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All sources NASA NTRS DOE OSTI

DOE OSTI2023

Intensity of sample processing methods impacts wastewater SARS-CoV-2 whole genome amplicon sequencing outcomes

Wastewater SARS-CoV-2 surveillance has been deployed since the beginning of the COVID-19 pandemic to monitor the dynamics in virus burden in local communities. Genomic surveillance of SARS-CoV-2 in wastewater, particularly efforts aimed at whole genome sequencing for variant tracking and identification, are still challenging due to low target concentration, complex microbial and chemical background, and lack of robust nucleic acid recovery experimental procedures. The intrinsic sample limitations are inherent to wastewater and are thus unavoidable. Here, we use a statistical approach that couples correlation analyses to a random forest-based machine learning algorithm to evaluate potentially important factors associated with wastewater SARS-CoV-2 whole genome amplicon sequencing outcomes, with a specific focus on the breadth of genome coverage. We collected 182 composite and grab wastewater samples from the Chicago area between November 2020 to October 2021. Samples were processed using a mixture of processing methods reflecting different homogenization intensities (HA + Zymo beads, HA + glass beads, and Nanotrap), and were sequenced using one of the two library preparation kits (the Illumina COVIDseq kit and the QIAseq DIRECT kit). Technical factors evaluated using statistical and machine learning approaches include sample types, certain sample intrinsic features, and processing and sequencing methods. The results suggested that sample processing methods could be a predominant factor affecting sequencing outcomes, and library preparation kits was considered a minor factor. Finally, a synthetic SARS-CoV-2 RNA spike-in experiment was performed to validate the impact from processing methods and suggested that the intensity of the processing methods could lead to different RNA fragmentation

60 APPLIED LIFE SCIENCES↗

DOE OSTI2022

Sequence-Controlled Secondary Structures and Stimuli Responsiveness of Bioinspired Polyampholytes

In this work, a comprehensive study focusing on the influence of the sequence charge pattern on the secondary structure preferences of annealed polyampholytes and their responsiveness to external stimuli is presented. Two sequences are designed composed entirely of ionizable amino acids (charge fraction f=1), and an equal number of positive and negative charges (f + =f - =0.5) with distinct charge-patterns consisting of lysine and glutamic acid monomers. The study reveals that the sequence charge pattern has a significant influence on the secondary structure preferences of polyampholytes at physiological pH. Furthermore, it shows that external stimuli such as pH, ionic strength and solvent dielectric constant can be used to modulate the secondary structure of the two designed sequences. The observed secondary structure transformations for the two sequences are also substantially different from those determined for uniformly charged polypeptides-homo-polypeptides under matching conditions.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2013

Moving Away from the Reference Genome: Evaluating a Peptide Sequencing Tagging Approach for Single Amino Acid Polymorphism Identifications in the Genus Populus

The genetic diversity across natural populations of the model organism, Populus, is extensive, containing a single nucleotide polymorphism roughly every 200 base pairs. When deviations from the reference genome occur in coding regions, they can impact protein sequences. Rather than relying on a static reference database to profile protein expression, we employed a peptide sequence tagging (PST) approach capable of decoding the plasticity of the Populus proteome. Using shotgun proteomics data from two genotypes of P. trichocarpa, a tag-based approach enabled the detection of 6,653 unexpected sequence variants. Through manual validation, our study investigated how the most abundant chemical modification (methionine oxidation) could masquerade as a sequence variant (AlaSer) when few site-determining ions existed. In fact, precise localization of an oxidation site for peptides with more than one potential placement was indeterminate for 70% of the MS/MS spectra. We demonstrate that additional fragment ions made available by high energy collisional dissociation enhances the robustness of the peptide sequence tagging approach (81% of oxidation events could be exclusively localized to a methionine). We are confident that augmenting fragmentation processes for a PST approach will further improve the identification of single amino acid polymorphism in Populus and potentially other species as well.

High energy collisional dissociation↗

DOE OSTI2012

THE YOUNG OPEN CLUSTERS KING 12, NGC 7788, AND NGC 7790: PRE-MAIN-SEQUENCE STARS AND EXTENDED STELLAR HALOS

The stellar contents of the open clusters King 12, NGC 7788, and NGC 7790 are investigated using MegaCam images. Comparisons with isochrones yield an age <20 Myr for King 12, 20-40 Myr for NGC 7788, and 60-80 Myr for NGC 7790 based on the properties of stars near the main-sequence turnoff (MSTO) in each cluster. The reddening of NGC 7788 is much larger than previously estimated. The luminosity functions (LFs) of King 12 and NGC 7788 show breaks that are attributed to the onset of pre-main-sequence (PMS) objects, and comparisons with models of PMS evolution yield ages that are consistent with those measured from stars near the MSTO. In contrast, the r' LF of main-sequence stars in NGC 7790 is matched to r' = 20 by a model that is based on the solar neighborhood mass function. The structural properties of all three clusters are investigated by examining the two-point angular correlation function of blue main-sequence stars. King 12 and NGC 7788 are each surrounded by a stellar halo that extends out to a radius of 5 arcmin ({approx}3.4 pc). It is suggested that these halos form in response to large-scale mass ejection early in the evolution of the clusters, as predicted by models. In contrast, blue main-sequence stars in NGC 7790 are traced out to a radius of {approx}7.5 arcmin ({approx}5.5 pc), with no evidence of a halo. It is suggested that all three clusters may have originated in the same star-forming complex, but not in the same giant molecular cloud.

79 ASTRONOMY AND ASTROPHYSICS↗

DOE OSTI2013

MAIN-SEQUENCE STAR POPULATIONS IN THE VIRGO OVERDENSITY REGION

We present deep color-magnitude diagrams (CMDs) for two Subaru Suprime-Cam fields in the Virgo Stellar Stream (VSS)/Virgo Overdensity (VOD) and compare them to a field centered on the highest concentration of Sagittarius (Sgr) Tidal Stream stars in the leading arm, Branch A of the bifurcation. A prominent population of main-sequence stars is detected in all three fields and can be traced as faint as g Almost-Equal-To 24 mag. Using theoretical isochrone fitting, we derive an age of 9.1{sup +1.0}{sub -1.1} Gyr, a median abundance of [Fe/H] = -0.70{sup +0.15}{sub -0.20} dex, and a heliocentric distance of 30.9 {+-} 3.0 kpc for the main sequence of the Sgr Stream Branch A. The dominant main-sequence populations in the two VSS/VOD fields ({Lambda}{sub Sun} Almost-Equal-To 265 Degree-Sign , B{sub Sun} Almost-Equal-To 13 Degree-Sign ) are located at a mean distance of 23.3 {+-} 1.6 kpc and have an age of {approx}8.2 Gyr, and an abundance of [Fe/H] = -0.67{sup +0.16}{sub -0.12} dex, similar to the Sgr Stream stars. These statistically robust parameters, derived from the photometry of 260 main-sequence stars, are also in good agreement with the age of the main population in the Sgr dwarf galaxy (8.0 {+-} 1.5 Gyr). They also agree with the peak in the metallicity distribution of 2-3 Gyr old M giants, [Fe/H] Almost-Equal-To -0.6 dex, in the Sgr north leading arm. We then compare the results from the VSS/VOD fields with the Sgr Tidal Stream model by Law and Majewski based on a triaxial Galactic halo shape that is empirically calibrated with Sloan Digital Sky Survey Sgr A-branch and Two Micron All Sky Survey M-giant stars. We find that the most prominent feature in the CMDs, the main-sequence population at 23 kpc, is not explained by the model. Instead the model predicts in these directions a low-density filamentary structure of Sgr debris stars at {approx}9 kpc and a slightly higher concentration of Sgr stars spread over a heliocentric distance range of 42-53 kpc. At best there is only marginal evidence for the presence of these populations in our data. Our findings then suggest that while there are probably some Sgr debris stars present, the dominant stellar population in the VOD originates from a different halo structure that has an almost identical age and metallicity as some sections of the Sgr tidal stream.

79 ASTRONOMY AND ASTROPHYSICS↗

DOE OSTI2013

FLAMES AND XSHOOTER SPECTROSCOPY ALONG THE TWO BLUE STRAGGLER STAR SEQUENCES OF M30

We present spectroscopic observations acquired with FLAMES and XSHOOTER at the Very Large Telescope for a sample of 15 blue straggler stars (BSSs) in the globular cluster (GC) M30. The targets have been selected to sample the two BSS sequences discovered, with seven BSSs along the blue sequence and eight along the red one. No difference in the kinematical properties of the two groups of BSSs has been found. In particular, almost all the observed BSSs have projected rotational velocities lower than {approx}30 km s{sup -1}, with only one (blue) fast rotating BSS (>90 km s{sup -1}), identified as a W UMa binary. This rotational velocity distribution is similar to those obtained in 47 Tucanae and NGC 6397, while M4 remains the only GC studied so far that harbors a large fraction of fast rotating BSSs. All stars hotter than {approx}7800 K (regardless of the parent BSS sequence) show iron abundances larger than those measured from normal cluster stars, with a clear-cut trend with the effective temperature. This behavior suggests that particle transport mechanisms driven by radiative levitation occur in the photosphere of these stars, as already observed for the BSSs in NGC 6397. Finally, four BSSs belonging to the red sequence (not affected by radiative levitation) show a strong depletion of [O/Fe], with respect to the abundance measured in red giant branch and horizontal branch stars. This O-depletion is compatible with the chemical signature expected in BSSs formed by mass-transfer processes in binary systems, in agreement with the mechanism proposed for the formation of BSSs in the red sequence.

79 ASTRONOMY AND ASTROPHYSICS↗

DOE OSTI2015

High-resolution imaging of PHIBSS z ∼ 2 main-sequence galaxies in CO J = 1 → 0

We present Karl Jansky Very Large Array observations of the CO J=1−0 transition in a sample of four z∼2 main-sequence galaxies. These galaxies are in the blue sequence of star-forming galaxies at their redshift, and are part of the IRAM Plateau de Bure HIgh-z Blue Sequence Survey which imaged them in CO J=3−2. Two galaxies are imaged here at high signal-to-noise, allowing determinations of their disk sizes, line profiles, molecular surface densities, and excitation. Using these and published measurements, we show that the CO and optical disks have similar sizes in main-sequence galaxies, and in the galaxy where we can compare CO J=1−0 and J=3−2 sizes we find these are also very similar. Assuming a Galactic CO-to-H{sub 2} conversion, we measure surface densities of Σ{sub mol}∼1200 M{sub ⊙} pc{sup −2} in projection and estimate Σ{sub mol}∼ 500–900 M{sub ⊙} pc{sup −2} deprojected. Finally, our data yields velocity-integrated Rayleigh–Jeans brightness temperature line ratios r{sub 31} that are approximately at unity. In addition to the similar disk sizes, the very similar line profiles in J=1−0 and J=3−2 indicate that both transitions sample the same kinematics, implying that their emission is coextensive. We conclude that in these two main-sequence galaxies there is no evidence for significant excitation gradients or a large molecular reservoir that is diffuse or cold and not involved in active star formation. We suggest that r{sub 31} in very actively star-forming galaxies is likely an indicator of how well-mixed the star formation activity and the molecular reservoir are.

79 ASTRONOMY AND ASTROPHYSICS↗

DOE OSTI2014

THE HABITABLE ZONES OF PRE-MAIN-SEQUENCE STARS

We calculate the pre-main-sequence habitable zone (HZ) for stars of spectral classes F-M. The spatial distribution of liquid water and its change during the pre-main-sequence phase of protoplanetary systems is important for understanding how planets become habitable. Such worlds are interesting targets for future missions because the coolest stars could provide habitable conditions for up to 2.5 billion years post-accretion. Moreover, for a given star type, planetary systems are more easily resolved because of higher pre-main-sequence stellar luminosities, resulting in larger planet-star separation for cool stars than is the case for the traditional main-sequence (MS) HZ. We use one-dimensional radiative-convective climate and stellar evolutionary models to calculate pre-main-sequence HZ distances for F1-M8 stellar types. We also show that accreting planets that are later located in the traditional MS HZ orbiting stars cooler than a K5 (including the full range of M stars) receive stellar fluxes that exceed the runaway greenhouse threshold, and thus may lose substantial amounts of water initially delivered to them. We predict that M-star planets need to initially accrete more water than Earth did, or, alternatively, have additional water delivered later during the long pre-MS phase to remain habitable. Our findings are also consistent with recent claims that Venus lost its water during accretion.

79 ASTRONOMY AND ASTROPHYSICS↗

DOE OSTI2023

Snekmer: a scalable pipeline for protein sequence fingerprinting based on amino acid recoding

Abstract Motivation The vast expansion of sequence data generated from single organisms and microbiomes has precipitated the need for faster and more sensitive methods to assess evolutionary and functional relationships between proteins. Representing proteins as sets of short peptide sequences (kmers) has been used for rapid, accurate classification of proteins into functional categories; however, this approach employs an exact-match methodology and thus may be limited in terms of sensitivity and coverage. We have previously used similarity groupings, based on the chemical properties of amino acids, to form reduced character sets and recode proteins. This amino acid recoding (AAR) approach simplifies the construction of protein representations in the form of kmer vectors, which can link sequences with distant sequence similarity and provide accurate classification of problematic protein families. Results Here, we describe Snekmer, a software tool for recoding proteins into AAR kmer vectors and performing either (i) construction of supervised classification models trained on input protein families or (ii) clustering for de novo determination of protein families. We provide examples of the operation of the tool against a set of nitrogen cycling families originally collected using both standard hidden Markov models and a larger set of proteins from Uniprot and demonstrate that our method accurately differentiates these sequences in both operation modes. Availability and implementation Snekmer is written in Python using Snakemake. Code and data used in this article, along with tutorial notebooks, are available at http://github.com/PNNL-CompBio/Snekmer under an open-source BSD-3 license. Supplementary information Supplementary data are available at Bioinformatics Advances online.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Comparison of the spatial statistics of random and defined-sequence photoresist films

The resolution-line edge roughness-sensitivity tradeoff has motivated the exploration of potential improvements using defined sequence polymers and polymer-bound photoacid generators and quenchers. We characterize the internal structures of positive tone photoresist polymer films formed from defined sequence polymers and compare them with random copolymers of the same composition. We model their imaging to connect initially to developable film structures. We use a polymer packing algorithm to simulate films of diverse compositions and locations of photoacid generators and quenchers, using the composition of an ESCAP photoresist. We use a simple extreme ultraviolet exposure-deprotection algorithm to model developable image formation within them. In all cases, the spatial distribution of chemical moieties in the film for defined sequence polymers is nearly indistinguishable from random copolymers. We evaluate several exposure-deprotection scenarios and find that a defined sequence copolymer has a distinctive developable image under certain circumstances. The use of defined sequence polymers within a photoresist layer does not automatically result in improved imaging; however, they do have some characteristics different from random polymers of the same composition. Further study of these characteristics may provide a route to improved control over the nanoscale imaging process.

36 MATERIALS SCIENCE↗

DOE OSTI2015

Identification and Resolution of Microdiversity through Metagenomic Sequencing of Parallel Consortia

To gain a predictive understanding of the interspecies interactions within microbial communities that govern community function, the genomic complement of every member population must be determined. Although metagenomic sequencing has enabled the de novo reconstruction of some microbial genomes from environmental communities, microdiversity confounds current genome reconstruction techniques. To overcome this issue, we performed short-read metagenomic sequencing on parallel consortia, defined as consortia cultivated under the same conditions from the same natural community with overlapping species composition. The differences in species abundance between the two consortia allowed reconstruction of near-complete (at an estimated >85% of gene complement) genome sequences for 17 of the 20 detected member species. Two Halomonas spp. indistinguishable by amplicon analysis were found to be present within the community. In addition, comparison of metagenomic reads against the consensus scaffolds revealed within-species variation for one of the Halomonas populations, one of the Rhodobacteraceae populations, and the Rhizobiales population. Genomic comparison of these representative instances of inter- and intraspecies microdiversity suggests differences in functional potential that may result in the expression of distinct roles in the community. In addition, isolation and complete genome sequence determination of six member species allowed an investigation into the sensitivity and specificity of genome reconstruction processes, demonstrating robustness across a wide range of sequence coverage (9× to 2,700×) within the metagenomic data set.

metagenomic sequencing↗

DOE OSTI2014

Targeted Analysis of Whole Genome Sequence Data to Diagnose Genetic Cardiomyopathy

Background—Cardiomyopathy is highly heritable but genetically diverse. At present, genetic testing for cardiomyopathy uses targeted sequencing to simultaneously assess the coding regions of more than 50 genes. New genes are routinely added to panels to improve the diagnostic yield. With the anticipated $1000 genome, it is expected that genetic testing will shift towards comprehensive genome sequencing accompanied by targeted gene analysis. Therefore, we assessed the reliability of whole genome sequencing and targeted analysis to identify cardiomyopathy variants in 11 subjects with cardiomyopathy. Methods and Results—Whole genome sequencing with an average of 37× coverage was combined with targeted analysis focused on 204 genes linked to cardiomyopathy. Genetic variants were scored using multiple prediction algorithms combined with frequency data from public databases. This pipeline yielded 1-14 potentially pathogenic variants per individual. Variants were further analyzed using clinical criteria and/or segregation analysis. Three of three previously identified primary mutations were detected by this analysis. In six subjects for whom the primary mutation was previously unknown, we identified mutations that segregated with disease, had clinical correlates, and/or had additional pathological correlation to provide evidence for causality. For two subjects with previously known primary mutations, we identified additional variants that may act as modifiers of disease severity. In total, we identified the likely pathological mutation in 9 of 11 (82%) subjects. We conclude that these pilot data demonstrate that ~30-40× coverage whole genome sequencing combined with targeted analysis is feasible and sensitive to identify rare variants in cardiomyopathy-associated genes.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2016

Identification of a Latin American-specific BabA adhesin variant through whole genome sequencing of Helicobacter pylori patient isolates from Nicaragua

In this study, Helicobacter pylori (H. pylori) is one of the most common bacterial infections in humans and this infection can lead to gastric ulcers and gastric cancer. H. pylori is one of the most genetically variable human pathogens and the ability of the bacterium to bind to the host epithelium as well as the presence of different virulence factors and genetic variants within these genes have been associated with disease severity. Nicaragua has particularly high gastric cancer incidence and we therefore studied Nicaraguan clinical H. pylori isolates for factors that could contribute to cancer risk. The complete genomes of fifty-two Nicaraguan H. pylori i isolates were sequenced and assembled de novo, and phylogenetic and virulence factor analyses were performed. The Nicaraguan isolates showed phylogenetic relationship with West African isolates in whole-genome sequence comparisons and with Western and urban South-and Central American isolates using MLSA (Multi-locus sequence analysis). A majority, 77 % of the isolates carried the cancer-associated virulence gene cagA and also the s1/i1/m1 vacuolating cytotoxin, vacA allele combination, which is linked to increased severity of disease. Specifically, we also found that Nicaraguan isolates have a blood group-binding adhesin (BabA) variant highly similar to previously reported BabA sequences from Latin America, including from isolates belonging to other phylogenetic groups. These BabA sequences were found to be under positive selection at several amino acid positions that differed from the global collection of isolates. In conclusion, the discovery of a Latin American BabA variant, independent of overall phylogenetic background, suggests hitherto unknown host or environmental factors within the Latin American population giving H. pylori isolates carrying this adhesin variant a selective advantage, which could affect pathogenesis and risk for sequelae through specific adherence properties.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Structural Models and Sequence Alignment Results of the Desulfovibrio vulgaris Proteome

This dataset contains the structural models for the primary transcripts of the Desulfovibrio vulgaris proteome as well as sequence alignment results for a subset of the encoded proteins. For each protein, the five models inferred from AlphaFold 2 are provided. The largest pTM-scoring model for each protein was energy minimized; this minimized structure as well as its AlphaFold pickle output file are also provided. This set of structures represent an alternate source of models for the D. vulgaris proteome to those available in the AlphaFold Protein Structure Database (AFDB). This is a bit more complicated since the proteins reporting in the AFDB originate from an outdated form of the D. vulgaris sequence. The different versions of the D. vulgaris gene annotation are collected in the Chronology subdirectory; further consideration of these changes on the structural space of the proteome are currently underway. For proteins that have been annotated as hypothetical, sequence alignment results from the HHblits and SAdLSA alignment methods are provided. These methods are often more capable to resolve sequence homology than other methods. Therefore, the results from both HHblits and SAdLSA are provided to identify possible homologs for these challenging proteins. Numerous sequence databases are utilized for these alignments. References AlphaFold v2 Multimer: https://doi.org/10.1101/2021.10.04.463034. References HHblits: hhtps://doi.org/10.1186/s12859-019-3019-7. References SAdLSA: hhtps://doi.org/10.3389/fbinf.2021.689960.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Classification of bacterial plasmid and chromosome derived sequences using machine learning

Plasmids are important genetic elements that facilitate horizonal gene transfer between bacteria and contribute to the spread of virulence and antimicrobial resistance. Most bacterial genome sequences in the public archives exist in draft form with many contigs, making it difficult to determine if a contig is of chromosomal or plasmid origin. Using a training set of contigs comprising 10,584 chromosomes and 10,654 plasmids from the PATRIC database, we evaluated several machine learning models including random forest, logistic regression, XGBoost, and a neural network for their ability to classify chromosomal and plasmid sequences using nucleotide k-mers as features. Based on the methods tested, a neural network model that used nucleotide 6-mers as features that was trained on randomly selected chromosomal and plasmid subsequences 5kb in length achieved the best performance, outperforming existing out-of-the-box methods, with an average accuracy of 89.38% ± 2.16% over a 10-fold cross validation. The model accuracy can be improved to 92.08% by using a voting strategy when classifying holdout sequences. In both plasmids and chromosomes, subsequences encoding functions involved in horizontal gene transfer—including hypothetical proteins, transporters, phage, mobile elements, and CRISPR elements—were most likely to be misclassified by the model. This study provides a straightforward approach for identifying plasmid-encoding sequences in short read assemblies without the need for sequence alignment-based tools.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2019

Short papers on current state of sequencing, metagenomics, and RNAseq for diagnostics

This report contains three papers on the current state of sequencing, metagenomics, and RNAseq for diagnostics. 1) There are many factors that must be evaluated before ascertaining what type of technology is most cost effective, and/or most technically effective. In general, long read technologies are primarily used for in field/in situ sequencing and for closing isolate genomes, while short read sequencing lends itself to rapid high quality determination of strain identity and to applications that require high depth, such as metagenomes and counting applications, like in RNAseq. 2) RNAseq can provide some important insights into the biology of active infections and provide advances in diagnostics approaches. Depth of sequencing coverage is critical to determine gene expression profiles. Full use of RNAseq data from both host and pathogen(s) for diagnostics and informed treatment options will require further investigation. 3) RNAseq can provide some important insights into the biology of active infections and provide advances in diagnostics approaches. Depth of sequencing coverage is critical to determine gene expression profiles. Full use of RNAseq data from both host and pathogen(s) for diagnostics and informed treatment options will require further investigation.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2006

Spitzer Space Telescope Sequencing Operations Software, Strategies, and Lessons Learned

The Space Infrared Telescope Facility (SIRTF) was launched in August, 2003, and renamed to the Spitzer Space Telescope in 2004. Two years of observing the universe in the wavelength range from 3 to 180 microns has yielded enormous scientific discoveries. Since this magnificent observatory has a limited lifetime, maximizing science viewing efficiency (ie, maximizing time spent executing activities directly related to science observations) was the key operational objective. The strategy employed for maximizing science viewing efficiency was to optimize spacecraft flexibility, adaptability, and use of observation time. The selected approach involved implementation of a multi-engine sequencing architecture coupled with nondeterministic spacecraft and science execution times. This approach, though effective, added much complexity to uplink operations and sequence development. The Jet Propulsion Laboratory (JPL) manages Spitzer s operations. As part of the uplink process, Spitzer s Mission Sequence Team (MST) was tasked with processing observatory inputs from the Spitzer Science Center (SSC) into efficiently integrated, constraint-checked, and modeled review and command products which accommodated the complexity of non-deterministic spacecraft and science event executions without increasing operations costs. The MST developed processes, scripts, and participated in the adaptation of multi-mission core software to enable rapid processing of complex sequences. The MST was also tasked with developing a Downlink Keyword File (DKF) which could instruct Deep Space Network (DSN) stations on how and when to configure themselves to receive Spitzer science data. As MST and uplink operations developed, important lessons were learned that should be applied to future missions, especially those missions which employ command-intensive operations via a multi-engine sequence architecture.

missions operations↗

NASA NTRS2018

Monitoring Astronaut Health with DNA Sequencing

In recent years microbe a plethora of microbe populations have been identified onboard the ISS (International Space Station). Approaches for real-time tracking of microbes for routine housekeeping and food/water safety monitoring will be critical for mission safety and crew health on future longer duration missions to the Moon or Mars. This work is a proof-of-concept study demonstrating an end-to-end phylogenetic identification and full genome sequencing effort of multiple microbial populations. Our methodology utilized the ISS flight-certified WetLab-2 molecular toolbox and the Biomolecule Sequencer projects for real-time end-to-end on-orbit microbial biological samples processing and molecular analysis with real time results generated utilizing only field "offline" analytic software. For this experiment we colony-cultured several ISS isolated microorganisms before generation of the pre-sequencing library via the automated VolTRAX device which enabled high library turnover with little wet-bench activity or potential future costly astronaut time. The pre-sequencing library is diluted in loading buffer and injected into the MinION sample port, drawn into the nanopore window by capillary action, and sequenced using the MinKnown. 16S and full genome alignment, nucleotide matching, gene identification, and phylogenetic sorting was accomplished utilizing the Epi2me software and the offline NCBI Blast viral, microbiome, and human somatic databases. In short, the methodologies developed herein replace the myriad of specific, often highly targeted microbiological tests used in the clinical laboratory, which would be difficult if not impossible to currently implement aboard the ISS or in deep space, with a single metagenomics test.

genomics↗