Assessment of Next Generation Sequencing Technologies for \u3ci\u3eDe novo\u3c/i\u3e and Hybrid Assemblies of Challenging Bacterial Genomes

In past decade, tremendous progress has been made in DNA sequencing methodologies in terms of throughput, speed, read-lengths, along with a sharp decrease in per base cost. These technologies, commonly referred to as next-generation sequencing (NGS) are complimented by the development of hybrid assembly approaches which can utilize multiple NGS platforms. In the first part of my dissertation I performed systematic evaluations and optimizations of nine de novo and hybrid assembly protocols across four novel microbial genomes. While each had strengths and weaknesses, via optimization using multiple strategies I obtained dramatic improvements in overall assembly size and quality. To select the best assembly, I also proposed the novel rDNA operon validation approach to evaluate assembly accuracy. Additionally, I investigated the ability of third-generation PacBio sequencing platform and achieved automated finishing of Clostridium autoethanogenum without any accessory data. These complete genome sequences facilitated comparisons which revealed rDNA operons as a major limitation for short read technologies, and also enabled comparative and functional genomics analysis. To facilitate future assessment and algorithms developments of NGS technologies we publically released the sequence datasets for C. autoethanogenum which span three generations of sequencing technologies, containing six types of data from four NGS platforms. To assess limitations of NGS technologies, assessment of unassembled regions within Illumina and PacBio assemblies was performed using eight microbial genomes. This analysis confirmed rDNA operons as major breakpoints within Illumina assembly while gaps within PacBio assembly appears to be an unaccounted for event and assembly quality is cumulative effect of read-depth, read-quality, sample DNA quality and presence of phage DNA or mobile genetic elements. In a final collaborative study an enrichment protocol was applied for isolation of live endophytic bacteria from roots of the tree Populus deltoides. This protocol achieved a significant reduction in contaminating plant DNA and enabled use these samples for single-cell genomics analysis for the first time. Whole genome sequencing of selected single-cell genomes was performed, assembly and contamination removal optimized, and followed by the bioinformatics, phylogenetic and comparative genomics analyses to identify unique characteristics of these uncultured microorganisms

Draft Genome Sequence for Desulfovibrio africanus Strain PCS.

Desulfovibrio africanus strain PCS is an anaerobic sulfate-reducing bacterium (SRB) isolated from sediment from Paleta Creek, San Diego, CA. Strain PCS is capable of reducing metals such as Fe(III) and Cr(VI), has a cell cycle, and is predicted to produce methylmercury. We present the D. africanus PCS genome sequence

Permeability Modeling of Symmetric Graphite Epoxy Laminates with Arbitrary Ply Orientations

Composites are extensively used for various aerospace applications and one of the important uses is as cryogenic fuel tank materials for RLV and ELV. Composites offer high strength to weight ratio and therefore are preferred to many other materials. In number of cases, arbitrarily orientated ply laminates are used to optimize various desirable properties and weight of the structure. However under structural mechanical loads and/or thermal loads due to change in temperature conditions, transverse micro-cracks are developed in the polymer matrix. These cracks along with interlaminar delaminations produced at the crack tips, lead to passage of cryogenic fuel permeation through the laminates. In this thesis, a mathematical model has been developed to find the delaminated crack opening for each ply throughout the thickness of laminate and permeability of symmetric arbitrarily orientated ply graphite epoxy laminates subjected to different load conditions. In this thesis, an expression for predicting delaminated crack opening displacement in symmetric arbitrarily oriented ply graphite epoxy laminates has been derived using first order shear deformation theory applied to five layer and three layer models. This expression for DCOD depends on crack length, delamination length, crack spacing and load conditions. The DCOD predicted by mathematical model showed good agreement with finite element analysis results. These predicted DCOD values are used to find out permeability of given laminate using Darcy's law of isothermal, viscous fluid flow of gases through porous media. Permeability can be predicted for more general symmetric ply laminate configurations with [Theta1/Theta2/Theta3/Theta4]s, various orientations and thickness. These observations have been made for IM7/PETI-5 graphite epoxy laminate system and conclusions may not be transferable to other types of laminates.Mechanical & Aerospace Engineerin

Complete Genome Sequence of Pelosinus fermentans JBW45, a Member of a Remarkably Competitive Group of Negativicutes in the Firmicutes Phylum.

The genome of Pelosinus fermentans JBW45, isolated from a chromium-contaminated site in Hanford, Washington, USA, has been completed with PacBio sequencing. Nine copies of the rRNA gene operon and multiple transposase genes with identical sequences resulted in breaks in the original draft genome and may suggest genomic instability of JBW45

Affinity purification of SARS-COV-2 spike protein receptor binding domain produced in a C1 fungal expression system

The Receptor Binding Domain (RBD)of the spike protein of SARS-CoV-2 has shown promise for diagnosis, treatment, and development of vaccines for COVID-19. However, two problems persist with large scale production of RBD: 1) lack of high productivity upstream cell culture, 2) absence of a commercial, highly selective affinity resin. In an effort to overcome these limitations, we evaluated two novel technologies for the production and purification of RBD. Briefly, RBD was expressed using C1, an engineered fungal strain of Thermothelomyces heterothallica (DyadicInternational1). The C1 platform expresses glycosylated antigens with high productivity, stability, and purity. RBD was purified using a novel affinity resin2 known to produce yields of 90% to 95% purity in one chromatography step. Affinity purification did not affect protein quality, as demonstrated by ACE-2 binding of RBD. The novel affinity resin showed excellent base stability, consistent product quality, and similar ACE-2 binding activity over 40 cycles. Please click Download on the upper right corner to see the full abstract

Draft Genome Sequence of Burkholderia sp. MR1, a Methylarsenate-Reducing Bacterial Isolate from Florida Golf Course Soil

To elucidate the environmental organoarsenical biocycle, we isolated a soil organism, Burkholderia sp. MR1, which reduces relatively nontoxic pentavalent methylarsenate to the more toxic trivalent methylarsenite, with the goal of identifying the gene for the reductase. Here, we report the draft genome sequence of Burkholderia sp. MR1

Generalized conductance sum rule in atomic break junctions

When an atomic-size break junction is mechanically stretched, the total conductance of the contact remains approximately constant over a wide range of elongations, although at the same time the transmissions of the individual channels (valence orbitals of the junction atom) undergo strong variations. We propose a microscopic explanation of this phenomenon, based on Coulomb correlation effects between electrons in valence orbitals of the junction atom. The resulting approximate conductance quantization is closely related to the Friedel sum rule.Comment: 4 pages, 1 figure, appears in Proceedings of the NATO Advanced Research Workshop ``Size dependent magnetic scattering'', Pecs, Hungary, May 28 - June 1, 200

Draft Genome Sequence of the Lignin-Degrading Burkholderia sp. Strain LIG30, Isolated from Wet Tropical Forest Soil

Burkholderia species are common soil Betaproteobacteria capable of degrading recalcitrant aromatic compounds and xenobiotics. Burkholderia sp. strain LIG30 was isolated from wet tropical forest soil and is capable of utilizing lignin as a sole carbon source. Here we report the draft genome sequence of Burkholderia sp. strain LIG30

Interactive flow behaviour and heat transfer enhancement in a microchannel with cross flow synthetic jet

This paper examines the effectiveness in combining a pulsating fluid jet for thermal enhancement in microchannel heat sinks. The proposed arrangement utilises an oscillating diaphragm to induce a high-frequency periodic fluid jet with zero net mass output at the jet orifice hence, termed "synthetic jet". The pulsed jet interacts with the fluid flow through microchannel passages altering their flow characteristics. The present study develops a 2-dimensional finite volume numerical simulation based on unsteady Reynolds-averaged Navier-Stokes equations for examining the microchannel-synthetic jet flow interaction. For a range of parametric conditions, the behaviour of this periodic flow with its special features is identified and the associated convective heat transfer rates are predicted. The results indicate that the pulsating jet leads to outstanding thermal performance in microchannel flow increasing its heat dissipation rate by about 4.3 times compared to a microchannel without jet interaction within the tested parametric range. The degree of thermal enhancement is seen to grow continuously to reach a steady value in the absence of fluid compressibility. The proposed strategy has an intrinsic ability for outstanding thermal characteristics without causing pressure drop increases in microchannel fluid passages, which is identified as a unique feature of the technique.The study also examines and presents the effects of fluid compressibility on the heat removal capacity of this arrangement. The technique is envisaged to have application potential in miniature electronic devices where localised cooling is desired over a base heat dissipation load

Surrogate model-based strategy for cryogenic cavitation model validation and sensitivity evaluation

The study of cavitation dynamics in cryogenic environment has critical implications for the performance and safety of liquid rocket engines, but there is no established method to estimate cavitation-induced loads. To help develop such a computational capability, we employ a multiple-surrogate model-based approach to aid in the model validation and calibration process of a transport-based, homogeneous cryogenic cavitation model. We assess the role of empirical parameters in the cavitation model and uncertainties in material properties via global sensitivity analysis coupled with multiple surrogates including polynomial response surface, radial basis neural network, kriging, and a predicted residual sum of squares-based weighted average surrogate model. The global sensitivity analysis results indicate that the performance of cavitation model is more sensitive to the changes in model parameters than to uncertainties in material properties. Although the impact of uncertainty in temperature-dependent vapor pressure on the predictions seems significant, uncertainty in latent heat influences only temperature field. The influence of wall heat transfer on pressure load is insignificant. We find that slower onset of vapor condensation leads to deviation of the predictions from the experiments. The recalibrated model parameters rectify the importance of evaporation source terms, resulting in significant improvements in pressure predictions. The model parameters need to be adjusted for different fluids, but for a given fluid, they help capture the essential fluid physics with different geometry and operating conditions. Copyright © 2008 John Wiley & Sons, Ltd.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/61228/1/1779_ftp.pd