First study determined the effects of genetic deletion of nNOS on the levels of spontaneous apoptosis in brain of young-adult (2-3 months) and aged (12-18 months) mice, using nNOS-knockout mice with age-matched B6129SF2/J mice as wild-type control. The results indicate that aging resulted in 11-fold increase in levels of apoptotic-DNA-fragmentation in B6129SF2/J mouse brain. nNOS-knockout mice demonstrated dramatic (72-fold) increases in levels of apoptotic-DNA-fragmentation in young-adult, but not aged, brains. Aging resulted in decreased number of nNOS-positive cells, increased number of iNOS-positive cells and no change of eNOS-positive cells in control mice. The data suggest that nNOS may serve an anti-apoptotic/neuroprotective role in young-adult mouse brain. However, because of diminished nNOS and increased iNOS with aging, this neuroprotective effect may become less effective in aged mice. / Fourth study showed that new microchip-electrophoresis-technology can be successfully used to identify and quantify levels of apoptosic-DNA-fragments in brain slice cultures, similar to our previous studies with CE-LIF. Because of the much greater throughput of microchip-electrophoresis-system, compared to CE-LIF, this new technology should help accelerate the progress of apoptosis research. / In second study, apoptotic effects of genetic deletion of either eNOS or iNOS were studied using young-adult (1-4 months) and aged (12-24 months) eNOS- or iNOS-knockout mice with age-matched C57BL/6J wild-type control mice. The data show that both young-adult and aged iNOS-knockout mice had dramatically (8- to 36-fold) higher levels of apoptotic-DNA-fragmentation compared to control, especially noticeable in hippocampus and medulla oblongata. Both young-adult and aged eNOS-knockout mice also had dramatically (18- to 35-fold) higher levels of apoptotic-DNA-fragmentation compared to control, especially in cerebral cortex, hippocampus and medulla oblongata. The data suggest that both iNOS and eNOS provide neuroprotective effects, helping to limit the extent of spontaneous apoptosis in brain of young-adult and aged mice. / Nitric oxide (NO) has either pro-apoptotic or anti-apoptotic effects on neuronal cells, depending on concentration of NO produced by different source of NO synthases (NOSs) including neuronal-NO-synthase (nNOS/NOS-1), inducible-NO-synthase (iNOS/NOS-2) or endothelial-NO-synthase (eNOS/NOS-3) and possibly age of the individual. The present study determines if genetic deletion of nNOS, iNOS or eNOS alters levels of aging-induced apoptosis in vivo and hydrogen peroxide (H2O2)-induced-apoptosis in organotypic brain slice cultures using NOS-knockout mice. The quantitative ultrasensitive techniques using capillary-electrophoresis with laser-induced-fluorescent detector (CE-LIF) and Cell-Death---Detection-ELISA were used as novel ways to accurately measure the levels of apoptotic-DNA-fragmentation. Expressions of different forms of NOSs were determined by immunohistochemical-staining. / Third study determined H2O2-induced apoptosis in hippocampal and cerebellar slices from young-adult (8-10 weeks) and aged (12-24 months) C57BL/6J control mice, as well as iNOS- and eNOS-knockout mice (determined by Cell-Death-Detection-ELISA measuring levels of apoptotic-DNA-fragmentation). The data show spontaneous onset of apoptosis occurred in both hippocampal and cerebellar slices during culturing, beginning at 24 hours and progressively increasing for 48--72 hours. Staurosporine (positive-control) and H2 O2 both caused time-dependent increases in apoptosis in both hippocampal and cerebellar slices, compared to time-matched controls. Lastly, genetic deletion of iNOS greatly reduced levels of spontaneous apoptosis in young hippocampus and aged cerebellum, suggesting iNOS had contributed to induction of spontaneous apoptosis. / Chow Wing Han Vivian. / "Dec 2005." / Advisers: Siew Boon Chew Cheng; Ray Ronald Fiscus. / Source: Dissertation Abstracts International, Volume: 67-11, Section: B, page: 6218. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 144-153). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Yan, Changqing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 92-97). / Abstracts in English and Chinese. / ABSTRACT (ENGLISH) --- p.iii / ABSTRACT (CHINESE) --- p.iv / ACKNOWLEDGMENTS --- p.v / LIST OF ABBREVIATIONS --- p.vi / TABLE OF CONTENTS --- p.vii / LIST OF FIGURES --- p.ix / LIST OF TABLES --- p.x / Chapter CHAPTER ONE: --- BACKGROUND INFORMATION --- p.1 / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Deoxyribonucleic Acid --- p.2 / Chapter 1.3 --- DNA Studies --- p.9 / Chapter 1.4 --- Cisplatin Studies --- p.11 / Chapter 1.5 --- Scope of the Thesis --- p.13 / Chapter CHAPTER TWO: --- METHODOLOY AND COMPUTATION --- p.16 / Chapter 2.1 --- Introduction --- p.16 / Chapter 2.2 --- Molecular Dynamics Simulation --- p.16 / Chapter 2.3 --- Quantum Mechanics Calculation --- p.23 / Chapter 2.4 --- Verification of Methodology --- p.25 / Chapter 2.4.1 --- Backbone Torsion Angles --- p.25 / Chapter 2.4.2 --- N7-N7 Distance --- p.30 / Chapter 2.4.3 --- Location of HOMO --- p.33 / Chapter 2.5 --- Summary --- p.35 / Chapter CHAPTER THREE: --- UNDERSTANDING OF THE CISPLATIN-DNA CROSSLINKS --- p.36 / Chapter 3.1 --- Introduction --- p.36 / Chapter 3.2 --- MO Analysis --- p.37 / Chapter 3.3 --- Potential Binding Products with the Ligand --- p.37 / Chapter 3.3.1 --- "1,2-d(GpG) Intrastrand Crosslink" --- p.43 / Chapter 3.3.2 --- "l,2-d(ApG) Intrastrand Crosslink" --- p.43 / Chapter 3.3.3 --- "l,3-d(GpXpG) Intrastrand Crosslink" --- p.44 / Chapter 3.3.4 --- d(GpC)d(GpC) Interstrand Crosslink --- p.44 / Chapter 3.3.5 --- d(GpXpC)d(GpXpC) Interstrand Crosslink --- p.44 / Chapter 3.3.6 --- Summary --- p.45 / Chapter 3.4 --- Potential Binding Products Analysis --- p.47 / Chapter 3.4.1 --- Site Identification Convention --- p.47 / Chapter 3.4.2 --- Potential Binding Products Analysis --- p.48 / Chapter 3.4.3 --- Applications --- p.53 / Chapter 3.5 --- Cisplatin-DNA Crosslink Products Analysis --- p.56 / Chapter 3.5.1 --- "1,2-d(GpG) and l,2-d(ApG) Intrastrand Crosslinks" --- p.61 / Chapter 3.5.2 --- "l,3-d(GpXpG) Intrastrand and d(GpXpC)d(GpXpC) Interstrand Crosslinks" --- p.62 / Chapter 3.5.3 --- d(GpC)d(GpC) Interstrand Crosslinks --- p.63 / Chapter 3.5.4 --- Platination at Terminal Positions --- p.65 / Chapter 3.6 --- Summary --- p.65 / Chapter CAHPTER FOUR: --- CONCLUDING REMARKS --- p.67 / APPENDIX I: BACKBONE TORSION ANGLES AND SUGAR RING CONFORMATIONS OF THE OPTIMIZED GEOMETRIES --- p.69 / APPENDIX II: BACKBONE TORSION ANGLES OF THE EXPERIMENTAL SEQUENCES FROM NUCLEIC ACID DATABASE (NDB) --- p.77 / REFERENCES --- p.92

In this thesis human skeletal remains believed to be the victims of the Batavia Mutiny of 1629 were subjected to DNA analysis. So far the remains of 10 individuals (of which 9 were available for this study) have been exhumed from Beacon Island, in the Houtman’s Abrolhos, off the coast of Western Australia. The remains are now stored in the Western Australia Maritime Museum (WAMM) in Fremantle. In this research an attempt is made to type ancient DNA (aDNA) from the remains of the Batavia Mutiny, which are almost 400 years old. Previous anthropological studies have been performed on these remains in order to assign sex, age and stature. The aim of the present project is to study the familial relationships of the remains and to determine their sex based on molecular genetic analysis. In order to protect the invaluable museum specimens and minimise the risk of contamination from exogenous contemporary DNA, a tooth sample from each available individual (designated A15507, A16316, A15831, M3901, SK5, SK6, SK7, SK8 and SK9) was subjected to DNA extraction. Comparison and optimisation of DNA extraction methods from more recent teeth samples was performed in order to determine the most suitable method for the DNA extraction of the ancient teeth samples. Three types of genetic markers were analysed in an attempt to study the familial relationships and determine the sex of each individual. Multiplex primers (Hummel, 2003) which simultaneously amplify the HV1 and HV2 regions of mitochondrial DNA (mtDNA) were used in this research to analyse familial relationships. These primers were selected because of their ability to amplify small fragments (131bp, 168bp and 217bp) of DNA template, which suit the nature of aDNA samples. Primers published by Sullivan et al.(1993), which amplify a 106bp region on chromosome X and 112bp on chromosome Y of the amelogenin gene, were used to determine sex. In addition, short tandem repeat (STR) marker were also analysed to determine familial and sex using the AmpFlSTR®Profiler PlusTMPCR kit from Applied Biosystems. The PCR conditions of all primers were optimised before usage on the Batavia remains. As aDNA analysis is prone to contamination, stringent precautions were undertaken throughout this research. Despite this, contamination is suspected in some of the mtDNA sequences obtained (particularly from SK5, SK7, A15507 and A15831), which most probably came from the positive control used in the optimisation analysis. For these samples the sequences for the HV2 region were poor and polymorphisms relative to a reference were similar to each other and to the positive control profile. However, some conclusions have been made on other individuals (SK8, SK9, M3901, A16316) based on the HV1 and HV2 sequences obtained. Based on two or more different polymorphisms observed in the individuals it was concluded that it is likely there is no maternal relationship between individuals A16316 and SK8, SK9 and M3901 and between individuals SK8, M3901 and SK9. However these results require repetition for confirmation. The attempt to type the amelogenin gene on chromosomes X and Y was unsuccessful most likely due to the poor preservation of the remains. It is apparent from this research that although it was possible to extract aDNA (especially multicopy mtDNA) from teeth material that were almost 400 years old, the main hurdle in this aDNA analysis was contamination and DNA degradation.

When offenders commit crime there is the potential that they may leave behind trace amounts of their DNA, even when there has been no apparent body fluid spill. During the examination of crime scenes, scene investigators try to identify areas that may be sampled to locate these traces. Specialist techniques are then required within the laboratory to enable such small amounts to be analysed to obtain a profile. These techniques are referred to as Low Template DNA analysis (LTDNA), of which Low Copy Number DNA (LCN DNA) is one instance. In 2008, following the Omagh Bombing trial, and comments made by Judge Weir, the UK Forensic Regulator commissioned a review of the science of LTDNA analysis. The subsequent report made specific mention of the fact that there was no available information on the success rate of the use of such DNA techniques and that there seemed to be confusion over what constituted a success. The report went on to state that there was no information on where such trace amounts of DNA were likely to be found, or what factors could influence the likelihood of obtaining a trace DNA profile (Caddy, 2008). This research considered the outcomes of LCN DNA analysis from 3,552 samples to try to establish where trace amounts of DNA could be found, whether some areas sampled were more successful in generating profiles than others, and the likelihood of the profiles obtained being of use to a criminal investigation. Analysis of results identified areas that were more successful in generating profiles of use to an investigation and highlighted significant differences in results across a variety of items from which samples were taken. DNA samples taken from items associated with communication such as mobile phones were much more likely to produce a profile useful to a criminal investigation than those taken from fixed surfaces within premises. The results obtained showed that obtaining a DNA profile did not necessarily correlate with the profile being of use to a criminal investigation. This was due to the fact that a large number of these profiles were anticipated eliminations from legitimate sources. Items that produced high numbers of profiles but were anticipated eliminations, and therefore of no value to an investigation, came from items associated with skin samples and clothing. The research went further to identify key factors that affected the profiling rates. Factors that had a positive influence on the ability to obtain a profile included: any area that had been in close proximity to saliva (direct contact was not required); samples that had been recovered from the inside of premises or vehicles and therefore protected from the elements; those that were dry; items that were of a porous nature; and those that had a rough texture. No differences were found between the actual surface materials (plastic, glass, wood, metal), as all showed a propensity to generate profiles. Other factors that were considered but proved to have no effect on the profiling rates included seasonal differences and whether the area targeted for sampling was clearly defined. Items that had had high contact with a victim, were recovered from outside or had been wet, all proved to be less useful to an nvestigation. A further finding of the research was that swabs that had been recovered and stored frozen appeared to deteriorate in their ability to profile. This was particularly notable if they were submitted later than 5 months after recovery. Items stored in dry conditions did not deteriorate in this way. Overall the research can be used to provide investigators with the knowledge of what areas of crime scenes are most likely to yield trace DNA material, the key factors that can affect the likelihood of obtaining a profile, and those areas that are more likely to produce profiles useful to criminal investigations.

The aim of this study was to design a resolution typing system for the HLA-B gene. This technique involves a one-step PCR reaction utilizing genomic DNA and sequence-specific primers to determine the specificity of each allele and to produce a larger primer data base ideal for serological analysis. The application of this technique to serological analysis can improve serology detection which is currently hindered by antibody cross-reactivity and the unavailability of useful typing reagents.

The goal of this project was to reduce processing time for forensic DNA testing without incurring significant added costs and/or the need for new instrumentation, while still generating high quality profiles. This was accomplished by: 1) extraction normalization using the ChargeSwitch® Forensic DNA Purification Kit such that a small range of DNA concentrations was consistently obtained, eliminating the need for sample quantification and dilution; 2) developing fast PCR protocols for STR primer sets using shorter amplification methods, low volume reactions and non-fast thermal cyclers; and 3) developing a quicker 3130xl Genetic Analyzer detection method using an alternative polymer/array length combination. Extraction normalization was achieved through a reduction in bead quantity, thereby forcing an increase in bead binding efficiency. Four products (AmpliTaq Gold® Fast PCR Master Mix, KAPA2G™ Fast Multiplex PCR Kit, SpeedSTAR™ HS DNA Polymerase and Type-it Microsatellite PCR Kit) were evaluated for low volume (3μl) fast PCR on a 384-well Veriti® thermal cycler with the Identifiler primer set. KAPA2G™ was selected for 3μl fast PCR protocols using PowerPlex 16 HS and Identifiler Plus primer sets (42-51min), as well as 5μl and 6μl Identifiler fast reactions on a 9700 thermal cycler (51-60min). Alternative detection (POP-6™/22cm) achieved 24-28min run times, but with decreased resolution as compared to traditional POP-4®/36cm detection for alleles >200bp; however, 1bp resolution was still obtainable for alleles <300bp. These modifications resulted in robust databasing processes with up to a 37% reduction in processing time for buccal swabs and Buccal DNA Collectors™ using the three primer sets evaluated (3μl fast PCR reactions) and generated high quality STR profiles with ≥90% pass rates.

Past sightings of red-tailed (Cercopithecus ascanius) x blue monkey (Cercopithecus mitis) hybrids in Uganda indicates the potential for hybridization between C. Ascanius and C. mitis individuals. Apart from Gombe Stream National Park, there is no of evidence suggestive of C. ascanius x C. mitis monkey hybridization at investigated East African locations. Phylogenetic analysis was examined using Mitochondrial DNA (mtDNA) sequence data of twelve C. mitis stuhlmanni samples (from two populations) in Kibale National Park (KNP), Uganda to test for any evidence of hybridization. Strict mono- phylogeny among two new C. mitis haplotypes were detected. Genetic diversity measurements support neither interspecific or intraspecific hybridization among C. mitis individuals from populations within Kibale National Park. To intensify the implications of this study further examination should include an increase in sample size(s), mtDNA comparison of C. mitis subspecies from additional populations at East African locations, and assessment of nuclear and genomic DNA. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection

A diversidade da seqüência do DNA de oito populações de Octopus cf. vulgaris da costa brasileira e de uma população de Octopus vulgaris proveniente de Portugal foi investigada pelo uso do gene Citocromo oxidase subunidade I (COI) do DNA mitocondrial. Aproximadamente 600 pb do gene COImt foram amplificados por meio dos primers LCO1490 e HCO2198, purificados e seqüenciados. As seqüências foram alinhadas pelo método Clustal W. A árvore filogenética gerada pelo alinhamento das seqüências do COImt revelou dois conjuntos principais, formando clados monofiléticos sustentados por bootstraps superiores a 93%. Um clado contendo os indivíduos provenientes das regiões Sudeste e Sul, similares aos haplótipos de Portugal, que são classificados como Octopus vulgaris, e outro conjunto formado pelos indivíduos coletados em várias localidades das regiões Norte e Nordeste. O nível de diferenciação genética encontrado sugere a presença de duas espécies de Octopus. Quanto à estruturação populacional, os resultados encontrados pelo uso do DNA nuclear e do DNA mitocondrial indicam que as populações estão estruturadas geneticamente. / The diversity of the sequence of the DNA of eight vulgaris populations of Octopus cf. vulgaris of the Brazilian coast and a population of Octopus vulgaris proceeding from Portugal was investigated by the use of the mitochondrial Cytochrome c oxidase subunit I (COI) gene. Approximately 600 bp of the mitochondrial COI gene were amplified by means of primers LCO1490 and HCO2198, purified and sequenced. The sequences were lined up by the ClustalW method. The phylogenetic tree generated by the alignment of the sequences of the COI revealed two main sets, forming monophyletic supported by bootstraps 93%. A clade containing the individuals proceeding from the Southeastern and South regions similar to the haplotypes of Portugal, which are classified as Octopus vulgaris, and another set formed by the individuals collected in several places of the North and Northeast regions. The level of the found genetic differentiation suggests the presence of two species of Octopus. As far as the population structure is concerned, the results found by the use of the nuclear DNA and the mitochondrial DNA indicates that the populations are genetically structured.

1997年，Dennis等證明胚胎DNA在孕婦母體中存在的事實開啟了產前無創診斷的大門。起初的應用包括性別鑒定和恒河猴血型系統的識別。隨著二代測序的出現和發展，對外周血游離DNA更加成熟的分析和應用應運而生。例如當孕婦懷孕十二周時， 應用二代測序技術在母體外周血DNA中預測胎兒21號染色體是否是三倍體， 其準確性達到98%。本論文的第一部分介紹如何應用母體外周血DNA構建胎兒的全基因組遺傳圖譜。這項研究極具挑戰，原因是孕後12周，胎兒對外周血DNA貢獻很小，大多數在10%左右，另外外周血中的胎兒DNA大多數短於200 bp。目前的演算法和程式都不適合於從母體外周血DNA中構建胎兒的遺傳圖譜。在這項研究中，根據母親和父親的基因型，用生物資訊學手段先構建胎兒可能有的遺傳圖譜，然後將母體外周血DNA的測序資訊比對到這張可能的遺傳圖譜上。如果在母親純和遺傳背景下，決定父親的特異遺傳片段，只要定性檢測父親的特異遺傳片段是否在母體外周血中存在。如果在母親雜合遺傳背景下，決定母親的遺傳特性，就要進行定量分析。我開發了單倍型相對劑量分析方案，統計學上判斷母親外周血中的兩條單倍型相對劑量水準，顯著增加的單倍型即為最大可能地遺傳給胎兒的單倍型。單倍型相對劑量分析方案可以加強測序資訊的分析效率，降低測序數據波動，比單個位點分析更加穩定，強壯。 / 隨著靶標富集測序出現，測序價格急劇下降。第一部分運用母親父親的多態位點基因型的組合加上測序的資訊可以計算出胎兒DNA在母體外周血中的濃度。但是該方法的局限是要利用母親父親的多態位點的基因型，而不能直接從測序的資訊中推測胎兒DNA在母體外周血中的濃度。本論文的第二部分，我開發了基於二項分佈的混合模型直接預測胎兒DNA在母體外周血中的濃度。當混合模型的似然值達到最大的時候，胎兒DNA在母體外周血中的濃度得到最優估算。由於靶標富集測序可以提供高倍覆蓋的測序資訊，從而有機會直接根據概率模型識別出母親是純和而且胎兒是雜合的有特異信息量的位點。 / 除了母體外周血DNA水準分析推動產前無創診斷外，表觀遺傳學的分析也不容忽視。 在本論文的第三部分，我開發了Methyl-Pipe軟體，專門用於全基因組的甲基化的分析。甲基化測序數據分析比一般的基因組測序分析更加複雜。由於重亞硫酸鹽測序文庫的沒有甲基化的胞嘧啶轉化成尿嘧啶，最後以胸腺嘧啶的形式存在PCR產物中， 但是對於甲基化的胞嘧啶則保持不變。 因此，為了實現將重亞硫酸鹽處理過的測序序列比對到參考基因組。首先，分別將Watson和Crick鏈的參考基因組中胞嘧啶轉化成全部轉化為胸腺嘧啶，同時也將測序序列中的胞嘧啶轉化成胸腺嘧啶。然後將轉化後的測序序列比對到參考基因組上。最後根據比對到基因組上的測序序列中的胞嘧啶和胸腺嘧啶的含量推到全基因組的甲基化水準和甲基化特定模式。Methyl-Pipe可以用於識別甲基化水平顯著性差異的基因組區別，因此它可以用於識別潛在的胎兒特異的甲基化位點用於產前無創診斷。 / The presence of fetal DNA in the cell-free plasma of pregnant women was first described in 1997. The initial clinical applications of this phenomenon focused on the detection of paternally inherited traits such as sex and rhesus D blood group status. The development of massively parallel sequencing technologies has allowed more sophisticated analyses on circulating cell-free DNA in maternal plasma. For example, through the determination of the proportional representation of chromosome 21 sequences in maternal plasma, noninvasive prenatal diagnosis of fetal Down syndrome can be achieved with an accuracy of >98%. In the first part of my thesis, I have developed bioinformatics algorithms to perform genome-wide construction of the fetal genetic map from the massively parallel sequencing data of the maternal plasma DNA sample of a pregnant woman. The construction of the fetal genetic map through the maternal plasma sequencing data is very challenging because fetal DNA only constitutes approximately 10% of the maternal plasma DNA. Moreover, as the fetal DNA in maternal plasma exists as short fragments of less than 200 bp, existing bioinformatics techniques for genome construction are not applicable for this purpose. For the construction of the genome-wide fetal genetic map, I have used the genome of the father and the mother as scaffolds and calculated the fractional fetal DNA concentration. First, I looked at the paternal specific sequences in maternal plasma to determine which portions of the father’s genome had been passed on to the fetus. For the determination of the maternal inheritance, I have developed the Relative Haplotype Dosage (RHDO) approach. This method is based on the principle that the portion of maternal genome inherited by the fetus would be present in slightly higher concentration in the maternal plasma. The use of haplotype information can enhance the efficacy of using the sequencing data. Thus, the maternal inheritance can be determined with a much lower sequencing depth than just looking at individual loci in the genome. This algorithm makes it feasible to use genome-wide scanning to diagnose fetal genetic disorders prenatally in a noninvasive way. / As the emergence of targeted massively parallel sequencing, the sequencing cost per base is reducing dramatically. Even though the first part of the thesis has already developed a method to estimate fractional fetal DNA concentration using parental genotype informations, it still cannot be used to deduce the fractional fetal DNA concentration directly from sequencing data without prior knowledge of genotype information. In the second part of this thesis, I propose a statistical mixture model based method, FetalQuant, which utilizes the maximum likelihood to estimate the fractional fetal DNA concentration directly from targeted massively parallel sequencing of maternal plasma DNA. This method allows fetal DNA concentration estimation superior to the existing methods in term of obviating the need of genotype information without loss of accuracy. Furthermore, by using Bayes’ rule, this method can distinguish the informative SNPs where mother is homozygous and fetus is heterozygous, which is potential to detect dominant inherited disorder. / Besides the genetic analysis at the DNA level, epigenetic markers are also valuable for noninvasive diagnosis development. In the third part of this thesis, I have also developed a bioinformatics algorithm to efficiently analyze genomewide DNA methylation status based on the massively parallel sequencing of bisulfite-converted DNA. DNA methylation is one of the most important mechanisms for regulating gene expression. The study of DNA methylation for different genes is important for the understanding of the different physiological and pathological processes. Currently, the most popular method for analyzing DNA methylation status is through bisulfite sequencing. The principle of this method is based on the fact that unmethylated cytosine residues would be chemically converted to uracil on bisulfite treatment whereas methylated cytosine would remain unchanged. The converted uracil and unconverted cytosine can then be discriminated on sequencing. With the emergence of massively parallel sequencing platforms, it is possible to perform this bisulfite sequencing analysis on a genome-wide scale. However, the bioinformatics analysis of the genome-wide bisulfite sequencing data is much more complicated than analyzing the data from individual loci. Thus, I have developed Methyl-Pipe, a bioinformatics program for analyzing the DNA methylation status of genome-wide methylation status of DNA samples based on massively parallel sequencing. In the first step of this algorithm, an in-silico converted reference genome is produced by converting all the cytosine residues to thymine residues. Then, the sequenced reads of bisulfite-converted DNA sequences are aligned to this modified reference sequence. Finally, post-processing of the alignments removes non-unique and low-quality mappings and characterizes the methylation pattern in genome-wide manner. Making use of this new program, potential fetal-specific hypomethylated regions which can be used as blood biomarkers can be identified in a genome-wide manner. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Jiang, Peiyong. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 100-105). / Abstracts also in Chinese. / Chapter SECTION I : --- BACKGROUND --- p.1 / Chapter CHAPTER 1: --- Circulating nucleic acids and Next-generation sequencing --- p.2 / Chapter 1.1 --- Circulating nucleic acids --- p.2 / Chapter 1.2 --- Next-generation sequencing --- p.3 / Chapter 1.3 --- Bioinformatics analyses --- p.9 / Chapter 1.4 --- Applications of the NGS --- p.11 / Chapter 1.5 --- Aims of this thesis --- p.12 / Chapter SECTION II : --- Mathematically decoding fetal genome in maternal plasma --- p.14 / Chapter CHAPTER 2: --- Characterizing the maternal and fetal genome in plasma at single base resolution --- p.15 / Chapter 2.1 --- Introduction --- p.15 / Chapter 2.2 --- SNP categories and principle --- p.17 / Chapter 2.3 --- Clinical cases and SNP genotyping --- p.20 / Chapter 2.4 --- Sequencing depth and fractional fetal DNA concentration determination --- p.24 / Chapter 2.5 --- Filtering of genotyping errors for maternal genotypes --- p.26 / Chapter 2.6 --- Constructing fetal genetic map in maternal plasma --- p.27 / Chapter 2.7 --- Sequencing error estimation --- p.36 / Chapter 2.8 --- Paternal-inherited alleles --- p.38 / Chapter 2.9 --- Maternally-derived alleles by RHDO analysis --- p.39 / Chapter 2.1 --- Recombination breakpoint simulation and detection --- p.49 / Chapter 2.11 --- Prenatal diagnosis of β- thalassaemia --- p.51 / Chapter 2.12 --- Discussion --- p.53 / Chapter SECTION III : --- Statistical model for fractional fetal DNA concentration estimation --- p.56 / Chapter CHAPTER 3: --- FetalQuant: deducing the fractional fetal DNA concentration from massively parallel sequencing of maternal plasma DNA --- p.57 / Chapter 3.1 --- Introduction --- p.57 / Chapter 3.2 --- Methods --- p.60 / Chapter 3.2.1 --- Maternal-fetal genotype combinations --- p.60 / Chapter 3.2.2 --- Binomial mixture model and likelihood --- p.64 / Chapter 3.2.3 --- Fractional fetal DNA concentration fitting --- p.66 / Chapter 3.3 --- Results --- p.71 / Chapter 3.3.1 --- Datasets --- p.71 / Chapter 3.3.2 --- Evaluation of FetalQuant algorithm --- p.75 / Chapter 3.3.3 --- Simulation --- p.78 / Chapter 3.3.4 --- Sequencing depth and the number of SNPs required by FetalQuant --- p.81 / Chapter 3.5 --- Discussion --- p.85 / Chapter SECTION IV : --- NGS-based data analysis pipeline development --- p.88 / Chapter CHAPTER 4: --- Methyl-Pipe: Methyl-Seq bioinformatics analysis pipeline --- p.89 / Chapter 4.1 --- Introduction --- p.89 / Chapter 4.2 --- Methods --- p.89 / Chapter 4.2.1 --- Overview of Methyl-Pipe --- p.90 / Chapter 4.3 --- Results and discussion --- p.96 / Chapter SECTION V : --- CONCLUDING REMARKS --- p.97 / Chapter CHAPTER 5: --- Conclusion and future perspectives --- p.98 / Chapter 5.1 --- Conclusion --- p.98 / Chapter 5.2 --- Future perspectives --- p.99 / Reference --- p.100

Thesis (Ph.D. (Zoology)) -- University of Limpopo, 2012