Contig assembly and plasmid analysis using DNA barcodes

Two methods of computational analysis of DNA barcodes are presented. A DNA barcode is formed by making GC-rich regions of a DNA molecule fluoresce while AT-rich regions remain dark, thus when stretched using nano-channels and viewed in a microscope, the DNA molecule will resemble a barcode with black and white stripes. Because of point-spread functions and pixellation the resolution will be roughly one data point per 200nm (or roughly 700 bp). This resolution is typically enough to distinguish between two different DNA molecules. First DNA barcodes are used for analyzing an antibiotic resistance outbreak. In the outbreak, antibiotic resistant bacteria infected newborn children at Sahlgrenska University Hospital. The bacteria were of different strains and it was suspected that the bacteria shared the antibiotic resistant gene with bacteria not containing it through the exchange of plasmids. A plasmid is a short circular DNA molecule, typical length between 2 kbp to 1 Mbp (base pairs), which bacteria use to store genes that benefit survival (such as antibiotic resistance genes). The second method is about matching short pieces of DNA sequence, called contigs, to a long intact barcode (from the same molecule as the contigs) to figure out the order of the pieces of sequence. In order to match a sequence to a barcode, the sequence has to be converted into a theoretical barcode first. After that it is compared to the long barcode, to find the optimal placement. Contigs are not supposed to overlap, and that is an assumption used in the methods presented in section 7. The matching in both methods is facilitated by the use of our new statistical tools in order to reduce the number of false positives in the matching process. The results for the plasmid tracing method show that the method can be used to trace plasmid spread. On the other hand, the results for the contig assembly show that the method has potential to be useful, but at the moment it has been unsuccessful at assembling real contigs into a full, correct, sequence

Setup and control of a test reactor for catalysis with optical access

CO2 Catalysis has been studied with Planar Laser Induced Fluorescence (PLIF) in combination with a mass spectrometer for a small, 23 cubic centimeters, cube shaped flow reactor. Both catalysis processes with one and with two catalysts inside the reactor have been carried out and analysed. Some results have been visualised by images showing concentration, given by the PLIF signal, of CO2 inside the reactor in two dimensions. This has the advantage of having two dimensional spatial resolution compared to only using the mass spectrometer. The experiments were also performed at almost ambient pressure (100 mbar).Med hjälp av laserljus i en kubisk kammare har halten CO2 runt en katalysator studerats. Denna typen av studier är viktiga för att kunna förbättra och effektivisera katalysatorer vilket är väldigt viktigt inom både industri och för miljömål. Använder man laser på det här sättet kan man se hur olika parametrar, så som flöde eller tryck, påverkar effektiviteten av katalysatorn. Andra metoder har inte den möjligheten utan istället studerar de antingen ytan eller enbart gasen efter att den har lämnat kammaren vilket gör att man förlorar information om hur gasen beter sig i kammaren

Facilitated sequence assembly using densely labeled optical DNA barcodes:A combinatorial auction approach

<div><p>The output from whole genome sequencing is a set of contigs, i.e. short non-overlapping DNA sequences (sizes 1-100 kilobasepairs). Piecing the contigs together is an especially difficult task for previously unsequenced DNA, and may not be feasible due to factors such as the lack of sufficient coverage or larger repetitive regions which generate gaps in the final sequence. Here we propose a new method for scaffolding such contigs. The proposed method uses densely labeled optical DNA barcodes from competitive binding experiments as scaffolds. On these scaffolds we position theoretical barcodes which are calculated from the contig sequences. This allows us to construct longer DNA sequences from the contig sequences. This proof-of-principle study extends previous studies which use sparsely labeled DNA barcodes for scaffolding purposes. Our method applies a probabilistic approach that allows us to discard “foreign” contigs from mixed samples with contigs from different types of DNA. We satisfy the contig non-overlap constraint by formulating the contig placement challenge as a combinatorial auction problem. Our exact algorithm for solving this problem reduces computational costs compared to previous methods in the combinatorial auction field. We demonstrate the usefulness of the proposed scaffolding method both for synthetic contigs and for contigs obtained using Illumina sequencing for a mixed sample with plasmid and chromosomal DNA.</p></div

Identification of principal chemical subsets of biofuel combustion : Ants walking in renewable fire

The work in this thesis was carried out to highlight important chemical pathways in skeletal mechanisms for the three smallest alcohol fuels (methanol, ethanol and npropanol), two representative fuel alkanes n-heptane and n-decane, and finally the biodiesel surrogates, methyl-decanoate, methyl-5-decenoate, and methyl-9-decenoate. This aim was set up to further the efforts of creating Computational Fluid Dynamics (CFD) suitable mechanisms, which in turn can be used by the industry to improve, for example, engines or gas turbines, or by academia to further understand turbulent combustion. The work in this thesis was divided into three parts: development of a novel reduction method, pathway analysis of biofuels, and pathway analysis of petroleum and biodiesel surrogates.Ant Colony Reduction (ACR) is a semi-stochastic reduction algorithm that has been applied successfully on kinetic mechanisms for several biofuels (methanol, ethanol, npropanol, methyl-decanoate and methyl-decenoate), alkanes, and volatile organic compound oxidation in atmospheric chemistry simulations. The method was developed within the framework of the present thesis work, as a new approach to the mechanism reduction problem, as much research has been carried out on various established reduction methods. It has been shown that in some cases the mechanisms reduced using the ACR method outperforms previously published reduced mechanisms by having a lower number of reactions or species while preserving the same accuracy compared to a chosen reference mechanism.The pathway analysis of the biofuel mechanisms was conducted in order to identify the principal reaction subsets for different combustion modes. It was then shown that similarities can be seen for each combustion mode for each of the smallest alcohols, methanol, ethanol and n-propanol. By understanding these subsets, an initial guess for the next alcohol fuel would be facilitated. Several trends were identified that was true for all the alcohol fuels, but there were also new reaction paths that was important for the new skeletal mechanism when the chain length of the alcohol was longer. Since the alcohol at some point has to decompose to two fragments, where only one can contain oxygen, alkane combustion chemistry became more important for n-propanol and ethanol than for methanol.A similar approach to the biofuel pathway analysis was conducted for n-heptane, for which separate reduced mechanisms were produced for the combustion phenomena ignition, flame propagation and extinction. The reference mechanisms for n-heptane are much larger than for the small alcohols, often resulting in larger skeletal mechanisms as well. After investigation, it was shown that for high temperature ignition and laminar burning velocity, it is not necessary to have too much detail in the chemistry. Even extinction and low temperature ignition mechanisms were below 150 reactions each, but when one mechanism for all conditions were constructed, the mechanism had 230 reactions. From the pathway analysis, the important reactions for each subset was identified and can serve as an initial guide for future reduction work on large hydrocarbon fuels.The methodology was also applied to biodiesel surrogates, methyl-decanoate, methyl-5-decenoate and methyl-9-decenoate. Once again, the mechanisms were larger in size due to the complexity of the reference mechanism. It was found that for the methyl-decenoates, the skeletal mechanisms were smaller since the number of possible, and important, intermediates are lower. Compared to other published skeletal mechanisms for the biodiesel surrogates, the sizes of these mechanisms are much smaller, between 200-861 reactions for low temperature ignition, while still retaining high predictability compared to their reference mechanism

Reduced kinetic mechanism for methanol combustion in spark-ignition engines

A reduced kinetic mechanism for methanol combustion at spark-ignition (SI) engine conditions is presented. The mechanism consists of 18 species and 55 irreversible reactions, small enough to be suitable for large eddy simulations (LES). The mechanism was reduced and optimized using the comprehensive mechanism (AramcoMech 2.0) as a starting point, to maintain performance at stoichiometric conditions for the pressure (10-50 bar) and temperature ranges relevant for SI-engine conditions. The mechanism was validated against experimental data for ignition delay at 1050-1650 K, flow reactor at 783 K and jet-stirred reactors at 800-1150 K, and simulated validation targets for laminar burning velocity under conditions where no experimental data are available. The mechanism performs well for pollutant formation (CO and CH2O), ignition delay, and laminar burning velocity, which are all important properties for LES of engines. Two other reduced mechanisms for methanol combustion, containing around the same number of species and reactions, were tested for comparison. The superior performance of the mechanism developed in the present work is likely a result of that it is specifically produced for the relevant conditions, while the other mechanisms were developed for a limited set of conditions compared to the present work. This highlights the importance of careful selection of reduced mechanisms for implementation in computational fluid dynamics simulations

Composition of Reduced Mechanisms for Ignition of Biodiesel Surrogates

Chemical kinetics mechanisms describing Fatty Acid Methyl Ester (FAME) biofuel combustion are quite extensive and cannot be implemented in Computational Fluid Dynamics simulations of real engine systems. Using the reduction methodology Ant Colony Reduction (ACR), skeletal reduction followed by optimization has been performed for the C-11 FAME biodiesel components methyl decanoate (MD), methyl 5-decenoate (MDe5), and methyl 9-decenoate (MDe9), and for the alkane n-decane. The aim of the present study was to produce small reduced mechanisms accurately describing ignition of the fuels over a wide range of conditions, and in addition to compare the size and composition of reduced mechanisms constructed from two parent mechanisms of different complexity. Reduction targets were ignition delay times over a wide range of equivalence ratios and pressures, for separate temperature ranges of 600&ndash;1100 K (LT) and 1100&ndash;1500 K (HT). One of the complex mechanisms was constructed to be simplified by a lumping approach and this one included MD and was also used to perform reduction for the alkane n-decane. The most detailed parent mechanism was used to create reduced mechanisms for all the three methyl esters. The lumped complex mechanisms resulted in more compact reduced mechanisms, 157 reactions for LT of MD, compared to 810 reactions for the more detailed mechanism. MD required the largest fuel breakdown subsets while the unsaturated methyl esters could be described by smaller subsets. All mechanisms had similar subsets for the smallest hydrocarbons and H/O chemistry, independent of the fuel and the choice of parent mechanism. The ACR approach for mechanism reduction created reduced mechanisms with high accuracy for all conditions included in the present study

Tailored chemical mechanisms for simulation of urban air pollution

A semi-stochastic, statistical reduction method for chemical kinetic schemes based on the ant colony optimization method, is developed for atmospheric chemistry simulations. The prime application is coupled dynamic and chemistry models for simulation of the dispersion and reactivity of chemical species on street scale, i.e. the modelling of urban air pollution in street canyons. The method is designed so that it will optimize the reduction process for any simulation case, as given by user-specific inputs, such as initial concentrations of reactive species, temperature, humidity, residence time, and solar radiation. These inputs will correspond to, or be deduced from, actual variables such as season, time-of-day, geographic location, proximity to volatile organic carbon or nitrogen oxides sources (e.g. forests, roads, industry, harbours etc.) and their source strengths, weather, composition of vehicle fleet, and traffic load inside the street canyon. The method is evaluated against three box model case studies (laboratory and atmospheric simulations) previously described in the literature. The method reduces the mechanism sizes with 62.5%, 84.7%, and 97.7% respectively, retaining the average accuracy for the prediction of the target compound (O3, NO2, and NO) concentrations by 94.1%, 90.3%, and 91.2% respectively. These preliminary results illustrate the potential for the method. Further developments, such as inclusion of lumping or short-cutting of reaction paths, can be considered

Comparative analysis of detailed and reduced kinetic models for CH 4 + H 2 combustion

Directed relation graph with error propagation (DRGEP) method combined with extensive validation for 0D, 1D and 2D CFD modeling supported by sensitivity and Rate-Of-Production (ROP) analyses are implemented for comparative study of detailed and reduced kinetic mechanisms for CH 4 + H 2 combustion. To this end, two detailed kinetic mechanisms, namely AramcoMech 2.0 and recently updated Konnov mechanism, were validated using available measurements of ignition delay times and laminar burning velocities for hydrogen, methane and hydrogen + methane fuel mixtures. For all experimental conditions visited, both detailed mechanisms demonstrated good and close to each other performance. Two-stage DRGEP method and reaction reduction based on computational singular perturbation (CSP) were then implemented to achieve two skeletal models: 25 species and 105 reactions for AramcoMech 2.0 and 27 species and 107 reactions for the Konnov model. The conditions for skeletal models generation cover ɸ = 0.5–2.0, temperature 900–2000 K, and pressure 1–50 bar. Turbulent non-premixed flames of CH 4 + H 2 in the Jet in Hot Co-flow (JHC) burner for two different oxygen concentrations in a co-flow were modeled using both skeletal models. 2-D RANS simulations with OpenFOAM code of the flame structure using the two skeletal kinetic mechanisms are similar except for the mass fraction of OH and CO. To elucidate the differences between two skeletal mechanisms generated using the same reduction method, extensive validation for 0D, 1D and 2D CFD modeling were supported by sensitivity analysis for detailed and skeletal reaction models. Good agreement between the skeletal and detailed mechanisms was found in top reactions as well as their sensitivity coefficients, which affect auto-ignition process and laminar flame propagation. Further chemical and sensitivity analysis of the structure of laminar flames demonstrate that three important reactions, i.e. CO + OH = CO 2 + H, H 2 + OH = H + H 2 O, and CH 4 + OH = CH 3 + H 2 O have different rate constants in the Aramco and Konnov models that may contribute to the differences in the prediction of CO concentration profiles. The simulation predictions for CO concentrations are improved for laminar flames and JHC flame by using a 25-species modified version in which these rate constants were taken from the Konnov mechanism. It was noted that DRGEP method applied to different detailed kinetic schemes generate skeletal models with different, non-overlapping lists of retained species. The presence of CH 2 CHO in the Aramco 25-species skeletal mechanism and its absence in the Konnov 27-species mechanism, and the presence of CH, CH 2 , CH 2 CO in the latter and their absence in the former mechanism were analysed and explained using Rate-Of-Production analysis for conditions found in the CFD simulations

Rapid tracing of resistance plasmids in a nosocomial outbreak using optical DNA mapping

Resistance to life-saving antibiotics increases rapidly worldwide, and multiresistant bacteria have become a global threat to human health. Presently, the most serious threat is the increasing spread of Enterobacteriaceae carrying genes coding for extended spectrum β-lactamases (ESBL) and carbapenemases on highly mobile plasmids. We here demonstrate how optical DNA maps of single plasmids can be used as fingerprints to trace plasmids, for example, during resistance outbreaks. We use the assay to demonstrate a potential transmission route of an ESBL-carrying plasmid between bacterial strains/species and between patients, during a polyclonal outbreak at a neonatal ward at Sahlgrenska University Hospital (Gothenburg, Sweden). Our results demonstrate that optical DNA mapping is an easy and rapid method for detecting the spread of plasmids mediating resistance. With the increasing prevalence of multiresistant bacteria, diagnostic tools that can aid in solving ongoing routes of transmission, in particular in hospital settings, will be of paramount importance

Contig scaffolding using Illumina contigs from a mixed sample of pUUH/chromosomal DNA.

<p>(Top) Optimal placement of the contig theory barcodes on the experimental pUUH barcode using our contig scaffolding method (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193900#sec002" target="_blank">Methods</a>). 220 contigs were obtained through Illumina sequencing of a mixed sample containing the pUUH plasmid and chromosomal DNA from the bacterium <i>Klebsiella pneumoniae</i>. Based on a sequence alignment 16 of the contigs are deemed to belong to the pUUH plasmid. Horizontal lines at the top corresponds to “true” contig positions based on a sequence comparison of the full pUUH sequence and the contig sequences. We find that 2 contig barcodes pass the length and p-value thresholds. The two contigs which were placed ended up at correct positions. (Bottom) The examples of removed contigs illustrates intensity profiles of a few typical non-matching barcodes: the four chromosomal contig barcodes with the smallest p-values and the third longest plasmid barcode (orange).</p