Orientador: Alexandre Reily Rocha / Dissertação (mestrado) - Universidade Federal do ABC. Programa de Pós-Graduação em Nanociências e Materiais Avançados, 2014.

Exome sequencing, the process of sequencing the set of all known exons simultaneously using next-generation sequencing technology, has dramatically changed the landscape of genetic research and genetic testing. The incredible volume of data produced by these experiments creates challenges in: 1) annotating the affects of observed variants, 2) filtering to remove noise, 3) identifying plausible disease-causing variants, and 4) validating experimental results. Here we will present a series of bioinformatic tools and techniques intended to address these challenges with exome sequencing and associated validation experiments. First, we will present the Automated Sequence Analysis Pipeline (ASAP), a tool for the efficient and automated management, detection and annotation of Sanger sequencing-based genetic testing and variant validation. This pipeline is extended to annotate exome-sequencing derived variants. Exome sequencing experiments produce a great number of variants that do not cause a patient's disease. One of the biggest challenges in exome sequencing experiments is sorting through these false positives to discover the true disease-causing variants. We have developed several techniques to aid in the reduction of these errors. The techniques described include: 1) the construction of a catalog of systematic errors by reprocessing thousands of publically available exomes, 2) a tool for the filtering of variants based on family structure and disease assumptions, and 3) a tool for discovering regions of autozygosity from the exomes of several affected patients in consanguineous pedigrees. Classes of variants that are undiscoverable using current analysis techniques gives rise to false negatives in exome sequencing experiments. We will present a tool, the Retrotransposon Insertion Detector for Exomes (RIDE) that uses the characteristic anomalies present in sequence alignments to detect the insertion of repetitive elements. The process of identifying a the cause of a patient's disease using exome sequencing data has been equated to finding a needle in a stack of needles. Only through the proper annotation of variants and the reduction of the error rates associated with exome sequencing experiments can this task be achieved in an efficient manner.

The investigation of genetic alterations in cancer-relatedgenes is useful for research, prognostic and therapeuticpurposes. However, the genetic heterogeneity that often occursduring tumour progression can make correct analysischallenging. The objective of this work has been to develop,evaluate and apply techniques that are sufficiently sensitiveand specific to detect and analyse genetic alterations in skintumours as well as in normal skin. Initially, a method based on laser-assisted microdissectionin combination with conventional dideoxy sequencing wasdeveloped and evaluated for the analysis of the p53 tumoursuppressor gene in small tissue samples. This method was shownto facilitate the analysis of single somatic cells fromhistologic tissue sections. In two subsequent studies themethod was used to analyse single cells to investigate theeffects of ultraviolet (UV) light on normal skin. Single p53immunoreactive and nonimmunoreactive cells from differentlayers of sunexposed skin, as well as skin protected fromexposure, were analysed for mutations in the p53 gene. Theresults revealed the structure of a clandestine p53 clone andprovided new insight into the possible events involved innormal differentiation by suggesting a role for allele dropout.The mutational effect of physiological doses of ultravioletlight A (UVA) on normal skin was then investigated by analysingthe p53 gene status in single immunoreactive cells at differenttime-points. Strong indications were found that UVA (even atlow doses) is indeed a mutagen and that its role should not bedisregarded in skin carcinogenesis. After slight modifications, the p53 mutation analysisstrategy was thenused to complement an x-chromosomeinactivation assay for investigation of basal cell cancer (BCC)clonality. The conclusion was that although the majority ofBCC’s are of monoclonal origin, an occasional tumour withapparently polyclonal origin exists. Finally, apyrosequencing-based mutation detection method was developedand evaluated for detection of hot-spot mutations in the N-rasgene of malignant melanoma. More than 80 melanoma metastasissamples were analysed by the standard approach of single strandconformation polymorphism analysis (SSCP)/DNA sequencing and bythis pyrosequencing strategy. Pyrosequencing was found to be agood alternative to SSCP/DNA sequencing and showed equivalentreproducibility and sensitivity in addition to being a simpleand rapid technique. <b>Keywords:</b>single cell, DNA sequencing, p53, mutation,UV, BCC, pyrosequencing, malignant melanoma, N-ras

In the present study, history of domestic sheep were investigated by mitochondrial DNA (mtDNA) based haplogroups (HPG) of 628 samples and mtDNA control region (CR) sequences of 240 samples from 13 Turkish sheep breeds which were located in the hearth of the first domestication center. Also, 30 Anatolian wild sheep (Ovis gmelinii anatolica) mtDNA CR sequences were obtained to contribute to the scenarios on initial domestication stages of sheep. Haplogroup compositions of breeds were identified with SSCP method by using mtDNA ND2 region. The genetic diversity and relationship between haplogroups were calculated. Phylogenetic analyses of haplogroups such as median joining networks and neighbor joining trees were constructed for mtDNA CR, cytochrome B (cytB) and combined CR-cytB sequences with sequences from the present study together with sequences retrieved from NCBI (http://www.ncbi.nlm.nih.gov/). Results of the present study showed that all previously observed haplogroups (HPG A-E) were present in Turkish sheep breeds. Two individuals from rare HPG D and eleven individuals from rare HPG E were detected and sequenced. With increased sample size, for HPG E, past population expansion was observed as was the case of HPG A, B and C with mismatch distributions and neutrality tests. Spatial autocorrelation analyses and synthetic map with respect to mtDNA (maternal) pattern revealed that Turkey was separated into two regions which may be attributed to the imprints of third migration of sheep associated with the arrivals of nomadic Turks to Anatolia nearly 1000 years before present. Finally, Ovis gmelinii anatolica samples exhibited two haplotypes / one of them belongs to HPG A (possibly feral domesticate), and the other one shows a distinct haplotype (close to HPG E and C) that was not observed before. Observed, low mtDNA diversity might be the result of isolation, fragmentation, extinction of fragments and bottlenecks. Ovis gmelinii anatolica can be part of the evolved descendants of the wild sheep which gave birth to the domestic sheep.

<p>The investigation of genetic alterations in cancer-relatedgenes is useful for research, prognostic and therapeuticpurposes. However, the genetic heterogeneity that often occursduring tumour progression can make correct analysischallenging. The objective of this work has been to develop,evaluate and apply techniques that are sufficiently sensitiveand specific to detect and analyse genetic alterations in skintumours as well as in normal skin.</p><p>Initially, a method based on laser-assisted microdissectionin combination with conventional dideoxy sequencing wasdeveloped and evaluated for the analysis of the p53 tumoursuppressor gene in small tissue samples. This method was shownto facilitate the analysis of single somatic cells fromhistologic tissue sections. In two subsequent studies themethod was used to analyse single cells to investigate theeffects of ultraviolet (UV) light on normal skin. Single p53immunoreactive and nonimmunoreactive cells from differentlayers of sunexposed skin, as well as skin protected fromexposure, were analysed for mutations in the p53 gene. Theresults revealed the structure of a clandestine p53 clone andprovided new insight into the possible events involved innormal differentiation by suggesting a role for allele dropout.The mutational effect of physiological doses of ultravioletlight A (UVA) on normal skin was then investigated by analysingthe p53 gene status in single immunoreactive cells at differenttime-points. Strong indications were found that UVA (even atlow doses) is indeed a mutagen and that its role should not bedisregarded in skin carcinogenesis.</p><p>After slight modifications, the p53 mutation analysisstrategy was thenused to complement an x-chromosomeinactivation assay for investigation of basal cell cancer (BCC)clonality. The conclusion was that although the majority ofBCC’s are of monoclonal origin, an occasional tumour withapparently polyclonal origin exists. Finally, apyrosequencing-based mutation detection method was developedand evaluated for detection of hot-spot mutations in the N-rasgene of malignant melanoma. More than 80 melanoma metastasissamples were analysed by the standard approach of single strandconformation polymorphism analysis (SSCP)/DNA sequencing and bythis pyrosequencing strategy. Pyrosequencing was found to be agood alternative to SSCP/DNA sequencing and showed equivalentreproducibility and sensitivity in addition to being a simpleand rapid technique.</p><p><b>Keywords:</b>single cell, DNA sequencing, p53, mutation,UV, BCC, pyrosequencing, malignant melanoma, N-ras</p>

<p>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.</p><p>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.</p><p>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.</p><p>Another project described in this thesis is the comparativesequencing of the 40 kb<i>cag</i>pathogenicity island (PAI) in four<i>Helicobacter pylori</i>strains. 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 the<i>cag</i>PAI into two parts. Further, information about thevariability of different genes could be obtained.</p><p><b>Keywords:</b>DNA sequencing, DNA purification, automation,solid-phase, streptavidin, biotin, modular probes,<i>Helicobacter pylori</i>,<i>cag</i>PAI.</p>

Recent advancements in massively parallel biosensing and sequencing technologies have revolutionized the field of molecular biology and paved the way to novel and exciting innovations in medicine, biology, and environmental monitoring. Among them, biosensor arrays (e.g., DNA and protein microarrays) have gained a lot of attention. DNA microarrays are parallel affinity biosensors that can detect the presence and quantify the amounts of nucleic acid molecules of interest. They rely on chemical attraction between target nucleic acid sequences and their Watson-Crick complements that serve as probes and capture the targets. The molecular binding between the probes and targets is a stochastic process and hence the number of captured targets at any time is a random variable. Detection in conventional DNA microarrays is based on a single measurement taken in the steady state of the binding process. Recently developed real-time DNA microarrays, on the other hand, acquire multiple temporal measurements which allow more precise characterization of the reaction and enable faster detection based on the early dynamics of the binding process. In this thesis, I study target estimation and limits of performance of real time affinity biosensors. Target estimation is mapped to the problem of estimating parameters of discretely observed nonlinear diffusion processes. Performance of the estimators is characterized analytically via Cramer-Rao lower bound on the mean-square error. The proposed algorithms are verified on both simulated and experimental data, demonstrating significant gains over state-of-the-art techniques. In addition to biosensor arrays, in this thesis I present studies of the signal processing aspects of next-generation sequencing systems. Novel sequencing technologies will provide significant improvements in many aspects of human condition, ultimately leading towards the understanding, diagnosis, treatment and prevention of diseases. Reliable decision-making in such downstream applications is predicated upon accurate base-calling, i.e., identification of the order of nucleotides from noisy sequencing data. Base-calling error rates are nonuniform and typically deteriorate with the length of the reads. I have studied performance limits of base-calling, characterizing it by means of an upper bound on the error rates. Moreover, in the context of shotgun sequencing, I analyzed how accuracy of an assembled sequence depends on coverage, i.e., on the average number of times each base in a target sequence is represented in different reads. These analytical results are verified using experimental data. Among many downstream applications of high-throughput biosensing and sequencing technologies, reconstruction of gene regulatory networks is of particular importance. In this thesis, I consider the gene network inference problem and propose a probabilistic graphical approach for solving it. Specifically, I develop graphical models and design message passing algorithms which are then verified using experimental data provided by the Dialogue for Reverse Engineering Assessment and Methods (DREAM) initiative. / text

0.7nm single wall carbon nanotubes have been synthesized within VPI-5 zeolite channels with sucrose as carbon precursor. VPI-5 molecular sieves are synthesized hydrothermally under conventional heating. X-ray powder diffraction, micro raman, scanning electron microscope (SEM), transmission electron microscope (TEM), Thermogravimetric analysis have been used to investigate the structure of zeolite and thermal decoposition process of carbon precursors. 0.4nm single wall carbon nanotubes have also been fabricated within AlPO4-5 nanopores. A key challenge is to produce high yield single wall carbon nanotubes with uniform diameter. In order to improve the carbon nanotube yield, different organic precursors are employed. Although the problem is still the repetition and low yield of CNTs, it is still an improvement for 0.7nm SWNTs synthesis with the new template prolysis method. The novel multilayer conductor/insulator/conductor structures have been fabricated. This structure might find potential application in DNA sequential reactions because each layer might be individually addressed with voltage. When bias is applied to the conductive layer, it can be chemically functionalized, which leads to membrane pore with multiple reaction sequences when the molecule traverses the membrane reactor. In this thesis, Carbon/polymer/carbon system and copper/polymer system will be introduced. O2 RIE was used to expose the edge of carbon/polymer/carbon structure. However, the conductivity of carbon layer is not high enough for electroplating. Copper pores etched by FeCl3 solution shows good conductivity, and can be electroplated with metal nanoparticles.

Exome sequencing, the process of sequencing the set of all known exons simultaneously using next-generation sequencing technology, has dramatically changed the landscape of genetic research and genetic testing. The incredible volume of data produced by these experiments creates challenges in: 1) annotating the affects of observed variants, 2) filtering to remove noise, 3) identifying plausible disease-causing variants, and 4) validating experimental results. Here we will present a series of bioinformatic tools and techniques intended to address these challenges with exome sequencing and associated validation experiments. First, we will present the Automated Sequence Analysis Pipeline (ASAP), a tool for the efficient and automated management, detection and annotation of Sanger sequencing-based genetic testing and variant validation. This pipeline is extended to annotate exome-sequencing derived variants. Exome sequencing experiments produce a great number of variants that do not cause a patient's disease. One of the biggest challenges in exome sequencing experiments is sorting through these false positives to discover the true disease-causing variants. We have developed several techniques to aid in the reduction of these errors. The techniques described include: 1) the construction of a catalog of systematic errors by reprocessing thousands of publically available exomes, 2) a tool for the filtering of variants based on family structure and disease assumptions, and 3) a tool for discovering regions of autozygosity from the exomes of several affected patients in consanguineous pedigrees. Classes of variants that are undiscoverable using current analysis techniques gives rise to false negatives in exome sequencing experiments. We will present a tool, the Retrotransposon Insertion Detector for Exomes (RIDE) that uses the characteristic anomalies present in sequence alignments to detect the insertion of repetitive elements. The process of identifying a the cause of a patient's disease using exome sequencing data has been equated to finding a needle in a stack of needles. Only through the proper annotation of variants and the reduction of the error rates associated with exome sequencing experiments can this task be achieved in an efficient manner.

Thanks to the development of next-generation sequencing (NGS) technology, whole genome data can be readily obtained from a variety of samples. Since the massive increase in available sequencing data, the development of efficient assembly algorithms has become the new bottleneck. Almost every new released tool is based on the De Brujin graph method, which focuses on assembling complete datasets with mathematical models. Although the decreasing sequencing costs made whole genome sequencing (WGS) the most straightforward and least laborious approach of gathering sequencing data, many research projects are only interested in the extranuclear genomes. Unfortunately, few of the available tools are specifically designed to efficiently retrieve these extranuclear genomes from WGS datasets. We developed a seed-and-extend algorithm that assembles organelle circular genomes from WGS data, starting from a single short seed sequence. The algorithm has been tested on several new (Gonioctena intermedia and Avicennia marina) and public (Arabidopsis thaliana and Oryza sativa) whole genome Illumina datasets and always outperformed other assemblers in assembly accuracy and contiguity. In our benchmark, NOVOPlasty assembled all genomes in less than 30 minutes with a maximum RAM memory requirement of 16 GB. NOVOPlasty is the only de novo assembler that provides a fast and straightforward manner to extract the extranuclear sequences from WGS data and generates one circular high quality contig.Heteroplasmy, the existence of multiple mitochondrial haplotypes within an individual, has been researched across different fields. Mitochondrial genome polymorphisms have been linked to multiple severe disorders and are of interest to evolutionary studies and forensic science. By utilizing ultra-deep sequencing, it is now possible to uncover previously undiscovered patterns of intra-individual polymorphism. However, it remains challenging to determine its source. Current available software can detect polymorphic sites but are not capable of determining the link between them. We therefore developed a new method to not only detect intra-individual polymorphisms within mitochondrial and chloroplast genomes, but also to look for linkage among polymorphic sites by assembling the sequence around each detected polymorphic site. Our benchmark study shows that this method can detect heteroplasmy more accurately than any method previously available and is the first tool that is able to completely or partially reconstruct the origin sequences for each intra-individual polymorphism. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished