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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 289 records · Page 16
All sourcesNASA NTRSDOE OSTI
DOE OSTI2013

Anchoring and ordering NGS contig assemblies by population sequencing (POPSEQ)

Next-generation whole-genome shotgun assemblies of complex genomes are highly useful, but fail to link nearby sequence contigs with each other or provide a linear order of contigs along individual chromosomes. Here, we introduce a strategy based on sequencing progeny of a segregating population that allows de novo production of a genetically anchored linear assembly of the gene space of an organism. We demonstrate the power of the approach by reconstructing the chromosomal organization of the gene space of barley, a large, complex and highly repetitive 5.1 Gb genome. We evaluate the robustness of the new assembly by comparison to a recently released physical and genetic framework of the barley genome, and to various genetically ordered sequence-based genotypic datasets. The method is independent of the need for any prior sequence resources, and will enable rapid and cost-efficient establishment of powerful genomic information for many species.

population sequencing↗
DOE OSTI2018

A novel ADCK4 mutation in a Chinese family with ADCK4-Associated glomerulopathy

Highlights: • ADCK4 is located on human chromosome 19q13.2 and encodes the ADCK4 protein, which interacts with members of the CoQ10 biosynthesis pathway. • Mutations in the ADCK4 gene can cause CoQ10 deficiency and mitochondrial nephropathy (ADCK4-associated glomerulopathy, ADCK4-GN). • ADCK4-GN mainly manifests as steroid resistant nephrotic syndrome clinically and focal segmental glomerulosclerosis pathologically. • The C-terminal portion in ADCK4 is important and indispensable. The upstream nonsense/frameshift mutations should be considered deleterious. • CoQ10 treatment may reduce proteinuria and postpone ADCK4-GN progression. AarF domain-containing kinase 4 (ADCK4)-associated glomerulopathy (ADCK4-GN) is an inherited mitochondrial nephropathy caused by mutations in the ADCK4 gene. Herein, we report a case of ADCK4-GN. The patient, a 14-year-old Chinese male, presented with asymptomatic proteinuria and steroid resistance. A renal biopsy showed focal segmental glomerulosclerosis (FSGS) and dysmorphic mitochondria in podocytes. Coenzyme Q10 treatment was partially effective. No adverse events were observed. Next generation and Sanger sequencing analyses revealed compound heterozygous mutations (c.625C > G, p.D209H and c.918G > T, p.C306X). Both inherited mutations are located in the highly conserved ABC1 domain. The unreported nonsense mutation causes a loss of 197 amino acids from the C-terminal portion of ADCK4, suggesting a deleterious effect of the truncating mutation by abolishing ADCK4 function. In conclusion, we identified a novel ABC1 domain-localized pathogenic mutation responsible for ADCK4-GN, further supporting the importance of the C-terminal portion of ADCK4. Next generation sequencing facilitated the early diagnosis. CoQ{sub 10} treatment may reduce proteinuria and postpone ADCK4-GN progression.

60 APPLIED LIFE SCIENCES↗
NASA NTRSDate not supplied

Guanine Oxidation in the Genome, not RNA Editing, Accounts for Single Nucleotide Variation in the Exome of Mice Flown on Board the ISS

We have conducted a further analysis of single nucleotide variation (somatic mutation) in mice flown aboard the ISS. We used data archived in GeneLab from a cohort of 18-week-old mice were flown to the ISS, housed in the Rodent Habitat and therefore subjected to microgravity for 37 days. Mice of similar age, sex and the same strain were used for ground controls housed in identical hardware and simulating, but not matching ISS environmental conditions (temperature, humidity and gas atmosphere). Primary data consists of next generation RNA sequencing for the tissues examined: eye, liver, skeletal muscle and kidney. We used novel software, developed at NASA Ames Research Center and deployed on the NASA Ames Supercomputer, to perform variant calling for single point mutations. Unexpectedly, we discovered a high degree of somatic mutation in ISS-flown mice, compared to controls. We found that the extent of somatic mutation correlated with the degree of gene expression in the four tissue types, with the highest degree of somatic mutation observed in genes with the highest degree of expression. Careful analysis that included measurement of specific nucleotide changes that occurred demonstrated that guanine substitutions were the most frequent, consistent with the hypothesis that reactive oxygen species-mediated guanine oxidation was responsible for the hypermutation events. By contrast, adenine substitutions would be expected if gene editing were responsible for the somatic mutation. These types of substitutions were much less frequent. The implication of these findings for astronaut health in a variety of mission scenarios will be discussed.

International Space Station↗
DOE OSTI2015

Impacts of degraded DNA on restriction enzyme associated DNA sequencing ( RADS eq)

Abstract Degraded DNA from suboptimal field sampling is common in molecular ecology. However, its impact on techniques that use restriction site associated next‐generation DNA sequencing ( RADS eq, GBS ) is unknown. We experimentally examined the effects of in situ DNA degradation on data generation for a modified double‐digest RADS eq approach (3 RAD ). We generated libraries using genomic DNA serially extracted from the muscle tissue of 8 individual lake whitefish ( Coregonus clupeaformis ) following 0‐, 12‐, 48‐ and 96‐h incubation at room temperature posteuthanasia. This treatment of the tissue resulted in input DNA that ranged in quality from nearly intact to highly sheared. All samples were sequenced as a multiplexed pool on an Illumina MiSeq. Libraries created from low to moderately degraded DNA (12–48 h) performed well. In contrast, the number of RAD tags per individual, number of variable sites, and percentage of identical RAD tags retained were all dramatically reduced when libraries were made using highly degraded DNA (96‐h group). This reduction in performance was largely due to a significant and unexpected loss of raw reads as a result of poor quality scores. Our findings remained consistent after changes in restriction enzymes, modified fold coverage values (2‐ to 16‐fold), and additional read‐length trimming. We conclude that starting DNA quality is an important consideration for RADS eq; however, the approach remains robust until genomic DNA is extensively degraded.

Graham, Carly F.↗
DOE OSTI2021

Discovery of photosynthesis genes through whole-genome sequencing of acetate-requiring mutants of Chlamydomonas reinhardtii

Large-scale mutant libraries have been indispensable for genetic studies, and the development of next-generation genome sequencing technologies has greatly advanced efforts to analyze mutants. In this work, we sequenced the genomes of 660 Chlamydomonas reinhardtii acetate-requiring mutants, part of a larger photosynthesis mutant collection previously generated by insertional mutagenesis with a linearized plasmid. We identified 554 insertion events from 509 mutants by mapping the plasmid insertion sites through paired-end sequences, in which one end aligned to the plasmid and the other to a chromosomal location. Nearly all (96%) of the events were associated with deletions, duplications, or more complex rearrangements of genomic DNA at the sites of plasmid insertion, and together with deletions that were unassociated with a plasmid insertion, 1470 genes were identified to be affected. Functional annotations of these genes were enriched in those related to photosynthesis, signaling, and tetrapyrrole synthesis as would be expected from a library enriched for photosynthesis mutants. Systematic manual analysis of the disrupted genes for each mutant generated a list of 253 higher-confidence candidate photosynthesis genes, and we experimentally validated two genes that are essential for photoautotrophic growth, CrLPA3 and CrPSBP4 . The inventory of candidate genes includes 53 genes from a phylogenomically defined set of conserved genes in green algae and plants. Altogether, 70 candidate genes encode proteins with previously characterized functions in photosynthesis in Chlamydomonas , land plants, and/or cyanobacteria; 14 genes encode proteins previously shown to have functions unrelated to photosynthesis. Among the remaining 169 uncharacterized genes, 38 genes encode proteins without any functional annotation, signifying that our results connect a function related to photosynthesis to these previously unknown proteins. This mutant library, with genome sequences that reveal the molecular extent of the chromosomal lesions and resulting higher-confidence candidate genes, will aid in advancing gene discovery and protein functional analysis in photosynthesis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Assessing impacts of unconventional natural gas extraction on microbial communities in headwater stream ecosystems in Northwestern Pennsylvania

Hydraulic fracturing and horizontal drilling have increased dramatically in Pennsylvania Marcellus shale formations, however the potential for major environmental impacts are still incompletely understood. High-throughput sequencing of the 16S rRNA gene was performed to characterize the microbial community structure of water, sediment, bryophyte, and biofilm samples from 26 headwater stream sites in northwestern Pennsylvania with different histories of fracking activity within Marcellus shale formations. Further, we describe the relationship between microbial community structure and environmental parameters measured. Approximately 3.2 million 16S rRNA gene sequences were retrieved from a total of 58 samples. Microbial community analyses showed significant reductions in species richness as well as evenness in sites with Marcellus shale activity. Beta diversity analyses revealed distinct microbial community structure between sites with and without Marcellus shale activity. For example, operational taxonomic units (OTUs) within the Acetobacteracea, Methylocystaceae, Acidobacteriaceae, and Phenylobacterium were greater than three log-fold more abundant in MSA+ sites as compared to MSA- sites. Further, several of these OTUs were strongly negatively correlated with pH and positively correlated with the number of well-pads in a watershed. It should be noted that many of the OTUs enriched in MSA+ sites are putative acidophilic and/or methanotrophic populations. This study revealed apparent shifts in the autochthonous microbial communities and highlighted potential members that could be responding to changing stream conditions as a result of nascent industrial activity in these aquatic ecosystems.

beta diversity↗
DOE OSTI2016

More of an art than a science: Using microbial DNA sequences to compose music

Bacteria are everywhere. Microbial ecology is emerging as a critical field for understanding the relationships between these ubiquitous bacterial communities, the environment, and human health. Next generation DNA sequencing technology provides us a powerful tool to indirectly observe the communities by sequencing and analyzing all of the bacterial DNA present in an environment. The results of the DNA sequencing experiments can generate gigabytes to terabytes of information however, making it difficult for the citizen scientist to grasp and the educator to convey this data. Here, we present a method for interpreting massive amounts of microbial ecology data as musical performances, easily generated on any computer and using only commonly available or freely available software and the ‘Microbial Bebop’ algorithm. Furthermore, using this approach citizen scientists and biology educators can sonify complex data in a fun and interactive format, making it easier to communicate both the importance and the excitement of exploring the planet earth’s largest ecosystem.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Improved assemblies using a source-agnostic pipeline for MetaGenomic Assembly by Merging (MeGAMerge) of contigs

Assembly of metagenomic samples is a very complex process, with algorithms designed to address sequencing platform-specific issues, (read length, data volume, and/or community complexity), while also faced with genomes that differ greatly in nucleotide compositional biases and in abundance. To address these issues, we have developed a post-assembly process: MetaGenomic Assembly by Merging (MeGAMerge). We compare this process to the performance of several assemblers, using both real, and in-silico generated samples of different community composition and complexity. MeGAMerge consistently outperforms individual assembly methods, producing larger contigs with an increased number of predicted genes, without replication of data. MeGAMerge contigs are supported by read mapping and contig alignment data, when using synthetically-derived and real metagenomic data, as well as by gene prediction analyses and similarity searches. Ultimately, MeGAMerge is a flexible method that generates improved metagenome assemblies, with the ability to accommodate upcoming sequencing platforms, as well as present and future assembly algorithms.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

A Case Study into Microbial Genome Assembly Gap Sequences and Finishing Strategies

This study characterized regions of DNA which remained unassembled by either PacBio and Illumina sequencing technologies for seven bacterial genomes. Two genomes were manually finished using bioinformatics and PCR/Sanger sequencing approaches and regions not assembled by automated software were analyzed. Gaps present within Illumina assemblies mostly correspond to repetitive DNA regions such as multiple rRNA operon sequences. PacBio gap sequences were evaluated for several properties such as GC content, read coverage, gap length, ability to form strong secondary structures, and corresponding annotations. Our hypothesis that strong secondary DNA structures blocked DNA polymerases and contributed to gap sequences was not accepted. PacBio assemblies had few limitations overall and gaps were explained as cumulative effect of lower than average sequence coverage and repetitive sequences at contig termini. An important aspect of the present study is the compilation of biological features that interfered with assembly and included active transposons, multiple plasmid sequences, phage DNA integration, and large sequence duplication. Furthermore, our targeted genome finishing approach and systematic evaluation of the unassembled DNA will be useful for others looking to close, finish, and polish microbial genome sequences.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

The past, present and future of microbiome analyses

Microbes evolved on Earth approximately 3.5 billion years ago and eventually occupied every habitable environment in its biosphere. While microorganisms are responsible for key functions on Earth including carbon and nutrient cycling and determining the health and disease of its inhabitants (i.e. plants and animals), >99% of the estimated trillions of microbes have yet to be discovered.1 In addition, high microbial diversity has made it difficult to study specific functions carried out by complex microbial communities within "microbiomes", such as those inhabiting the soil and human gut.2,3 Fortunately, technological advances over the last few decades have greatly facilitated studies of complex microbiomes and their functions. Here we will specifically discuss advances related to nucleic acid sequencing and mass spectrometry analyses that have enabled exploration and understanding of complex microbiomes in a range of environments and our own bodies.4,5,6,7

Next Generation sequencing and Mass Spec↗
DOE OSTI2015

Insights from 20 years of bacterial genome sequencing

Since the first two complete bacterial genome sequences were published in 1995, the science of bacteria has dramatically changed. Using third-generation DNA sequencing, it is possible to completely sequence a bacterial genome in a few hours and identify some types of methylation sites along the genome as well. Sequencing of bacterial genome sequences is now a standard procedure, and the information from tens of thousands of bacterial genomes has had a major impact on our views of the bacterial world. In this review, we explore a series of questions to highlight some insights that comparative genomics has produced. To date, there are genome sequences available from 50 different bacterial phyla and 11 different archaeal phyla. However, the distribution is quite skewed towards a few phyla that contain model organisms. But the breadth is continuing to improve, with projects dedicated to filling in less characterized taxonomic groups. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system provides bacteria with immunity against viruses, which outnumber bacteria by tenfold. How fast can we go? Second-generation sequencing has produced a large number of draft genomes (close to 90 % of bacterial genomes in GenBank are currently not complete); third-generation sequencing can potentially produce a finished genome in a few hours, and at the same time provide methlylation sites along the entire chromosome. The diversity of bacterial communities is extensive as is evident from the genome sequences available from 50 different bacterial phyla and 11 different archaeal phyla. Genome sequencing can help in classifying an organism, and in the case where multiple genomes of the same species are available, it is possible to calculate the pan- and core genomes; comparison of more than 2000 Escherichia coli genomes finds an E. coli core genome of about 3100 gene families and a total of about 89,000 different gene families. Why do we care about bacterial genome sequencing? There are many practical applications, such as genome-scale metabolic modeling, biosurveillance, bioforensics, and infectious disease epidemiology. In the near future, high-throughput sequencing of patient metagenomic samples could revolutionize medicine in terms of speed and accuracy of finding pathogens and knowing how to treat them.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRSDate not supplied

Elevating the Quality of Space Omics Sequencing Data: Innovations and Methodologies from NASA GeneLab Sample Processing Laboratory

NASA’s GeneLab, part of the NASA Open Science Data Repository, is a space-related database that hosts a diverse range of transcriptomics, proteomics, epigenomics and genomics data. The NASA GeneLab Sample Processing Laboratory (SPL) generates omics data from biological experiments conducted aboard the International Space Station, Space Shuttle and space related ground experiments, this omics data then hosted on the GeneLab repository. Samples generated such experiments pose numerous technical challenges such as small experimental sample size, variance in dissection times, limited tissue preservation methods, prolonged storage time, and more. GeneLab SPL team had developed specialized expertise in nucleic acid extraction, library preparation and sequencing of such biological samples via extensive training and years of experience. In order to ensure data accuracy and consistency across experiments, SPL has developed standardized protocols for each species and tissue type. These protocols in conjunction with quality control metrics and data standards are crucial in generating of high-quality data. SPL protocols and standards have been developed in collaboration with the scientific community and had been made publicly available on the GeneLab portal, guaranteeing comparability of datasets across spaceflight experiments. To ensure reliability of data generation, SPL leverages cutting-edge innovations in laboratory automation for sample processing. By leveraging these state-of-the-art platforms, SPL achieves high levels of data reproducibility while significantly minimizing sources of bias and variability, especially across experiments with large numbers of samples. Over the past few years, the space biology investigator community has accessed SPL-generated data from the Open Science Data Repository for a myriad of data re-analysis and re-use studies. We observe a trend that in-house SPL-generated data consistently outperforms outsourced sequencing data in terms of technical standards, quality control metrics, timeliness of data delivery, and sequencing and reagent efficiency. Superior data generation has and will continue to enable discoveries in disease, diagnostic tools, and the biological effects of long duration spaceflight.

GeneLab↗
DOE OSTI2024

Ongoing Cooperative Engagement Facilitates Agile Pandemic and Outbreak Response: Lessons Learned Through Cooperative Engagement Between Uganda and the United States

Pathogens threaten human lives and disrupt economies around the world. This has been clearly illustrated by the current COVID-19 pandemic and outbreaks in livestock and food crops. Here, to manage pathogen emergence and spread, cooperative engagement programs develop and strengthen biosafety, biosecurity, and biosurveillance capabilities among local researchers to detect pathogens. In this case study, we describe the efforts of a collaboration between the Los Alamos National Laboratory and the Uganda Virus Research Institute, the primary viral diagnostic laboratory in Uganda, to implement and ensure the sustainability of sequencing for biosurveillance. We describe the process of establishing this capability along with the lessons learned from both sides of the partnership to inform future cooperative engagement efforts in low- and middle-income countries. We found that by strengthening sequencing capabilities at the Uganda Virus Research Institute before the COVID-19 pandemic, the institute was able to successfully sequence SARS-CoV-2 samples and provide data to the scientific community. We highlight the need to strengthen and sustain capabilities through in-country training, collaborative research projects, and trust.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Comparative Sequence Analysis of Multidrug-Resistant IncA/C Plasmids from Salmonella enterica

Determinants of multidrug resistance (MDR) are often encoded on mobile elements, such as plasmids, transposons, and integrons, which have the potential to transfer among foodborne pathogens, as well as to other virulent pathogens, increasing the threats these traits pose to human and veterinary health. Our understanding of MDR among Salmonella has been limited by the lack of closed plasmid genomes for comparisons across resistance phenotypes, due to difficulties in effectively separating the DNA of these high-molecular weight, low-copy-number plasmids from chromosomal DNA. To resolve this problem, we demonstrate an efficient protocol for isolating, sequencing and closing IncA/C plasmids from Salmonella sp. using single molecule real-time sequencing on a Pacific Biosciences (Pacbio) RS II Sequencer. We obtained six Salmonella enterica isolates from poultry, representing six different serovars, each exhibiting the MDR-Ampc resistance profile. Salmonella plasmids were obtained using a modified mini preparation and transformed with Escherichia coli DH10Br. A Qiagen Large-Construct kitTM was used to recover highly concentrated and purified plasmid DNA that was sequenced using PacBio technology. These six closed IncA/C plasmids ranged in size from 104 to 191 kb and shared a stable, conserved backbone containing 98 core genes, with only six differences among those core genes. The plasmids encoded a number of antimicrobial resistance genes, including those for quaternary ammonium compounds and mercury. We then compared our six IncA/C plasmid sequences: first with 14 IncA/C plasmids derived from S. enterica available at the National Center for Biotechnology Information (NCBI), and then with an additional 38 IncA/C plasmids derived from different taxa. These comparisons allowed us to build an evolutionary picture of how antimicrobial resistance may be mediated by this common plasmid backbone. Our project provides detailed genetic information about resistance genes in plasmids, advances in plasmid sequencing, and phylogenetic analyses, and important insights about how MDR evolution occurs across diverse serotypes from different animal sources, particularly in agricultural settings where antimicrobial drug use practices vary.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Improved Microbial Community Characterization of 16S rRNA via Metagenome Hybridization Capture Enrichment

Environmental microbial diversity is often investigated from a molecular perspective using 16S ribosomal RNA (rRNA) gene amplicons and shotgun metagenomics. While amplicon methods are fast, low-cost, and have curated reference databases, they can suffer from amplification bias and are limited in genomic scope. In contrast, shotgun metagenomic methods sample more genomic regions with fewer sequence acquisition biases, but are much more expensive (even with moderate sequencing depth) and computationally challenging. Here, we develop a set of 16S rRNA sequence capture baits that offer a potential middle ground with the advantages from both approaches for investigating microbial communities. These baits cover the diversity of all 16S rRNA sequences available in the Greengenes (v. 13.5) database, with no sequence having <78% sequence identity to at least one bait for all segments of 16S. The use of our baits provide comparable results to 16S amplicon libraries and shotgun metagenomic libraries when assigning taxonomic units from 16S sequences within the metagenomic reads. We demonstrate that 16S rRNA capture baits can be used on a range of microbial samples (i.e., mock communities and rodent fecal samples) to increase the proportion of 16S rRNA sequences (average > 400-fold) and decrease analysis time to obtain consistent community assessments. Furthermore, our study reveals that bioinformatic methods used to analyze sequencing data may have a greater influence on estimates of community composition than library preparation method used, likely due in part to the extent and curation of the reference databases considered. Thus, enriching existing aliquots of shotgun metagenomic libraries and obtaining modest numbers of reads from them offers an efficient orthogonal method for assessment of bacterial community composition.

59 BASIC BIOLOGICAL SCIENCES↗