Melanoma is a heterogeneous neoplasm at the histomorphologic, immunophenotypic, and molecular levels. Melanoma with extreme histomorphologic heterogeneity can pose a diagnostic challenge in which the diagnosis may predominantly rely on its immunophenotypic profile. However, tumor survival and response to therapy are linked to tumor genetic heterogeneity rather than tumor morphology. Therefore, understating the molecular characteristics of such melanomas become indispensable. In this study, DNA was extracted from 11 morphologically distinct regions in eight formalin-fixed, paraffin-embedded melanomas. In each region, mutations in 50 cancer-related genes were tested using next-generation sequencing (NGS). A tumor was considered genetically heterogeneous if at least one non-overlapping mutation was identified either between the histologically distinct regions of the same tumor (intratumor heterogeneity) or among the histologically distinct regions of the paired primary and metastatic tumors within the same patient (intertumor heterogeneity). Our results revealed that genetic heterogeneity existed in all tumors as non-overlapping mutations were detected in every tested tumor (n = 5, 100%; intratumor: n = 2, 40%; intertumor: n = 3, 60%). Conversely, overlapping mutations were also detected in all the tested regions (n = 11, 100%). Melanomas exhibiting histomorphologic heterogeneity are often associated with genetic heterogeneity, which might contribute to tumor survival and poor response to therapy.

Introduction: The surprising presence of P. vivax in West Africa and their ability to infect a Duffy negative population is one more threat to public health. In order to contribute to malaria elimination efforts, there is a need to investigate the origin and characteristics of P. vivax population isolates in Northern Mali. Next Generation Sequence Analysis (NGSA) can help us understand parasite genetic characteristics although low parasite density is a challenge for whole genome sequencing (WGS). In the present work, we investigated if selective whole genome amplification (sWGA) can enrich P. vivax DNA extracted from Rapid Diagnostic Tests (RDTs) for Whole Genome Sequencing. We also investigated the origin and the susceptibility to antimalarial drugs of the strains isolated in Northern Mali. Methods: Parasite DNA was extracted from 267 RDTs using the QIAamp DNA mini kit, then nested PCR and 7 samples were positive for P. vivax. After sWGA, the whole genomes were sequenced using the Illumina platform. Next Generation Sequences Analysis was done followed by population differentiation analyses. Twenty-two additional P. vivax whole genomes from other parts of the World were downloaded from the European Nucleotide Archive for further Neighbour Joining analysis. Results: The sequences extracted from RDTs showed high contamination with human DNA (80%). From the parasite DNA, in total 69529 SNPs were found in the seven P. vivax strains of Northern Mali. The most significant p-values per SNP were carried by the chromosomes 2, 3, 4, 5, 12, 13 and 14. With regard to variant effects, the Transition/Transversion ratio was 1.1. The density of variants with a high effect was 1.62%. There was no mutation associated with antimalarial drugs resistance on pvcrt-o or pvmdr-1 genes. Pairwise differentiation suggests a high degree of relatedness between P. vivax strains isolated in Northern Mali. The NeighboursJoining analysis shows clearly that strains from Mali cluster together and are genetically distinct from those from Mauritania, which shares a border with Mali. The strains isolated in Northern Mali are genetically closer to those from Madagascar, India and Latina America. Conclusion: We did not identify mutations associated to the resistance to antimalarial drugs in pvcrt-o and pvmdr-1 genes. This study confirms that P. vivax strains genetically distinct from those of Mauritania are circulating in Mali. Finally, we conclude that sWGA is a feasible approach for P. vivax DNA enrichment for WGS despite the high proportion of human contamination.

Indiana University-Purdue University Indianapolis (IUPUI) / Several experimental methods has been developed for the study of the central dogma since late 20th century. Protein mass spectrometry and next generation sequencing (including DNA-Seq and RNA-Seq) forms a triangle of experimental methods, corresponding to the three vertices of the central dogma, i.e., DNA, RNA and protein. Numerous RNA sequencing and protein mass spectrometry experiments has been carried out in attempt to understand how the expression change of known genes affect biological functions in various of organisms, however, it has been once overlooked that the result data of these experiments are in fact holograms which also reveals other delicate biological mechanisms, such as RNA splicing and the expression of long non-coding RNAs. In this dissertation, we carried out five studies based on high-throughput sequencing data, in an attempt to understand how RNA splicing and differential expression of long non-coding RNAs is associated biological functions. In the first two studies, we identified and characterized 197 stimulant induced and 477 developmentally regulated alternative splicing events from RNA sequencing data. In the third study, we introduced a method for identifying novel alternative splicing events that were never documented. In the fourth study, we introduced a method for identifying known and novel RNA splicing junctions from protein mass spectrometry data. In the fifth study, we introduced a method for identifying long non-coding RNAs from poly-A selected RNA sequencing data. Taking advantage of these methods, we turned RNA sequencing and protein mass spectrometry data into an information gold mine of splicing and long non-coding RNA activities. / 2019-05-06

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第18122号 / 理博第4000号 / 新制||理||1577(附属図書館) / 30980 / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 村山 美穂, 教授 幸島 司郎, 教授 伊谷 原一 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

Advances in next-generation sequencing (NGS) technologies have resulted in significant reduction of cost per sequenced base pair and increase in sequence data volume. On the other hand, most currently used NGS technologies produce relatively short sequence reads (50 - 150 bp) compared to Sanger sequencing (~700 bp). This represents an additional challenge in data analysis, because shorter reads are more difficult to assemble. At this point, production of sequencing data outpaces our capacity to analyze them. Newer NGS technologies capable of producing longer reads are emerging, which should simplify and speed up genome assembly. However, this will only increase the number of sequenced genomes without structural and functional annotation. In addition to multiple scientific initiatives to sequence thousands of genomes, personalized medicine centered on sequencing and analysis of individual human genomes will become more available. This poses a challenge for computer science and emphasizes the importance of developing new computational algorithms, methodology, tools, and pipelines. This dissertation focuses on development of these software tools, methodologies, and resources to help address the need for processing of volumes of data generated by new sequencing technologies. The research concentrated on genome structure analysis, individual variation, and comparative biology. This dissertation presents: (1) the Short Read Classification Pipeline (SRCP) for preliminary genome characterization of unsequenced genomes; (2) a novel methodology for phylogenetic analysis of closely related organisms or strains of the same organism without a sequenced genome; (3) a centralized online resource for standardized gene nomenclature. Utilizing the SRCP and the methodology for initial phylogenetic analysis developed in this dissertation enables positioning the organism in the evolutionary context. This should facilitate identification of orthologs between the species and paralogs within the species even in the initial stage of the analysis when only exome is sequenced and, thus, enable functional annotation by transferring gene nomenclature from well-annotated 1:1 orthologs, as required by the online standardized gene nomenclature resource developed in this dissertation. Thus, the tools, methodology, and resources presented here are tied together in following the initial analysis workflow for structural and functional annotation.

Emerging sequencing approaches have revolutionized the way we can collect DNA sequence data for applications in bioforensics and biosurveillance. In this research, we present an approach to construct a database of known biological agents and use this database to develop a statistical framework to analyze raw reads from next-generation sequence data for species identification and strain attribution. Our method capitalizes on a Bayesian statistical framework that accommodates information on sequence quality, mapping quality and provides posterior probabilities of matches to a known database of target genomes. Importantly, our approach also incorporates the possibility that multiple species can be present in the sample or that the target strain is not even contained within the reference database. Furthermore, our approach can accurately discriminate between very closely related strains of the same species with very little coverage of the genome and without the need for genome assembly - a time consuming and labor intensive step. We demonstrate our approach using genomic data from a variety of known bacterial agents of bioterrorism and agents impacting human health.

Yes / Introduction: Mobile phones are used the world over, including in healthcare settings. This study aimed to investigate the viable microbial colonisation of mobile phones used by healthcare personnel. Methods: Swabs collected on the same day from 30 mobile phones belonging to healthcare workers from three separate paediatric wards of an Australian hospital were cultured on five types of agar plate, then colonies from each phone were pooled, extracted and sequenced by shotgun metagenomics. Questionnaires completed by staff whose phones were sampled assisted in the analysis and interpretation of results. Results and discussion: All phones sampled cultured viable bacteria. Overall, 399 bacterial operational taxonomic units were identified from 30 phones, with 1432 cumulative hits. Among these were 58 recognised human pathogenic and commensal bacteria (37 Gram-negative, 21 Gram-positive). The total number of virulence factor genes detected was 347, with 1258 cumulative hits. Antibiotic resistance genes (ARGs) were detected on all sampled phones and overall, 133 ARGs were detected with 520 cumulative hits. The most important classes of ARGs detected encoded resistance to beta-lactam, aminoglycoside and macrolide antibiotics and efflux pump mediated resistance mechanisms. Conclusion: Mobile phones carry viable bacterial pathogens and may act as fomites by contaminating the hands of their users and indirectly providing a transmission pathway for hospital-acquired infections and dissemination of antibiotic resistance. Further research is needed, but meanwhile adding touching mobile phones to the five moments of hand hygiene is a simple infection control strategy worth considering in hospital and community settings. Additionally, the implementation of practical and effective guidelines to decontaminate mobile phone devices would likely be beneficial to the hospital population and community at large.

Introduction: Sepsis is a major source of morbidity and mortality in neonates; however, identification of the causative pathogens can be challenging. Next generation sequencing (NGS) is a high-throughput, parallel sequencing technique for DNA. Pathogen-targeted enrichment followed by NGS has the potential to be more sensitive and faster than current gold-standard blood culture. In this pilot study, we will test the feasibility and pathogen detection patterns of pathogen-targeted NGS in neonates with suspected sepsis. Additionally, the distribution and diagnostic accuracy of cell-free DNA and protein C levels at two time points will be explored. Methods: We will conduct a prospective, pilot observational study. Neonates over 1 kg with suspected sepsis from a single tertiary care children’s hospital will be recruited for the study. Recruitment will be censored at 200 events or 6 months duration. Two blood study samples will be taken: the first simultaneous to the blood culture (time = 0 hr, for NGS and biomarkers) via an exception to consent (deferred consent) and another 24 hours later after prospective consent (biomarkers only). Neonates will be adjudicated into those with clinical sepsis, culture-proven sepsis and without sepsis based on clinical criteria. Feasibility parameters (e.g. recruitment) and NGS process time will be reported. Analysis: NGS results will be described in aggregate, compared to the simultaneous blood culture (sensitivity and specificity) and reviewed via expert panel for plausibility. Pilot data for biomarker distribution and diagnostic accuracy (sensitivity and specificity) for distinguishing between septic and non-septic neonates will be reported. Study amendment and interim results: After obtaining ethics approval, study enrolment started October 15, 2020. Interim feasibility results showed successful deferred consent, but low enrolment. A study amendment was used to increase enrolment, create pre-packaged blood kits and implement a substitute decision maker Notification form. / Thesis / Master of Health Sciences (MSc)

Chaperone proteins are essential components in the maintenance and turnover of the proteome. Many chaperones play integral functions in the folding and unfolding of cellular substrates under many conditions, including heat stress. Most chaperones can be characterized into two categories; the typical ATP dependent chaperones and the ATP independent chaperones. One ATP independent chaperone class it the Small Heat Shock Proteins (sHSPs), which as molecular life vests and are thought to protect misfolding proteins from irreversible aggregation. One such organism, the cyanobacterium Synechocystis sp. PCC 6803, is an excellent model for the study and understanding of these proteins and their functions in vivo. The genome of Synechocystis encodes only one sHSP, Hsp16.6, and it has be shown to be essential for acquired thermotolerance. Two mutant derivatives of Hsp16.6 with single amino acid substitutions in the N-terminal arm (L9P and E25K) have loss-of-function phenotypes similar to knock out strains, but each has very different biochemical properties. The mutant L9P has an inability to interact with putative substrates during heat stress in vivo, while the mutant E25K appears unable to release substrates. Using a directed evolution approach, suppressors have been isolated that recover the lost thermotolerance of their respective parent strains, either L9P (16 suppressors) or E25K (10 suppressors). Illumina sequencing and comparative genomics have been used to identify alterations in the genomes of the suppressor strains in order to define genetic circuits involved in thermotolerance.

The epigenome carries dynamic information that controls gene expression and maintains cell identity during both disease and normal development. The inherent plasticity of the epigenome paves new avenues for developing diagnostic and therapeutic tools for human diseases. Microfluidic technology has improved the sensitivity and resolution of epigenomic analysis due to its outstanding ability to manipulate nanoliter-scale liquid volumes. In this thesis, I report three projects focusing on low-input, cell-type-specific and spatially resolved histone modification profiling on microfluidic platforms. First, I applied Microfluidic Oscillatory Washing-based Chromatin Immunoprecipitation followed by sequencing (MOWChIP-seq) to study the effect of culture dimensionality, hypoxia stress and bacterium infection on histone modification landscapes of brain tumor cells. I identified differentially marked regions between different culture conditions. Second, I adapted indexed ChIPmentation and introduced mu-CM, a low-input microfluidic device capable of performing 8 assays in parallel on different histone marks using as few as 20 cells in less than 7 hours. Last, I investigated the spatially resolved epigenome and transcriptome of neuronal and glial cells from coronal sections of adult mouse neocortex. I applied unsupervised clustering to identify distinct spatial patterns in neocortex epigenome and transcriptome that were associated with central nervous system development. I demonstrated that our method is well suited for scarce samples, such as biopsy samples from patients in the context of precision medicine. / Doctor of Philosophy / Epigenetic is the study of alternations in organisms not caused by alternation of the genetic codes. Epigenetic information plays pivotal role during growth, aging and disease. Epigenetic information is dynamic and modifiable, and thus serves as an ideal target for various diagnostic and therapeutic strategies of human diseases. Microfluidics is a technology that manipulates liquids with extremely small volumes in miniaturized devices. Microfluidics has improved the sensitivity and resolution of epigenetic analysis. In this thesis, I report three projects focusing on low-input, cell-type-specific and spatially resolved histone modification profiling on microfluidic platforms. Histone modification is one type of epigenetic information and regulates gene expression. First, we studied the influence of culture condition and bacterium infection on histone modification profile of brain tumor cells. Second, we introduced mu-CM, combining a low-input microfluidic device with indexed ChIPmentation and is capable of performing 8 assays in parallel using as few as 20 cells. Last, we investigated spatial variations in the epigenome and transcriptome across adult mouse neocortex, the outer layer of brain involving in higher-order function, such as cognition. I identified distinct spatial patterns responsible for central nervous system development using machine learning algorithm. Our method is well suited for studying scarce samples, such as cells populations isolated from patients in the context of precision medicine.