characterize microbial communities generally employ sequencing of the 16S rRNA gene (<500 bp) with high accuracy (∼99%) but limited phylogenetic resolution. However, long-read sequencing now allows for the profiling of near-full-length ribosomal operons (16S-ITS-23S rRNA genes) on platforms such as the Oxford Nanopore MinION. Here, we describe an rRNA operon database with >300 ,000 entries, representing >10 ,000 prokaryotic species and ∼ 150, 000 strains. Additionally, BLAST parameters were identified for strain-level resolution using in silico mutated, mock rRNA operon sequences (70-95% identity) from four bacterial phyla and two members of the Euryarchaeota, mimicking MinION reads. MegaBLAST settings were determined that required <3 s per read on a Mac Mini with strain-level resolution for sequences with >84% identity. These settings were tested on rRNA operon libraries from the human respiratory tract, farm/forest soils and marine sponges ( n = 1, 322, 818 reads for all sample sets). Most rRNA operon reads in this data set yielded best BLAST hits (95 ± 8%). However, only 38-82% of library reads were compatible with strain-level resolution, reflecting the dominance of human/biomedical-associated prokaryotic entries in the database. Since the MinION and the Mac Mini are both portable, this study demonstrates the possibility of rapid strain-level microbiome analysis in the field.

Impact of Porous Matrices and Concentration by Lyophilization on Cell-Free Expression.

Cell-free expression systems have drawn increasing attention as a tool to achieve complex biological functions outside of the cell. Several applications of the technology involve the delivery of functionality to challenging environments, such as field-forward diagnostics or point-of-need manufacturing of pharmaceuticals. To achieve these goals, cell-free reaction components are preserved using encapsulation or lyophilization methods, both of which often involve an embedding of components in porous matrices like paper or hydrogels. Previous work has shown a range of impacts of porous materials on cell-free expression reactions. Here, we explored a panel of 32 paperlike materials and 5 hydrogel materials for the impact on reaction performance. The screen included a tolerance to lyophilization for reaction systems based on both cell lysates and purified expression components. For paperlike materials, we found that (1) materials based on synthetic polymers were mostly incompatible with cell-free expression, (2) lysate-based reactions were largely insensitive to the matrix for cellulosic and microfiber materials, and (3) purified systems had an improved performance when lyophilized in cellulosic but not microfiber matrices. The impact of hydrogel materials ranged from completely inhibitory to a slight enhancement. The exploration of modulating the rehydration volume of lyophilized reactions yielded reaction speed increases using an enzymatic colorimetric reporter of up to twofold with an optimal ratio of 2:1 lyophilized reaction to rehydration volume for the lysate system and 1.5:1 for the purified system. The effect was independent of the matrices assessed. Testing with a fluorescent nonenzymatic reporter and no matrix showed similar improvements in both yields and reaction speeds for the lysate system and yields but not reaction speeds for the purified system. We finally used these observations to show an improved performance of two sensors that span reaction types, matrix, and reporters. In total, these results should enhance efforts to develop field-forward applications of cell-free expression systems.

Rapid Visual Authentication Based on DNA Strand Displacement.

Novel ways to track and verify items of a high value or security is an ever-present need. Taggants made from deoxyribonucleic acid (DNA) have several advantageous properties, such as high information density and robust synthesis; however, existing methods require laboratory techniques to verify, limiting applications. Here, we leverage DNA nanotechnology to create DNA taggants that can be validated in the field in seconds to minutes with a simple equipment. The system is driven by toehold-mediated strand-displacement reactions where matching oligonucleotide sequences drive the generation of a fluorescent signal through the potential energy of base pairing. By pooling different "input" oligonucleotide sequences in a taggant and spatially separating "reporter" oligonucleotide sequences on a paper ticket, unique, sequence-driven patterns emerge for different taggant formulations. Algorithmically generated oligonucleotide sequences show no crosstalk and ink-embedded taggants maintain activity for at least 99 days at 60 °C (equivalent to nearly 2 years at room temperature). The resulting fluorescent signals can be analyzed by the eye or a smartphone when paired with a UV flashlight and filtered glasses.

barCoder: a tool to generate unique, orthogonal genetic tags for qPCR detection.

BACKGROUND: Tracking dispersal of microbial populations in the environment requires specific detection methods that discriminate between the target strain and all potential natural and artificial interferents, including previously utilized tester strains. Recent work has shown that genomic insertion of short identification tags, called "barcodes" here, allows detection of chromosomally tagged strains by real-time PCR. Manual design of these barcodes is feasible for small sets, but expansion of the technique to larger pools of distinct and well-functioning assays would be significantly aided by software-guided design. RESULTS: Here we introduce barCoder, a bioinformatics tool that facilitates the process of creating sets of uniquely identifiable barcoded strains. barCoder utilizes the genomic sequence of the target strain and a set of user-specified PCR parameters to generate a list of suggested barcode "modules" that consist of binding sites for primers and probes, and appropriate spacer sequences. Each module is designed to yield optimal PCR amplification and unique identification. Optimal amplification includes metrics such as ideal melting temperature and G+C content, appropriate spacing, and minimal stem-loop formation; unique identification includes low BLAST hits against the target organism, previously generated barcode modules, and databases (such as NCBI). We tested the ability of our algorithm to suggest appropriate barcodes by generating 12 modules for Bacillus thuringiensis serovar kurstaki-a simulant for the potential biowarfare agent Bacillus anthracis-and three each for other potential target organisms with variable G+C content. Real-time PCR detection assays directed at barcodes were specific and yielded minimal cross-reactivity with a panel of near-neighbor and potential contaminant materials. CONCLUSIONS: The barCoder algorithm facilitates the generation of synthetically barcoded biological simulants by (a) eliminating the task of creating modules by hand, (b) minimizing optimization of PCR assays, and (c) reducing effort wasted on non-unique barcode modules.

Quantification of Interlaboratory Cell-Free Protein Synthesis Variability.

Cell-free protein synthesis (CFPS) platforms, once primarily a research tool to produce difficult to express proteins, are increasingly being pursued by the synthetic biology community for applications including biomanufacturing, rapid screening systems, and field-ready sensors. While consistency within individual studies is apparent in the literature, challenges with reproducing results between laboratories, or even between individuals within a laboratory, are discussed openly by practitioners. As the field continues to grow and move toward applications, a quantitative understanding of expected variability for CFPS and the relative contribution of underlying sources will become increasingly important. Here we offer the first quantitative assessment of interlaboratory variability in CFPS. Three laboratories implemented a single CFPS protocol and performed a series of exchanges, both of material and personnel, designed to quantify relative contributions to variability associated with the site, operator, cell extract preparation, and supplemental reagent preparation. We found that materials prepared at each laboratory, exchanged pairwise, and tested at each site resulted in 40.3% coefficient of variation compared to 7.64% for a single operator across days using a single set of materials. Reagent preparations contributed significantly to observed variability; extract preparations, however, surprisingly did not explain any of the observed variability, even when prepared in different laboratories by different operators. Subsequent exchanges showed that both the site and the operator each contributed to observed interlaboratory variability. In addition to providing the first quantitative assessment of interlaboratory variability in CFPS, these results establish a baseline for individual operator variability across days that can be used as an initial benchmark for community-driven standardization efforts. We anticipate that our results will narrow future avenues of investigation to develop best practices that will ultimately drive down interlaboratory variability, accelerating research progress and informing the suitability of CFPS for real-world applications.

Tracking a serial killer: Integrating phylogenetic relationships, epidemiology, and geography for two invasive meningococcal disease outbreaks.

BACKGROUND: While overall rates of meningococcal disease have been declining in the United States for the past several decades, New York City (NYC) has experienced two serogroup C meningococcal disease outbreaks in 2005-2006 and in 2010-2013. The outbreaks were centered within drug use and sexual networks, were difficult to control, and required vaccine campaigns. METHODS: Whole Genome Sequencing (WGS) was used to analyze preserved meningococcal isolates collected before and during the two outbreaks. We integrated and analyzed epidemiologic, geographic, and genomic data to better understand transmission networks among patients. Betweenness centrality was used as a metric to understand the most important geographic nodes in the transmission networks. Comparative genomics was used to identify genes associated with the outbreaks. RESULTS: Neisseria meningitidis serogroup C (ST11/ET-37) was responsible for both outbreaks with each outbreak having distinct phylogenetic clusters. WGS did identify some misclassifications of isolates that were more distant from the outbreak strains, as well as those that should have been included based on high genomic similarity. Genomes for the second outbreak were more similar than the first and no polymorphism was found to either be unique or specific to either outbreak lineage. Betweenness centrality as applied to transmission networks based on phylogenetic analysis demonstrated that the outbreaks were transmitted within focal communities in NYC with few transmission events to other locations. CONCLUSIONS: Neisseria meningitidis is an ever changing pathogen and comparative genomic analyses can help elucidate how it spreads geographically to facilitate targeted interventions to interrupt transmission.

Use of Unamplified RNA/cDNA-Hybrid Nanopore Sequencing for Rapid Detection and Characterization of RNA Viruses.

Nanopore sequencing, a novel genomics technology, has potential applications for routine biosurveillance, clinical diagnosis, and outbreak investigation of virus infections. Using rapid sequencing of unamplified RNA/cDNA hybrids, we identified Venezuelan equine encephalitis virus and Ebola virus in 3 hours from sample receipt to data acquisition, demonstrating a fieldable technique for RNA virus characterization.

Whole-Genome Sequencing in Microbial Forensic Analysis of Gamma-Irradiated Microbial Materials.

Effective microbial forensic analysis of materials used in a potential biological attack requires robust methods of morphological and genetic characterization of the attack materials in order to enable the attribution of the materials to potential sources and to exclude other potential sources. The genetic homogeneity and potential intersample variability of many of the category A to C bioterrorism agents offer a particular challenge to the generation of attributive signatures, potentially requiring whole-genome or proteomic approaches to be utilized. Currently, irradiation of mail is standard practice at several government facilities judged to be at particularly high risk. Thus, initial forensic signatures would need to be recovered from inactivated (nonviable) material. In the study described in this report, we determined the effects of high-dose gamma irradiation on forensic markers of bacterial biothreat agent surrogate organisms with a particular emphasis on the suitability of genomic DNA (gDNA) recovered from such sources as a template for whole-genome analysis. While irradiation of spores and vegetative cells affected the retention of Gram and spore stains and sheared gDNA into small fragments, we found that irradiated material could be utilized to generate accurate whole-genome sequence data on the Illumina and Roche 454 sequencing platforms.

Pathosphere.org: pathogen detection and characterization through a web-based, open source informatics platform.

BACKGROUND: The detection of pathogens in complex sample backgrounds has been revolutionized by wide access to next-generation sequencing (NGS) platforms. However, analytical methods to support NGS platforms are not as uniformly available. Pathosphere (found at Pathosphere.org) is a cloud - based open - sourced community tool that allows for communication, collaboration and sharing of NGS analytical tools and data amongst scientists working in academia, industry and government. The architecture allows for users to upload data and run available bioinformatics pipelines without the need for onsite processing hardware or technical support. RESULTS: The pathogen detection capabilities hosted on Pathosphere were tested by analyzing pathogen-containing samples sequenced by NGS with both spiked human samples as well as human and zoonotic host backgrounds. Pathosphere analytical pipelines developed by Edgewood Chemical Biological Center (ECBC) identified spiked pathogens within a common sample analyzed by 454, Ion Torrent, and Illumina sequencing platforms. ECBC pipelines also correctly identified pathogens in human samples containing arenavirus in addition to animal samples containing flavivirus and coronavirus. These analytical methods were limited in the detection of sequences with limited homology to previous annotations within NCBI databases, such as parvovirus. Utilizing the pipeline-hosting adaptability of Pathosphere, the analytical suite was supplemented by analytical pipelines designed by the United States Army Medical Research Insititute of Infectious Diseases and Walter Reed Army Institute of Research (USAMRIID-WRAIR). These pipelines were implemented and detected parvovirus sequence in the sample that the ECBC iterative analysis previously failed to identify. CONCLUSIONS: By accurately detecting pathogens in a variety of samples, this work demonstrates the utility of Pathosphere and provides a platform for utilizing, modifying and creating pipelines for a variety of NGS technologies developed to detect pathogens in complex sample backgrounds. These results serve as an exhibition for the existing pipelines and web-based interface of Pathosphere as well as the plug-in adaptability that allows for integration of newer NGS analytical software as it becomes available.

Bacterial and viral identification and differentiation by amplicon sequencing on the MinION nanopore sequencer.

BACKGROUND: The MinION™ nanopore sequencer was recently released to a community of alpha-testers for evaluation using a variety of sequencing applications. Recent reports have tested the ability of the MinION™ to act as a whole genome sequencer and have demonstrated that nanopore sequencing has tremendous potential utility. However, the current nanopore technology still has limitations with respect to error-rate, and this is problematic when attempting to assemble whole genomes without secondary rounds of sequencing to correct errors. In this study, we tested the ability of the MinION™ nanopore sequencer to accurately identify and differentiate bacterial and viral samples via directed sequencing of characteristic genes shared broadly across a target clade. RESULTS: Using a 6 hour sequencing run time, sufficient data were generated to identify an E. coli sample down to the species level from 16S rDNA amplicons. Three poxviruses (cowpox, vaccinia-MVA, and vaccinia-Lister) were identified and differentiated down to the strain level, despite over 98% identity between the vaccinia strains. The ability to differentiate strains by amplicon sequencing on the MinION™ was accomplished despite an observed per-base error rate of approximately 30%. CONCLUSIONS: While nanopore sequencing, using the MinION™ platform from Oxford Nanopore in particular, continues to mature into a commercially available technology, practical uses are sought for the current versions of the technology. This study offers evidence of the utility of amplicon sequencing by demonstrating that the current versions of MinION™ technology can accurately identify and differentiate both viral and bacterial species present within biological samples via amplicon sequencing.

Pathoadaptive mutations in Salmonella enterica isolated after serial passage in mice.

How pathogenic bacteria adapt and evolve in the complex and variable environment of the host remains a largely unresolved question. Here we have used whole genome sequencing of Salmonella enterica serovar Typhimurium LT2 populations serially passaged in mice to identify mutations that adapt bacteria to systemic growth in mice. We found unique pathoadaptive mutations in two global regulators, phoQ and stpA, which increase the competitive indexes of the bacteria 3- to 5-fold. Also, all mouse-adapted lineages had changed the orientation of the hin invertable element, resulting in production of a FliC type of flagellum. Competition experiments in mice with locked flagellum mutants showed that strains expressing the FliC type of flagellum had a 5-fold increase in competitive index as compared to those expressing FljB type flagellum. Combination of the flagellum cassette inversion with the stpA mutation increased competitive indexes up to 20-fold. These experiments show that Salmonella can rapidly adapt to a mouse environment by acquiring a few mutations of moderate individual effect that when combined confer substantial increases in growth.

Comparative genomics of 2009 seasonal plague (Yersinia pestis) in New Mexico.

Plague disease caused by the gram-negative bacterium Yersinia pestis routinely affects animals and occasionally humans, in the western United States. The strains native to the North American continent are thought to be derived from a single introduction in the late 19(th) century. The degree to which these isolates have diverged genetically since their introduction is not clear, and new genomic markers to assay the diversity of North American plague are highly desired. To assay genetic diversity of plague isolates within confined geographic areas, draft genome sequences were generated by 454 pyrosequencing from nine environmental and clinical plague isolates. In silico assemblies of Variable Number Tandem Repeat (VNTR) loci were compared to laboratory-generated profiles for seven markers. High-confidence SNPs and small Insertion/Deletions (Indels) were compared to previously sequenced Y. pestis isolates. The resulting panel of mutations allowed clustering of the strains and tracing of the most likely evolutionary trajectory of the plague strains. The sequences also allowed the identification of new putative SNPs that differentiate the 2009 isolates from previously sequenced plague strains and from each other. In addition, new insertion points for the abundant insertion sequences (IS) of Y. pestis are present that allow additional discrimination of strains; several of these new insertions potentially inactivate genes implicated in virulence. These sequences enable whole-genome phylogenetic analysis and allow the unbiased comparison of closely related isolates of a genetically monomorphic pathogen.

Genomic signatures of strain selection and enhancement in Bacillus atrophaeus var. globigii, a historical biowarfare simulant.

BACKGROUND: Despite the decades-long use of Bacillus atrophaeus var. globigii (BG) as a simulant for biological warfare (BW) agents, knowledge of its genome composition is limited. Furthermore, the ability to differentiate signatures of deliberate adaptation and selection from natural variation is lacking for most bacterial agents. We characterized a lineage of BGwith a long history of use as a simulant for BW operations, focusing on classical bacteriological markers, metabolic profiling and whole-genome shotgun sequencing (WGS). RESULTS: Archival strains and two "present day" type strains were compared to simulant strains on different laboratory media. Several of the samples produced multiple colony morphotypes that differed from that of an archival isolate. To trace the microevolutionary history of these isolates, we obtained WGS data for several archival and present-day strains and morphotypes. Bacillus-wide phylogenetic analysis identified B. subtilis as the nearest neighbor to B. atrophaeus. The genome of B. atrophaeus is, on average, 86% identical to B. subtilis on the nucleotide level. WGS of variants revealed that several strains were mixed but highly related populations and uncovered a progressive accumulation of mutations among the "military" isolates. Metabolic profiling and microscopic examination of bacterial cultures revealed enhanced growth of "military" isolates on lactate-containing media, and showed that the "military" strains exhibited a hypersporulating phenotype. CONCLUSIONS: Our analysis revealed the genomic and phenotypic signatures of strain adaptation and deliberate selection for traits that were desirable in a simulant organism. Together, these results demonstrate the power of whole-genome and modern systems-level approaches to characterize microbial lineages to develop and validate forensic markers for strain discrimination and reveal signatures of deliberate adaptation.