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1
Chen, Ying-Ja. "DNA sequencing by denaturation". Diss., [La Jolla] : University of California, San Diego, 2009. http://wwwlib.umi.com/cr/ucsd/fullcit?p3359122.
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Thesis (Ph. D.)--University of California, San Diego, 2009.Title from first page of PDF file (viewed July 14, 2009). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 87-91).
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Leah, Labib. "Helicase Purification for DNA Sequencing". Thesis, Université d'Ottawa / University of Ottawa, 2014. http://hdl.handle.net/10393/31341.
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BACKGROUND: A method to increase accuracy and ease-of-use, while decreasing time and cost in deoxyribonucleic acid (DNA) sequence identification, is sought after. Helicase, which unwinds DNA, and avidin, which strongly attracts biotin for potential attraction of biotinylated DNA segments, were investigated for use in a novel DNA sequencing method.
AIM: This study aimed to (1) purify bacteriophage T7 gene product 4 helicase and helicase-avidin fusion protein in a bacterial host and (2) characterize their functionality.
METHODS: Helicase and helicase-avidin were cloned for purification from bacteria. Helicase-avidin was solubilised via urea denaturation/renaturation. DNA and biotin binding were assessed using Electrophoretic Mobility Shift Assays and biotinylated resins, respectively.
RESULTS: (1) Helicase and helicase-avidin proteins were successfully purified. (2) Helicase protein was able to bind DNA and avidin protein strongly bound biotin.
CONCLUSION: Helicase and helicase-avidin can be purified in a functional form from a bacterial host, thus supporting further investigation for DNA sequencing purposes.
3
Boufounos, Petros T. 1977. "Signal processing for DNA sequencing". Thesis, Massachusetts Institute of Technology, 2002. http://hdl.handle.net/1721.1/17536.
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Thesis (M.Eng. and S.B.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2002.Includes bibliographical references (p. 83-86).
DNA sequencing is the process of determining the sequence of chemical bases in a particular DNA molecule-nature's blueprint of how life works. The advancement of biological science in has created a vast demand for sequencing methods, which needs to be addressed by automated equipment. This thesis tries to address one part of that process, known as base calling: it is the conversion of the electrical signal-the electropherogram--collected by the sequencing equipment to a sequence of letters drawn from ( A,TC,G ) that corresponds to the sequence in the molecule sequenced. This work formulates the problem as a pattern recognition problem, and observes its striking resemblance to the speech recognition problem. We, therefore, propose combining Hidden Markov Models and Artificial Neural Networks to solve it. In the formulation we derive an algorithm for training both models together. Furthermore, we devise a method to create very accurate training data, requiring minimal hand-labeling. We compare our method with the de facto standard, PHRED, and produce comparable results. Finally, we propose alternative HMM topologies that have the potential to significantly improve the performance of the method.
by Petros T. Boufounos.
M.Eng.and S.B.
4
Blomstergren, Anna. "Strategies for de novo DNA sequencing". Doctoral thesis, KTH, Biotechnology, 2003. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-3649.
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The development of improved sequencing technologies hasenabled the field of genomics to evolve. Handling andsequencing of large numbers of samples require an increasedlevel of automation in order to obtain high throughput andconsistent quality. Improved performance has lead to thesequencing of numerous microbial genomes and a few genomes fromhigher eukaryotes and the benefits of comparing sequences bothwithin and between species are now becoming apparent. Thisthesis describes both the development of automated purificationmethods for DNA, mainly sequencing products, and a comparativesequencing project.
The initially developed purification technique is dedicatedto single stranded DNA containing vector specific sequences,exemplified by sequencing products. Specific capture probescoupled to paramagnetic beads together with stabilizing modularprobes hybridize to the single stranded target. After washing,the purified DNA can be released using water. When sequencingproducts are purified they can be directly loaded onto acapillary sequencer after elution. Since this approach isspecific it can be applied to multiplex sequencing products.Different probe sets are used for each sequencing product andthe purifications are performed iteratively.
The second purification approach, which can be applied to anumber of different targets, involves biotinylated PCR productsor sequencing products that are captured using streptavidinbeads. This has been described previously, buthere theinteraction between streptavidin and biotin can be disruptedwithout denaturing the streptavidin, enabling the re-use of thebeads. The relatively mild elution conditions also enable therelease of sensitive biotinylated molecules.
Another project described in this thesis is the comparativesequencing of the 40 kbcagpathogenicity island (PAI) in fourHelicobacter pyloristrains. The results included thediscovery of a novel gene, present in approximately half of theSwedish strains tested. In addition, one of the strainscontained a major rearrangement dividing thecagPAI into two parts. Further, information about thevariability of different genes could be obtained.
Keywords:DNA sequencing, DNA purification, automation,solid-phase, streptavidin, biotin, modular probes,Helicobacter pylori,cagPAI.
5
Hu, Yue. "Microbial DNA Sequencing in Environmental Studies". Doctoral thesis, KTH, Skolan för bioteknologi (BIO), 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-204897.
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The field of microbial ecology has just entered a new era of rapid technological development and generation of big data. The high-throughput sequencing techniques presently available provide an opportunity to extensively inventorize the blueprints of life. Now, millions of microbes of natural microbial communities can be studied simultaneously without prior cultivation. New species and new functions (genes) can be discovered just by mining sequencing data. However, there is still a tremendous number of microorganisms not yet examined, nor are the ecosystem functions these carry out. The modern genomic technologies can contribute to solve environmental problems and help us understand ecosystems, but to most efficiently do so, methods need to be continuously optimised. During my Ph. D. studies, I developed a method to survey eukaryotic microbial diversity with a higher accuracy, and applied various sequencing-based approaches in an attempt to answer questions of importance in environmental research and ecology. In PAPER-I, we developed a set of 18S rRNA gene PCR primers with high taxonomic coverage, meeting the requirements of currently popular sequencing technologies and matching the richness of 18S rRNA reference sequences accumulated so far. In PAPER-II, we conducted the first sequencing-based spatial survey on the combined eukaryotic and bacterial planktonic community in the Baltic Sea to uncover the relationship of microbial diversity and environmental conditions. Here, the 18S primers designed in PAPER-I and a pair of broad-coverage 16S primers were employed to target the rRNA genes of protists and bacterioplankton for amplicon sequencing. In PAPER-III, we integrated metagenomic, metabarcoding, and metatranscriptomic data in an effort to scrutinise the protein synthesis potential (i.e., activity) of microbes in the sediment at a depth of 460 m in the Baltic Sea and, thus, disclosing microbial diversity and their possible ecological functions within such an extreme environment. Lastly, in PAPER-IV, we compared the performance of E. coli culturing, high-throughput sequencing, and portable real-time sequencing in tracking wastewater contamination in an urban stormwater system. From the aspects of cost, mobility and accuracy, we evaluated the usage of sequencing-based approaches in civil engineering, and for the first time, validated the real-time sequencing device in use within water quality monitoring. In summary, these studies demonstrate how DNA sequencing of microbial communities can be applied in environmental monitoring and ecological research.Yue Hu was supported by a scholarship from the China Scholarship Council (CSC #201206950024)
Yue Hu has been publishing papers under the name "Yue O. O. Hu".
QC 20170403
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Peng, Hongbo. "Towards hybridization-assisted nanopore DNA sequencing". View abstract/electronic edition; access limited to Brown University users, 2008. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3318349.
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Haan, Nicholas. "Statistical models and algorithms for DNA sequencing". Thesis, University of Cambridge, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.396070.
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Dezfouli, Mahya. "Barcoded DNA Sequencing for Parallel Protein Detection". Doctoral thesis, KTH, Genteknologi, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-159506.
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The work presented in this thesis describes methodologies developed for integration and accurate interpretation of barcoded DNA, to empower large-scale-omics analysis. The objectives mainly aim at enabling multiplexed proteomic measurements in high-throughput format through DNA barcoding and massive parallel sequencing. The thesis is based on four scientific papers that focus on three main criteria; (i) to prepare reagents for large-scale affinity-proteomics, (ii) to present technical advances in barcoding systems for parallel protein detection, and (iii) address challenges in complex sequencing data analysis. In the first part, bio-conjugation of antibodies is assessed at significantly downscaled reagent quantities. This allows for selection of affinity binders without restrictions to accessibility in large amounts and purity from amine-containing buffers or stabilizer materials (Paper I). This is followed by DNA barcoding of antibodies using minimal reagent quantities. The procedure additionally enables efficient purification of barcoded antibodies from free remaining DNA residues to improve sensitivity and accuracy of the subsequent measurements (Paper II). By utilizing a solid-phase approach on magnetic beads, a high-throughput set-up is ready to be facilitated by automation. Subsequently, the applicability of prepared bio-conjugates for parallel protein detection is demonstrated in different types of standard immunoassays (Papers I and II). As the second part, the method immuno-sequencing (I-Seq) is presented for DNAmediated protein detection using barcoded antibodies. I-Seq achieved the detection of clinically relevant proteins in human blood plasma by parallel DNA readout (Paper II). The methodology is further developed to track antibody-antigen interaction events on suspension bead arrays, while being encapsulated in barcoded emulsion droplets (Paper III). The method, denoted compartmentalized immuno-sequencing (cI-Seq), is potent to perform specific detections with paired antibodies and can provide information on details of joint recognition events. Recent progress in technical developments of DNA sequencing has increased the interest in large-scale studies to analyze higher number of samples in parallel. The third part of this thesis focuses on addressing challenges of large-scale sequencing analysis. Decoding of a huge DNA-barcoded data is presented, aiming at phase-defined sequence investigation of canine MHC loci in over 3000 samples (Paper IV). The analysis revealed new single nucleotide variations and a notable number of novel haplotypes for the 2nd exon of DLA DRB1. Taken together, this thesis demonstrates emerging applications of barcoded sequencing in protein and DNA detection. Improvements through the barcoding systems for assay parallelization, de-convolution of antigen-antibody interactions, sequence variant analysis, as well as large-scale data interpretation would aid biomedical studies to achieve a deeper understanding of biological processes. The future perspectives of the developed methodologies may therefore stem for advancing large-scale omics investigations, particularly in the promising field of DNA-mediated proteomics, for highly multiplex studies of numerous samples at a notably improved molecular resolution.QC 20150203
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Hu, Yuwei S. M. Massachusetts Institute of Technology y Chin Soon Lim. "Scheduling of biological samples for DNA sequencing". Thesis, Massachusetts Institute of Technology, 2009. http://hdl.handle.net/1721.1/54214.
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Thesis (S.M.)--Massachusetts Institute of Technology, Computation for Design and Optimization Program, 2009.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student submitted PDF version of thesis.
Includes bibliographical references (p. 95-97).
In a DNA sequencing workflow, a biological sample has to pass through multiple process steps. Two consecutive steps are hydroshearing and library construction. Samples arrive randomly into the inventory and are to complete both processes before their due dates. The research project is to decide the optimal sequence of samples to go through these two processes subject to operational constraints. Two approaches, namely, heuristic and integer programming have been pursued in this thesis. A heuristic algorithm is proposed to solve the scheduling problem. A variant of the problem involving deterministic arrivals of samples is also considered for comparison purposes. Comparison tests between the two approaches are carried out to investigate the performance of the proposed heuristic for the original problem and its variant. Sensitivity analysis of the schedule to parameters of the problem is also conducted when using both approaches.
by Yuwei Hu and Chin Soon Lim.
S.M.
10
Santiago, Garcia Eric y Aspåker Hannes Salomonsson. "Temporal Convolutional Networks for Nanopore DNA Sequencing". Thesis, KTH, Skolan för elektroteknik och datavetenskap (EECS), 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-295625.
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Nanopore DNA sequencing is a novel method forsequencing DNA where an electronic signal is modulated bynucleotides passing through nanosized pores embedded in a mem-brane. While current state-of-the-art solutions employ recurrentneural networks to analyse the signal, temporal convolutionalnetworks have recently been shown to match or outperformrecurrent networks in signal processing tasks. In this project, weinvestigate the performance of temporal convolutional networkson a simplified version of the sequencing task, where thegoal is to predict the nucleotides passing through the pore ateach instance in time, without reconstructing the correspondingDNA sequence. The impact of several network parameters onpredictive performance is analysed to determine an optimalarchitecture. While the implemented networks are shown tobe proficient at predicting nucleotides within the pore, thecurrent implementation is unlikely to outperform state-of-the-art solutions without further improvement.En nyligen utvecklad metod för att sekvensera DNA innefattar att en elektrisk signal moduleras genom att nukleotider passerar genom porer i nanostorlek. I kommersiella lösningar analyseras denna signal med hjälp av maskininlärning via Recurrent Neural Networks, men en variant av neruala nätverk som kallas Temporal Convolution Networks har nyligen har visat sig ha bättre prestanda jämfört med Recurrent Networks för olika typer av signalbehandlingsproblem. Målet med detta projekt är att undersöka användbarheten av Temporal Convolutional Networks för en förenklad version av DNA-sekvensering, där uppdraget endast är att identifera de nukleotider som passerar genom poren vid varje given tidpunkt, istället för att rekonstruera en komplett DNA-sekvens. För att kunna bestämma en optimal arkitektur på nätverket så undersöks effekten av flera olika parametrar. De implementerade nätverken visas ha god förmåga att klassificera nukleotider, men är troligtvis i behov av ytterligare förbättringar för att kunna konkurrera med nuvarande kommersiella lösningar.
Kandidatexjobb i elektroteknik 2020, KTH, Stockholm
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Arner, Erik. "Solving repeat problems in shotgun sequencing /". Stockholm, 2006. http://diss.kib.ki.se/2006/91-7140-996-3/.
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Davies, Stephen William. "Application of communication theory to automatic DNA sequencing". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape8/PQDD_0005/NQ41137.pdf.
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Lundin, Sverker. "Methods to Prepare DNA for Efficient Massive Sequencing". Doctoral thesis, KTH, Genteknologi, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-105116.
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Massive sequencing has transformed the field of genome biology due to the continuous introduction and evolution of new methods. In recent years, the technologies available to read through genomes have undergone an unprecedented rate of development in terms of cost-reduction. Generating sequence data has essentially ceased to be a bottleneck for analyzing genomes instead to be replaced by limitations in sample preparation and data analysis. In this work, new strategies are presented to increase both the throughput of library generation prior to sequencing, and the informational content of libraries to aid post-sequencing data processing. The protocols developed aim to enable new possibilities for genome research concerning project scale and sequence complexity. The first two papers that underpin this thesis deal with scaling library production by means of automation. Automated library preparation is first described for the 454 sequencing system based on a generic solid-phase polyethylene-glycol precipitation protocol for automated DNA handling. This was one of the first descriptions of automated sample handling for producing next generation sequencing libraries, and substantially improved sample throughput. Building on these results, the use of a double precipitation strategy to replace the manual agarose gel excision step for Illumina sequencing is presented. This protocol considerably improved the scalability of library construction for Illumina sequencing. The third and fourth papers present advanced strategies for library tagging in order to multiplex the information available in each library. First, a dual tagging strategy for massive sequencing is described in which two sets of tags are added to a library to trace back the origins of up to 4992 amplicons using 122 tags. The tagging strategy takes advantage of the previously automated pipeline and was used for the simultaneous sequencing of 3700 amplicons. Following that, an enzymatic protocol was developed to degrade long range PCR-amplicons and forming triple-tagged libraries containing information of sample origin, clonal origin and local positioning for the short-read sequences. Through tagging, this protocol makes it possible to analyze a longer continuous sequence region than would be possible based on the read length of the sequencing system alone. The fifth study investigates commonly used enzymes for constructing libraries for massive sequencing. We analyze restriction enzymes capable of digesting unknown sequences located some distance from their recognition sequence. Some of these enzymes have previously been extensively used for massive nucleic acid analysis. In this first high throughput study of such enzymes, we investigated their restriction specificity in terms of the distance from the recognition site and their sequence dependence. The phenomenon of slippage is characterized and shown to vary significantly between enzymes. The results obtained should favor future protocol development and enzymatic understanding. Through these papers, this work aspire to aid the development of methods for massive sequencing in terms of scale, quality and knowledge; thereby contributing to the general applicability of the new paradigm of sequencing instruments.QC 20121126
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Liang, Lijun. "Computational studies of DNA sequencing with graphene nanopores". Doctoral thesis, KTH, Teoretisk kemi och biologi, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-157664.
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The aim of DNA sequencing is to obtain the order of DNA composition comprising the base pairs A (adenine) T (thymine), and C (cytosine) G (guanine). The fast development of DNA sequencing technology allows us to better understand the relationships among diseases, inheritance, and individuality. Solid state nanopores have been recommended as the next generation platform for DNA sequencing due to its low-cost and high-throughput. In particular, nanopores fabricated from graphene sheets are extremely thin and structurally robust and have been extensively used in DNA detection in recent years. In DNA sequencing, the translocation of a DNA molecule through a nanopore is known to be a very complicated issue and is affected by many factors, such as ion concentration, thickness of the nanopore, and the nanopore diameter. The technique of molecular dynamic simulations has been a complementary tool to study DNA translocation through nanopores. In this thesis, I summarize my work of computational studies of DNA sequencing using graphene nanopores. These studies include: DNA translocation through single-layer graphene nanopores of different diameters under conditions of various ion concentrations and applied voltages; DNA translocation through multilayer graphene nanopores varied from a single to a few layers; pulling out single strand DNA molecules from small graphene nanopores of different geometries. The major contributions of this work include: 1. Effects of bias voltage on DNA translocation time were investigated leading to the insight that lower applied voltages can extend the time of DNA translocation through monolayer graphene nanopores. The effect of salt concentration on the corresponding ionic current was studied. At a low ionic concentration (< 0.3M), the current increases as DNA translocates through a nanopore. However, at a high ionic concentration (>0.5M), the current decreases as DNA translocates through the nanopore. A theoretical model was proposed to explore the relationship between the current and the occupied nanopore area. We demonstrated that the DNA translocation time can be prolonged by narrowing the diameter of a nanopore properly and the reduction of the blockade current depends on the ratio of the unoccupied nanopore area to the total nanopore area. 2. DNA translocation through multilayer graphene nanopores was studied by molecular dynamics simulations with the aim to achieve single-base resolution. We show that the DNA translocation time can be extended by increasing the graphene layers up to a moderate number (7) and that the current in DNA translocation undergoes a stepwise change upon DNA going through an multi-layer graphene (MLG) nanopore. A model was built to account for the relationship between the current change and the unoccupied volume of the MLG nanopore. We demonstrate that the blockade current is closely related to the unoccupied volume. The dynamics of DNA translocation depends specifically on the interaction of nucleotides with the graphene sheet. Thus, our study indicates that the resolution of DNA detection can be improved by increasing the number of graphene layers in a certain range and by modifying the surface of graphene nanopores. 3. The effect of graphene nanopore geometry on DNA sequencing has been assessed by steered molecular dynamics simulations. DNA fragments including A, T, C, G and 5-methylcytosine (MC) were pulled through graphene nanopores of different geometries with diameters down to ~1nm by steered molecular dynamics simulations. We demonstrated that the bases (A, T, C, G, and MC) can be indentified in single-base resolution by the characteristic force peak values in a circular graphene nanopore but not in graphene nanopores of other geometries. Symmetric nanopores are thus better suited to DNA sequence detection via force curves than asymmetric nanopores. This implies that the graphene nanopore surface should be modified as symmetric as possible to sequence DNA by an atomic force microscope or optical tweezers. This helps us to understand low-cost and time-efficient DNA sequencing in narrow nanopores. 4. The translocation time for different nucleotides to pass through graphene nanopores with certain diameters was investigated. It was found that the translocation times are different for different bases under a low electric field. The results indicate that DNA can be sequenced by the translocation time to pass through a graphene nanopore. 5. Inspired by the structure of K+ channel proteins, a series of oxygen doped graphene nanopores of different size were designed to discriminate the transport of K+ and Na+ ions. The results indicate that the ion selectivity of such biomimetic graphene nanopores can be simply controlled by the size of the nanopore. Compared to K+, the smaller radius of Na+ leads to a much higher free energy barrier in the nanopore of a certain size.QC 20141212
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Arram, James. "FPGA acceleration of DNA sequencing analysis and storage". Thesis, Imperial College London, 2017. http://hdl.handle.net/10044/1/56867.
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In this work we explore how Field-Programmable Gate Arrays (FPGAs) can be used to alleviate the data processing bottlenecks in DNA sequencing. We focus our efforts on accelerating the FM-index, a data structure used to solve the computationally intensive string matching problems found in DNA sequencing analysis such as short read alignment. The main contributions of this work are: 1) We accelerate the FM-index using FPGAs and develop several novel methods for reducing the memory bottleneck of the search algorithm. These methods include customising the FM-index structure according to the memory architecture of the FPGA platform and minimising the number of memory accesses through both architectural and algorithmic optimisations. 2) We present a new approach for accelerating approximate string matching using the backtracking FM-index. This approach makes use of specialised approximate string matching modules and a run-time reconfigurable architecture in order to achieve both high sensitivity and high performance. 3) We extend the FM-index search algorithm for reference-based compression and accelerate it using FPGAs. This accelerated design is integrated into fastqZip and fastaZip, two new tools that we have developed for the fast and effective compression of sequence data stored in the FASTQ and FASTA formats respectively. We implement our designs on the Maxeler Max4 Platform and show that they are able to outperform state-of-the-art DNA sequencing analysis software. For instance, our hardware-accelerated compression tool for FASTQ data is able to achieve a higher compression ratio than the best performing tool, fastqz, whilst the average compression and decompression speeds are 25 and 43 times faster respectively.
16
Quick, Joshua. "Real-time pathogen surveillance systems using DNA sequencing". Thesis, University of Birmingham, 2018. http://etheses.bham.ac.uk//id/eprint/8135/.
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Microbiological research has uncovered the basis of fermentation, infectious disease, vaccination and antibiotics. Now, a technological revolution leveraging DNA, the code of life, has allowed us to unravel cellular and evolutionary processes in exquisite detail. Today our need for new innovation is still great. The modern world is a challenging environment: over-population, climate change and highly mobile populations create a high risk of pandemic disease especially from viruses and many bacteria are now resistant to our life saving antibiotic drugs due to overuse. In hospitals, the spread of pathogens can be rapid and life threatening. Whole-genome sequencing has the power to identify the source of infections and determine whether clusters of cases belong to an outbreak. Portable, real-time nanopore sequencing enables sequencing to be performed near the patient, even in resource-limited settings. Integrating with existing datasets allows digital surveillance able to detect outbreaks earlier while they can still be contained. Early demonstrations of the power of whole-genome sequencing for outbreak surveillance have made it an area of intense interest and further development in laboratory methods and infrastructure will make it an important tool that can be deployed in response to future outbreaks.
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Elmouelhi, Ahmed (Ahmed M. ). 1979. "Genome scanning : an AFM-based DNA sequencing technique". Thesis, Massachusetts Institute of Technology, 2003. http://hdl.handle.net/1721.1/34149.
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Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2003.Includes bibliographical references (p. 157-160).
Genome Scanning is a powerful new technique for DNA sequencing. The method presented in this thesis uses an atomic force microscope with a functionalized cantilever tip to sequence single stranded DNA immobilized to a mica surface. The functionalized cantilever tip hybridizes with only one base type (A, C, T, or G) and results in distinct peaks in the AFM-produced image. Genome Scanning has been successful at identifying 40 base strands of synthesized DNA and has been shown to detect a particular base type on 48 kilobase strands of lambda DNA. Currently, Genome Scanning is only accurate to 3-26 bases at a time, however, it can achieve a sequencing speed of 6000 bases/sec. In other words, Genome Scanning can be used to sequence the 3 billion bases of the human genome in 5.78 days.
by Ahmed Elmouelhi.
S.M.
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Schwartz, Jerrod Joseph. "Technologies for high throughput single molecule DNA sequencing /". May be available electronically:, 2009. http://proquest.umi.com/login?COPT=REJTPTU1MTUmSU5UPTAmVkVSPTI=&clientId=12498.
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Sharma, Prashant. "Hardware accelerator for SOM based DNA sequencing Algorithm". Thesis, KTH, Skolan för elektroteknik och datavetenskap (EECS), 2018. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-251808.
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The prevalent experience based diagnosis of health problem are often incorrect. Different aspect of this problem are microorganism’s adaptation of antibiotics and effectiveness of the generic medicines on each individual etc. The DNA sequencing based diagnosis is evolving to deal with this problem. The algorithmic part of these techniques is difficult to speedup and therefore have a high latency. As a solution to this problem, machine learning based methods, such as BioNN, uses self organizing maps(SOM) which do not need an explicit assembly process and categorizes bacteria with smaller sample data. For a memory and computation intensive process, such as BioNN, it is undesirable to implement on CPUs. The generic architectures, such as GPUs, are designed to handle varying range of needs, thus may not be the most power and performance efficient. Furthermore, the cloud based solutions will provide even worse results. Therefore, the customized hardware has to be designed. Moreover, the design and verification of an architecture from the scratch from the ASIC methodology requires considerable engineering effort. This project plans to use a coarse grain re-configurable architecture (CGRA) platform also known as the SiLago. The SiLago methodology is aimed to reduce the design and verification effort of the custom design repective to the ASIC methodology, without compromising much on the design trade-offs. The algorithm has been implemented on two coarse grain reconfigurable fabrics, providing a kick start to the ambitious project. The parametric-SiLago implementation of BioSOMs, where trained for two E Coli strains of bacteria with 40K training vectors. The results of SiLago implementation were benchmarked against a GPU GTX 1070 implementation in the CUDA framework. The comparison reveals 4 to 140 X speed up and 4 to 5 orders improvement in energy-delay product compared to implementation on GPU.
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Clark, Graeme T. "A study of DNA repair". Thesis, University of Southampton, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.302000.
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Mitra, Robi David. "Polony sequencing : DNA sequencing technology and a computational analysis reveals chromosomal domains of gene expression". Thesis, Massachusetts Institute of Technology, 2000. http://hdl.handle.net/1721.1/8797.
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Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2000.Includes bibliographical references.
The first part of this thesis describes the development of polony sequencing, a sequencing technology in which DNA is cloned, amplified and sequenced in a polymer matrix. A complex library of one to ten million linear DNA molecules is amplified by performing polymerase chain reaction (PCR) in a thin polyacrylamide film poured on a glass microscope slide. The polyacrylamide matrix retards the diffusion of the DNA molecules so that each amplification product remains localized near its parent molecule. At the end of the reaction, a number of polymerase colonies, or "polonies", have formed, each one grown from a single template molecule. As many as 5 million clones can be amplified in parallel on a single slide. By including an acrydite modification at the 5' end of one of the PCR primers, the amplified DNA will be covalently attached to the polyacrylamide matrix, allowing further enzymatic manipulations to be performed on all clones simultaneously. Also described in this thesis is my progress in development of a protocol to sequence the polonies by repeated cycles of extension with fluorescent deoxynucleotide. Because polony sequencing is inherently parallel, and sub-picoliter volumes are used for each reaction, the technology should be substantially faster and cheaper than existing methods. Applications for polony sequencing such as gene expression analysis, SNP discovery, and SNP screening will also be discussed. The second part of this thesis describes a computational analysis that tests the hypothesis that chromosomal position affects gene expression. It is shown that, throughout the genome, genes lying close together on the same chromosome often show significant coexpression. This coexpression is independent of the orientation of genes to each other, but is dependent on the distance between genes. In several cases where adjacent genes show highly correlated expression, the promoter of only one of the genes contains an upstream activating sequence (UAS) known to be associated with the expression pattern. These results suggest that in certain regions of the genome a single transcription factor binding site may regulate several genes. It is also shown that evolution may take advantage of this phenomenon by keeping genes with similar functions in adjacent positions along the chromosomes. The techniques that are presented provide a computational method to delineate the locations of chromosomal domains and identify the boundary elements that flank them.
Robi David Mitra.
Ph.D.
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Voss, Karl O. "Capillary electrophoresis for DNA sequencing and cytosine methylation analysis". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp02/NQ29120.pdf.
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FERNANDES, ERALDO LUIS REZENDE. "HEURISTICS FOR THE PROBLEM OF DNA SEQUENCING BY HYBRIDIZATION". PONTIFÍCIA UNIVERSIDADE CATÓLICA DO RIO DE JANEIRO, 2005. http://www.maxwell.vrac.puc-rio.br/Busca_etds.php?strSecao=resultado&nrSeq=6412@1.
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COORDENAÇÃO DE APERFEIÇOAMENTO DO PESSOAL DE ENSINO SUPERIORO seqüenciamento por hibridação é uma alternativa interessante para a tarefa de seqüenciamento de DNA. Este método ainda está sendo aperfeiçoado e pode superar as técnicas utilizadas em termos de tempo e custo. Uma etapa crucial do método consiste em resolver um problema combinatório que pode ser formulado como um caso especial do problema do caixeiro viajante com coleta de prêmios. Neste trabalho, propõe-se uma nova heurística construtiva multi-partida para resolver este problema. Uma estratégia de aprendizado baseada em uma memória adaptativa e um procedimento de construção de vocabulário são utilizados para melhorar o desempenho da heurística multi-partida. A memória adaptativa é utilizada para intensificar as construções de novas soluções com os elementos que aparecem com uma freqüência maior nas melhores soluções encontradas anteriormente pela heurística multi-partida. O procedimento de construção de vocabulário consiste em construir novas soluções através da combinação de partes comuns a boas soluções. Testes computacionais mostraram que estas duas estratégias aumentam significativamente o desempenho da heurística multi-partida e são particularmente indicadas para problemas de escalonamento nos quais as melhores soluções são na maioria dos casos formadas por blocos de elementos que aparecem juntos com muita freqüência. A heurística proposta supera os resultados dos melhores algoritmos encontrados na literatura, tanto em termos da qualidade das soluções encontradas, como do tempo de computação.
Sequencing by hybridization is an attractive alternative for DNA sequencing. This novel method can be less time and cost consuming than the techniques applied nowadays. A very important step of this method is to solve a combinatorial problem formulated as a special case of the prize-collecting traveling salesman problem. In this work, we propose a new multistart construtive heuristic to solve this problem. A learning strategy based on adaptive memory and a vocabulary building procedure are used to improve the performance of the multistart heuristic. The adaptive memory is used to intensify the construction of new solutions with the elements that appear frequently in the best solutions previously found by the multistart heuristic. The objective of the vocabulary building procedure is to construct new solutions combining parts of good solutions. Computational experiments have shown that these two methods significantly improves the performance of the multistart heuristic and are particularly suitable for scheduling problems whose best solutions are in most cases built by blocks of elements that appear together very often. The proposed heuristic obtains systematically better solutions and is less time consuming than the best algorithms found in the literature.
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Herena, Louis E. (Louis Eduardo) 1963. "Application of manufacturing tools in the DNA sequencing process". Thesis, Massachusetts Institute of Technology, 1999. http://hdl.handle.net/1721.1/80760.
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Thesis (M.B.A.)--Massachusetts Institute of Technology, Sloan School of Management; and, (S.M.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering; in conjunction with the Leaders for Manufacturing Program at MIT, 1999.Includes bibliographical references.
by Louis E. Herena.
S.M.
M.B.A.
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Fritz, Markus Hsi-Yang. "Exploiting high throughput DNA sequencing data for genomic analysis". Thesis, University of Cambridge, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.610819.
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Stoddart, David. "Progress towards ultra-rapid DNA sequencing with protein nanopores". Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:597b217c-c995-401d-b772-4cb373d195af.
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The sequencing of individual DNA strands with nanopores is being developed as a rapid, low-cost platform in which bases are identified in order as a DNA strand is transported through a pore under an electrical potential. Several challenges remain and this thesis focuses on one major area, the base identification properties of the a-hemolysin (aHL) nanopore. Under the potentials required for threading, DNA translocates too fast for single bases to be identified. However, immobilization of the DNA within the pore increases the residence time and therefore improves the precision of the electrical current reading and allows for the small differences in current flow, associated with different sequences, to be observed. DNA molecules with a 3'-terminal biotin-tag were complexed with streptavidin. Streptavidin is too large to be transported through the aHL pore and therefore the DNA-btnestreptavidin complex is not fully translocated; thus, the DNA strand is immobilized within the pore. Using this approach the nucleobase recognition properties of the aHL pore were mapped. The data suggest that the transmembrane 13 barrel domain of the pore contains at least three nucleobase recognition sites, termed R1, R2 and R3. Additional sequence information can be gained when multiple recognition sites are employed within a single aHL pore, as compared to the simple case of a single recognition site. Recognition site R1, which is located near the central constriction, can be modified by site-directed mutagenesis of Met-113. It was observed that amino acids with related side chains produce similar patterns of nucleobase recognition. Amino acids that provide an energy barrier to ion flow (e.g. bulky or hydrophobic residues) strengthen base identification, while amino acids that lower the barrier, weaken identification. Deletion and site-directed mutagenesis were used to remove one recognition site and generate an αHL pore. With truncated β barrel domain that contains only two recognition sites.
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Xu, D. Q. "Automatic interpretation of MWPC images of DNA sequencing gels". Thesis, University of Manchester, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.712123.
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Stymne, Jakob y Odeback Oliver Welin. "Evaluation of Temporal Convolutional Networks for Nanopore DNA Sequencing". Thesis, KTH, Skolan för elektroteknik och datavetenskap (EECS), 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-295624.
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Nanopore sequencing, a recently developed methodfor DNA sequencing, involves applying a constant electricfield over a membrane and translocating single-stranded DNAmolecules through membrane pores. This results in an electricalsignal, which is dependent on the structure of the DNA. The aimof this project is to train and evaluate a non-causal temporalconvolution neural network in order to accurately translate suchelectrical raw signal into the corresponding nucleotide sequence.The training dataset is sampled from the E. coli bacterial genomeand the phage Lambda virus. We implemented and evaluatedseveral different temporal convolutional architectures. Using anetwork with five residual blocks with five convolutional layersin each block yields maximum performance, with a predictionaccuracy of 76.1% on unseen test data. This result indicates thata temporal convolution network could be an effective way tosequence DNA data.Nanopore sequencing är en nyligen utvecklad metod för DNA-sekvensering som innebär att man applicerar ett konstant elektriskt fält över ett membran och translokerar enkelsträngade DNA-molekyler genom membranporer. Detta resulterar i en elektrisk signal som beror på DNA-strukturen. Målet med detta projekt är att träna och evaluera icke-kausula ”temporal convolutional networks” som ska kunna översätta denna ofiltrerade elektriska signalen till den motsvarande nukleotidsekvensen. Träningsdatan är ett urval av genomen från bakterien E. coli och viruset phage Lambda. Vi implementerade och utvärderade ett antal olika nätverksstrukturer. Ett nätverk med fem residuala block med fem faltande lager i varje block gav maximal prestation, med en precision på 76.1% på testdata. Detta resultat indikerar att ett ”temporal convolution network” skulle kunna vara ett effektivt sätt att sekvensera DNA.
Kandidatexjobb i elektroteknik 2020, KTH, Stockholm
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Dutson, Claire L. "C5-modified 2'-deoxyuridine-5'-triphosphates for DNA sequencing". Thesis, University of Sheffield, 2015. http://etheses.whiterose.ac.uk/9172/.
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Allen, Esther. "C5-modified 2'-deoxycytidine-5'-triphosphates for DNA sequencing". Thesis, University of Sheffield, 2016. http://etheses.whiterose.ac.uk/12135/.
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Koh, Kyung Duk. "Repair, consequence, and profile of ribonucleotides in DNA". Diss., Georgia Institute of Technology, 2015. http://hdl.handle.net/1853/53534.
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Ribonucleotides, also known as ribonucleoside monophosphates (rNMPs), are the most abundant non-canonical nucleotides incorporated into genomic DNA. Despite the relevance, information about their repair pathways, consequences, and profiles is still lacking. Exploiting the use of oligonucleotides containing rNMPs in a molecular approach to generate various RNA/DNA hybrids of chosen sequence and structure at the chromosomal level in cells, we show that mispaired rNMPs embedded into genomic DNA are not only targeted by ribonucleases H (RNases H) but also by the mismatch repair (MMR) system both in E. coli and S. cerevisiae cells. In addition, we discovered that paired rNMPs in DNA are targets of both RNase H type 2 and nucleotide excision repair (NER) in yeast. Also, we report atomic force microscopy (AFM)-based single molecule elasticity measurement, molecular dynamics simulation, and nuclear magnetic resonance spectroscopy results, showing that rNMPs in short DNA duplexes can change the elastic and structural properties of DNA. Lastly, we developed ribose-seq, a method for capturing rNMPs embedded in DNA. High-throughput sequencing of rNMP-captured molecules from the yeast S. cerevisiae revealed widespread but non-random rNMP distribution with preferences in base composition of rNMPs and neighboring DNA sequence context in both nuclear and mitochondrial DNA. With ribose-seq, systematic profiling of rNMP incorporation into genomic DNA is achieved, potentially allowing determination of specific signatures of rNMPs in DNA which could help to better understand the nature of rNMP repair mechanisms, effect of rNMPs on DNA mechanical properties and structure, and eventually rNMP impact on genome integrity.
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Smith, Victoria. "A molecular genetic analysis of yeast chromosome IX". Thesis, University of Cambridge, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.239206.
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Hawkins, Trevor. "Genome sequencing and analysis of 260Kb covering chromosome III of Caenorhabditis elegans". Thesis, University of Sussex, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.332544.
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Adams, Maria. "Genetic and functional analysis of the human thyroid hormones receptor beta". Thesis, University of Hertfordshire, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.262796.
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Bowler, Frank Ray. "Reading DNA with PNA : a dynamic chemical approach to DNA sequence analysis". Thesis, University of Edinburgh, 2011. http://hdl.handle.net/1842/5270.
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Single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) constitute important sources of genetic variation which provide insight into disease aetiology and idiosyncratic differences in drug response. The analysis of such genetic variation relies upon the generation of allele-specific products, typically by enzymatic extension or the hybridization of allele-specific DNA probes. Herein, a distinct enzyme-free, dynamic chemistry-based method of producing allele-specific products for genotyping was developed. The approach was initially demonstrated in model systems using synthetic DNA, which was used as a template in a base-filling reductive amination reaction on a PNA backbone. The templated dynamic reaction between a free secondary amine at a ‘blank’ position on the PNA strand and four aldehyde-modified nucleobases drove selective formation of the ‘correct’ iminium intermediate according to Watson-Crick base-pairing rules. In a blind trial, the method was extended to genotype twelve cystic fibrosis patients for two mutations (one SNP and one indel) linked to this disease. Enzyme-free dynamic chemistry thus permitted successful genotyping in both singleplex and duplex formats, demonstrating the application of dynamic chemistry as a distinct method of allelediscrimination with certain advantages over those reported previously. The application of this method as a tool for the discovery of non-natural nucleobases with improved properties for antisense and genotyping applications was also investigated. Furthermore, progress was made towards the use of dynamic chemistry as a means of full nucleic acid sequence analysis, through the templated sequence-selective extension of PNA probes by reductive amination.
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Bauer, David L. V. "Preparing and sequencing ultra-long DNA molecules from single chromosomes". Thesis, University of Oxford, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.640151.
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In this thesis, I describe the development of a single-molecule platform for analysing long DNA molecules that captures haplotype and large-scale structural variation (SV) in addition to DNA sequence. Cunent DNA sequencing methods cannot adequately examine haplotype and SV - both contribute to biological function and disease and are candidates for the location of "missing heritability" in the genome. Both haplotype and SV fundamentally relate to the structure of single chromosomes. Using a lab-on-a-chip nanofluidic device, SV was analysed on stretched (> 2 Mb) DNA fragments. In order to integrate this larger-scale SV information with the base-by-base sequence of the molecule being analysed, the DNA molecule was amplified and sequenced. I developed algorithms to handle the unique features of sequence data from amplified single DNA molecules. I obtained sequence and genotyping data, confirmed successful isolation of single DNA fragments from the chip, and validated the barcoding method used to detect SV. This lab-on-a-chip device for handling long DNAs can also serve as a 'reaction chamber' to answer more fundamental biological questions regarding chromosome structure as a whole. Using a microfluidic chip, I was able to provide the first direct images of DNA catenation within metaphase chromosomes and demonstrate that DNA catenation, in addition to proteins, plays a crucial role in metaphase chromosome architecture. The fluidic platform can be adapted to future 'third-generation' single-molecule sequencing applications that intenogate single DNA molecules directly. I have demonstrated this potential in two ways: First, I used intercalating dyes to form an optical waveguide along DNA to improve single-molecule detection. Secondly, I I engineered E. coli RNA Polymerase to detect single base translocation events along a DNA substrate. Such a polymerase could be used in future third-generation sequencing schemes based upon base-stepping motion or energy transfer to dye-modified nucleotides as the polymerase processes on a long DNA template.
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Graham, Joseph (Joseph Arthur). "An analysis of the next generation DNA sequencing technology market". Thesis, Massachusetts Institute of Technology, 2007. http://hdl.handle.net/1721.1/42360.
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Thesis (S.M.)--Massachusetts Institute of Technology, System Design and Management Program, 2007.Includes bibliographical references (p. 57-60).
While there is no shortage of successful and failed biotechnology ventures, it is still very difficult to gage, a priori, how a new company will fare in this industry. In many cases new biotechnology ventures are driven by rapidly evolving technology and emergent customer needs, both unpredictable by nature. Also, the Biotech Industry faces increased public and federal scrutiny as companies attempt to navigate murky ethical and legal waters. This thesis will explore the ongoing development of the next generation DNA sequencing market in an effort to predict exactly which factors will play a role in determining who will ultimately succeed. This will be accomplished through an analysis incorporating a combination of historical precedents in this industry and traditional market theories. The goal is to produce a set of dimensions along which to judge the current and future participants in this market in order to determine which are most likely to succeed.
by Joseph Graham.
S.M.
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Hogstrom, Larson J. "Return on investment and library complexity analysis for DNA sequencing". Thesis, Massachusetts Institute of Technology, 2016. http://hdl.handle.net/1721.1/104556.
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Thesis: S.M., Massachusetts Institute of Technology, Computation for Design and Optimization Program, 2016.Cataloged from PDF version of thesis.
Includes bibliographical references (page 49).
Understanding the profiles of information acquisition during DNA sequencing experiments is critical to the design and implementation of large-scale studies in medical and population genetics. One known technical challenge and cost driver in next-generation sequencing data is the occurrence of non-independent observations that are created from sequencing artifacts and duplication events from polymerase chain reaction (PCR). The current study demonstrates improved return on investment (ROI) modeling strategies to better anticipate the impact of non-independent observations in multiple forms of next-generation sequencing data. Here, a physical modeling approach based on Pó1ya urn was evaluated using both multi-point estimation and duplicate set occupancy vectors. The results of this study can be used to reduce sequencing costs by improving aspects of experimental design including sample pooling strategies, top-up events, and termination of non-informative samples.
by Larson J. Hogstrom.
S.M.
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Song, Jamie 1977. "Optimization of data acquisition system for novel DNA sequencing instrument". Thesis, Massachusetts Institute of Technology, 2000. http://hdl.handle.net/1721.1/86500.
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Thesis (S.B. and M.Eng.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2000.Includes bibliographical references (leaf 61).
by Jamie Song.
S.B.and M.Eng.
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Kumar, Mayank S. M. Massachusetts Institute of Technology. "Development of a microfluidic platform for integrated DNA sequencing protocols". Thesis, Massachusetts Institute of Technology, 2007. http://hdl.handle.net/1721.1/40371.
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Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2007.Includes bibliographical references.
This thesis describes the design and development of a microfluidic platform to reduce costs and improve the quality of in the DNA sequencing methodology currently implemented at the Broad Institute in Cambridge, Massachusetts. The Sequencing Center at the Broad Institute currently generates an average of 130 million bases per day with an average read length of 800. This is enabled by the successful preparation and detection of over 97,000 unique samples. Most of the cost per sample is tied up in expensive proprietary reagents utilized in the various reactions comprising the preparation process. Through the application of microfluidics, the possibility of drastically scaling down the amount of proprietary reagents is explored. Stamp-sized elastomeric polydimethylsiloxane (PDMS) microfluidic devices were developed and microfluidic sample manipulation techniques were standardized. Using these devices and techniques, an attempt was made to adapt the various components of the sequencing process to the microfluidic platform. Work within the scope of this thesis is focused on the adaptation of the commercial sequencing protocols, which are labor intensive, consume costly reagents and serve as limitations for high-throughput parallelization of the process.
(cont.) The first is the amplification reaction. By scaling down the process from a plate-based format to an integrated microfluidic device, amplification reagent consumption was reduced by two orders of magnitude while maintaining the quality and length of the sequencing reads (with the subsequent sequencing reaction run off chip). As a follow up project, an attempt was made to scale down the Sequencing Reaction, which, in spite of limitations, suggested a good path toward the eventual development of an integrated microfluidic device for the preparation of running the complete sequencing reaction protocol on-chip.
by Mayank Kumar.
S.M.
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Sigurgeirsson, Benjamín. "Analysis of RNA and DNA sequencing data : Improved bioinformatics applications". Doctoral thesis, KTH, Genteknologi, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-184158.
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Massively parallel sequencing has rapidly revolutionized DNA and RNA research. Sample preparations are steadfastly advancing, sequencing costs have plummeted and throughput is ever growing. This progress has resulted in exponential growth in data generation with a corresponding demand for bioinformatic solutions. This thesis addresses methodological aspects of this sequencing revolution and applies it to selected biological topics. Papers I and II are technical in nature and concern sample preparation and data anal- ysis of RNA sequencing data. Paper I is focused on RNA degradation and paper II on generating strand specific RNA-seq libraries. Paper III and IV deal with current biological issues. In paper III, whole exomes of cancer patients undergoing chemotherapy are sequenced and their genetic variants associ- ated to their toxicity induced adverse drug reactions. In paper IV a comprehensive view of the gene expression of the endometrium is assessed from two time points of the menstrual cycle. Together these papers show relevant aspects of contemporary sequencing technologies and how it can be applied to diverse biological topics.QC 20160329
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Camerlengo, Terry Luke. "Techniques for Storing and Processing Next-Generation DNA Sequencing Data". The Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1388502159.
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Manara, Richard. "Free energy calculations of DNA translocation through protein nanopores and nanopore design for DNA sequencing". Thesis, University of Southampton, 2015. https://eprints.soton.ac.uk/374791/.
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DNA sequencing has vastly opened up the world of molecular biology, leading to new areas of interest, especially in medical research. Unfortunately the methods of DNA sequencing have only ever seen gradual improvements, as Sanger sequencing is still very much the norm despite its high cost and slow speed. Nanopores present an exciting opportunity for DNA sequencing, however, despite the concept being presented in 1996 several problems have prevented the creation of a publicly available sequencing device. The two main focuses of research into nanopores so far have been improving the resolution between bases and the slowing down of DNA translocation through the pore so modern ammeters can read the sequence accurately. The simulation work presented in this thesis largely focuses on the energetics associated with DNA translocation. This is performed in several parts; an investigation into the probability of pore entry, study into the free energy of translocation for two proteins in addition to solvent contribution to this free energy, finally a theoretical project was undertaken to investigate bottom up nanopore design.
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Buschmann, Tilo. "The Systematic Design and Application of Robust DNA Barcodes". Doctoral thesis, Universitätsbibliothek Leipzig, 2016. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-209812.
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High-throughput sequencing technologies are improving in quality, capacity, and costs, providing versatile applications in DNA and RNA research. For small genomes or fraction of larger genomes, DNA samples can be mixed and loaded together on the same sequencing track. This so-called multiplexing approach relies on a specific DNA tag, index, or barcode that is attached to the sequencing or amplification primer and hence accompanies every read. After sequencing, each sample read is identified on the basis of the respective barcode sequence.
Alterations of DNA barcodes during synthesis, primer ligation, DNA amplification, or sequencing may lead to incorrect sample identification unless the error is revealed and corrected. This can be accomplished by implementing error correcting algorithms and codes. This barcoding strategy increases the total number of correctly identified samples, thus improving overall sequencing efficiency. Two popular sets of error-correcting codes are Hamming codes and codes based on the Levenshtein distance.
Levenshtein-based codes operate only on words of known length. Since a DNA sequence with an embedded barcode is essentially one continuous long word, application of the classical Levenshtein algorithm is problematic. In this thesis we demonstrate the decreased error correction capability of Levenshtein-based codes in a DNA context and suggest an adaptation of Levenshtein-based codes that is proven of efficiently correcting nucleotide errors in DNA sequences. In our adaptation, we take any DNA context into account and impose more strict rules for the selection of barcode sets. In simulations we show the superior error correction capability of the new method compared to traditional Levenshtein and Hamming based codes in the presence of multiple errors.
We present an adaptation of Levenshtein-based codes to DNA contexts capable of guaranteed correction of a pre-defined number of insertion, deletion, and substitution mutations. Our improved method is additionally capable of correcting on average more random mutations than traditional Levenshtein-based or Hamming codes. As part of this work we prepared software for the flexible generation of DNA codes based on our new approach. To adapt codes to specific experimental conditions, the user can customize sequence filtering, the number of correctable mutations and barcode length for highest performance.
However, not every platform is susceptible to a large number of both indel and substitution errors. The Illumina “Sequencing by Synthesis” platform shows a very large number of substitution errors as well as a very specific shift of the read that results in inserted and deleted bases at the 5’-end and the 3’-end (which we call phaseshifts). We argue in this scenario that the application of Sequence-Levenshtein-based codes is not efficient because it aims for a category of errors that barely occurs on this platform, which reduces the code size needlessly. As a solution, we propose the “Phaseshift distance” that exclusively supports the correction of substitutions and phaseshifts. Additionally, we enable the correction of arbitrary combinations of substitution and phaseshift errors. Thus, we address the lopsided number of substitutions compared to phaseshifts on the Illumina platform.
To compare codes based on the Phaseshift distance to Hamming Codes as well as codes based on the Sequence-Levenshtein distance, we simulated an experimental scenario based on the error pattern we identified on the Illumina platform. Furthermore, we generated a large number of different sets of DNA barcodes using the Phaseshift distance and compared codes of different lengths and error correction capabilities. We found that codes based on the Phaseshift distance can correct a number of errors comparable to codes based on the Sequence-Levenshtein distance while offering the number of DNA barcodes comparable to Hamming codes. Thus, codes based on the Phaseshift distance show a higher efficiency in the targeted scenario. In some cases (e.g., with PacBio SMRT in Continuous Long Read mode), the position of the barcode and DNA context is not well defined. Many reads start inside the genomic insert so that adjacent primers might be missed. The matter is further complicated by coincidental similarities between barcode sequences and reference DNA. Therefore, a robust strategy is required in order to detect barcoded reads and avoid a large number of false positives or negatives.
For mass inference problems such as this one, false discovery rate (FDR) methods are powerful and balanced solutions. Since existing FDR methods cannot be applied to this particular problem, we present an adapted FDR method that is suitable for the detection of barcoded reads as well as suggest possible improvements.
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Van, der Vaart Maniesh. "Characterization of circulating free DNA in healthy and diseased individuals / Maniesh van der Vaart". Thesis, North-West University, 2009. http://hdl.handle.net/10394/5074.
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Thornley, David John. "Analysis of trace data from fluorescence based Sanger sequencing". Thesis, Imperial College London, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.286265.
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Bourin, Stephanie. "Novel strategies for DNA detection assay". Thesis, University of London, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.265939.
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Rose, B. A. (Beverley Ann). "The characterisation and partial sequencing of the grapevine chloroplast genome". Thesis, Stellenbosch : Stellenbosch University, 2004. http://hdl.handle.net/10019.1/53763.
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Thesis (MSc)--University of Stellenbosch, 2004.ENGLISH ABSTRACT: A number of proteins essential for the survival of a plant are encoded by the chloroplast genome. The characterization and sequencing of a number of algal and plant chloroplast genomes has facilitated researchers understanding of cellular functions and metabolism. Chloroplast DNA (cpDNA) has also been used to determine inter- and intraspecies evolutionary relationships and this organelle offers an alternative means of expressing foreign genes. Although a number of species' chloroplast genomes have been characterized and sequenced, no previous attempts of this kind have been made for a chloroplast genome of the family Vitaceae. In this study, attempts were made to characterize and partially sequence the chloroplast genome of Vilis vinifera. Chloroplast DNA was isolated from the Sultana and Sugra 1 cultivars and digested with restriction enzymes that produced cpDNA fragments of a suitable size for cloning. The fragments were shotgun-cloned into a plasmid vector and white colonies were screened by means of PCR and colony blotting. Three EcoRI-digested clones and one PstI-digested clone were obtained in this manner. Walking outwards from a previously sequenced grapevine rrn 16 gene region by means of PCR also allowed us to sequence a further -3310 bp region of the Sultana chloroplast genome. BAC clones containing V. vinifera cv L. Cabernet Sauvignon cpDNA inserts became available later in the project. It was decided to use these clones for further library construction instead of isolated cpDNA. The 5' and 3' end sequences of seven of the 24 BAC clones were obtained. These were compared to sequences found in the NCBI database to find - homologous chloroplast regions and determine the size of each BAC insert. One clone appeared to contain the entire grapevine chloroplast genome, apart from a 500 bp region. This clone was selected for further analysis. The BAC clone DNA was isolated and restriction-digested fragments were shotgun-cloned into a plasmid vector. White colonies were screened by isolating the plasmid DNA and digesting it with appropriate restriction enzy~es. The 5' and 3' ends of putative positive clones were sequenced and mapped onto the Atropa belladonna chloroplast genome. A total of 15 clones were obtained in this project. Five of these contain cpDNA isolated from grapevine leaves and 10 contain fragments sub-cloned from the BAC clone. The biggest problem encountered with both methods used for library construction was genomic DNA contamination. Genomic DNA either originated from the plant nuclear genome or from the bacterial host cells in which the BAC clones were maintained. Many of the clones screened contained genomic DNA, and these could only be identified and removed once the clones had been sequenced. Even when a commercial kit was used for BAC clone isolation, 31% of the clones screened contained genomic DNA. This kit was specifically designed for the isolation of genomic DNA-free large constructs. The clones obtained from the two strategies provided a good representation of the grapevine chloroplast genome. The only region not represented was the Small Single Copy (SSC) region. Approximately 40% of the grapevine chloroplast genome was covered by these clones. This provides a basis for further genome characterization, physical mapping and sequencing of the grapevine chloroplast genome.
AFRIKAANSE OPSOMMING: Die chloroplasgenoom kodeer VIr 'n hele aantal proteïene wat essensieel is VIr die voortbestaan van 'n plant. Die karakterisering en volgorde bepaling van 'n aantal alg en plant chloroplasgenome het dit. vir navorsers moontlik gemaak om sellulêre funksies en metabolisme van plante te ontrafel. Chloroplas DNA (cpDNA) is ook gebruik om intra- en interspecies evolusionêre verwantskappe vas te stel. Dié organel verskaf ook 'n alternatiewe manier vir die uitdrukking van transgene. Alhoewel die chloroplasgenome van 'n hele aantal species al gekarakteriseer is en die DNA volgorde daarvan bepaal is, is daar nog geen navorsing van bogenoemde aard op die chloroplasgenoom van die Vitaceae familie gedoen rue. In hierdie studie is beoog om die chloroplasgenoom van Vitis vinifera te karakteriseer en gedeeltelike volgordebepaling daarvan te doen. Chloroplas DNA is geïsoleer vanaf Sultana en Sugra 1 kultivars en restriksie-ensiem vertering is gedoen met ensieme wat cpDNA fragmente, met geskikte grootte vir klonering, produseer. Dié fragmente is in 'n plasmiedvektor gekloneer met die haelgeweer-metode en wit kolonies is gesif deur middel van PKR en die kolonieklad metode. Op hierdie manier is drie EcoRI-verteerde klone en een PstI-verteerde kloon verkry. Deur uitwaarts te loop, deur middel van PKR, vanaf 'n druif rrnl6 geenstreek, waarvan die volgorde voorafbepaal is, was dit vir ons moontlik om ook die volgorde te bepaal van 'n verdere ~3310 bp streek van die Sultana chloroplasgenoom. BAC klone wat V. vinifera cv L. Cabernet Sauvignon cpDNA fragmente bevat, het later in die projek beskikbaar geraak. Daar is besluit om hierdie klone, i.p.v. die geïsoleerde cpDNA, te gebruik vir verdere biblioteek konstruksie. Die 5' en 3' entpuntvolgordes van sewe uit die 24 BAC ~lone is verkry. Hierdie volgordes is vergelyk met volgordes in die NCB Idatabasis om homoloë chloroplas streke te identifiseer, en die grootte van elke BAC fragment te bepaal. Die het geblyk dat die hele druif chloroplasgenoom in een van die klone vervat is, behalwe vir 'n 500 bp streek. Die BAC-kloon DNA is geïsoleer en die restriksie-verteerde fragmente is in 'n plasmiedvektor gekloon d.m.V. die haelgeweer-metode. Wit kolonies is gesif deur die isolering van plasmied DNA en die vertering daarvan met geskikte restriksie-ensieme. Die volgorde van die 5' en 3' entpunte van skynbare positiewe klone is bepaal en gekarteer op die Atropa belladonna chloroplasgenoom. In hierdie studie is 'n totaal van 15 klone verkry. Vyf hiervan bevat cpDNA wat vanaf druifblare geïsoleer is, en 10 bevat fragmente wat vanaf die BAC-klone gesubkloneer is. Genorniese DNA kontaminasie was die grootste probleem wat ondervind is tydens beide metodes wat gebruik is vir biblioteek konstruksie. Genomiese DNA was afkomstig vanaf óf die plant nukleêre genoom óf die bakteriële gasheerselle waarin die BAC-klone gehou is. Baie van die klone wat gesif is, het genomiese DNA bevat, en dit kon eers geïdentifiseer en verwyder word nadat die volgorde van die klone bepaal is. Selfs al is 'n kommersiële produk vir BAC-kloon isolasie gebruik, het 31% van die gesifde klone steeds genomiese DNA bevat. Dié kommersiële produk is spesifiek vir die isolasie van groot konstrukte, wat genomiese DNA vry is, ontwerp. Die klone wat deur die twee strategeë verkry is, het 'n goeie verteenwoordiging van die druif chloroplasgenoom gegee. Die enigste streek wat die verteenwoordig is nie, was die Klein Enkelkopie (SSC) streek. Ongeveer 40% van die druif chloroplasgenoom is deur hierdie klone gedek. Dit verskaf 'n basis vir verdere genoomkarakterisering, fisiese kartering en volgordebepaling van die druif chloroplasgenoom.
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Lin, Cheng-Hsien Kenny. "An ASIC application for DNA sequencing by Smith-Waterman algorithm (DNASSWA) /". [St. Lucia, Qld.], 2004. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe18716.pdf.
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Yuwaraj, Murugathas. "A local processor for improving the accuracy of automated DNA sequencing". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp05/NQ63645.pdf.
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