In this thesis the processing mechanisms of short non-coding RNAs (ncRNAs) is investigated by using data generated by the current method of high-throughput sequencing (HTS). The recently adapted short RNA-seq protocol allows the sequencing of RNA fragments of microRNA-like length (∼18-28nt). Thus, after mapping the data back to a reference genome, it is possible to not only measure, but also visualize the expression of all ncRNAs that are processed to fragments of this specific length. Short RNA-seq data was used to show that a highly abundant class of small RNAs, called microRNA-offset-RNAs (moRNAs), which was formerly detected in a basal chordate, is also produced from human microRNA precursors. To simplify the search, the blockbuster tool that automatically recognizes blocks of reads to detect specific expression patterns was developed. By using blockbuster, blocks from moRNAs were detected directly next to the miR or miR* blocks and could thus easily be registered in an automated way. When further investigating the short RNA-seq data it was realized that not only microRNAs give rise to short ∼22nt long RNA pieces, but also almost all other classes of ncRNAs, like tRNAs, snoRNAs, snRNAs, rRNAs, Y-RNAs, or vault RNAs. The formed read patterns that arise after mapping these RNAs back to a reference genome seem to reflect the processing of each class and are thus specific for the RNA transcripts of which they are derived from. The potential of this patterns in classification and identification of non-coding RNAs was explored. Using a random forest classifier which was trained on a set of characteristic features of the individual ncRNA classes, it was possible to distinguish three types of ncRNAs, namely microRNAs, tRNAs, and snoRNAs. To make the classification available to the research community, the free web service ‘DARIO’ that allows to study short read data from small RNA-seq experiments was developed. The classification has shown that read patterns are specific for different classes of ncRNAs. To make use of this feature, the tool deepBlockAlign was developed. deepBlockAlign introduces a two-step approach to align read patterns with the aim of quickly identifying RNAs that share similar processing footprints. In order to find possible exceptions to the well-known microRNA maturation by Dicer and to identify additional substrates for Dicer processing the small RNA sequencing data of a Dicer knockdown experiment in MCF-7 cells was re-evaluated. There were several Dicer-independent microRNAs, among them the important tumor supressor mir-663a. It is known that many aspects of the RNA maturation leave traces in RNA sequencing data in the form of mismatches from the reference genome. It is possible to recover many well- known modified sites in tRNAs, providing evidence that modified nucleotides are a pervasive phenomenon in these data sets.

Were he alive today, would Louis Pasteur still champion culture methods he pioneered over 150 years ago for identifying bacterial pathogens? Or, might he suggest that new molecular techniques may prove a better way forward for quickly detecting the true microbial diversity of wounds? As modern clinicians faced with treating complex patients with diabetic foot infections (DFI), should we still request venerated and familiar culture and sensitivity methods, or is it time to ask for newer molecular tests, such as 16S rRNA gene sequencing? Or, are molecular techniques as yet too experimental, non-specific and expensive for current clinical use? While molecular techniques help us to identify more microorganisms from a DFI, can they tell us ‘who done it?', that is, which are the causative pathogens and which are merely colonizers? Furthermore, can molecular techniques provide clinically relevant, rapid information on the virulence of wound isolates and their antibiotic sensitivities? We herein review current knowledge on the microbiology of DFI, from standard culture methods to the current era of rapid and comprehensive ‘crime scene investigation' (CSI) techniques.

Doctor of Philosophy / Department of Plant Pathology / Ari Jumpponen / Megan Kennelly / Soil microbial communities play fundamental and complex roles in the productivity of agriculture. However, we still have a limited understanding of the response of microbial communities to different farming systems, such as organic and conventional fertility management regimens. We applied high-throughput sequencing to develop a better understanding of how soil microbial communities (bacteria and fungi) in vegetable production respond to organic or conventional soil fertility management. Specifically, my three studies examined the following questions: 1. How do soil microbial communities from cDNA and DNA samples compare in organic and conventional fertility treatments? 2. How do soil microbial communities in a tomato cropping season respond to long-term organic vs. conventional soil fertility treatments? 3. How do soil bacterial and fungal communities respond to high tunnels, plastic mulch and organic amendments across a tomato cropping season? The first two questions were addressed at the Kansas State University Horticulture and Extension Center in Olathe, KS, using organic and conventional field plots with three levels of fertilizer. We sampled the plots during the development of a tomato crop. The third question was addressed at a commercial farm in Lawrence, KS, during its transition to organic vegetable production, during a tomato crop. The Lawrence experiment included as treatments field plots versus high tunnels, and three organic nutrient amendments. We used 454-pyrosequencing of bacterial and fungal ribosomal markers to compare total resident (DNA) and active microbial communities (cDNA, which is DNA synthesized from a single stranded RNA template) for our first question. We used Illumina MiSeq metabarcoding of bacterial and fungal ribosomal markers for our second and third questions. In all three studies we evaluated bacterial and fungal community responses using Simpson´s diversity index, Simpson´s evenness and richness for each experiment. For the first question, when we compared DNA and cDNA, bacterial diversity was higher in cDNA samples from organic compared to conventional management. In addition, fungal diversity from cDNA samples was higher than from DNA samples. In contrast, in the second question, bacterial and fungal diversity indices did not differ in the tomato crop under organic and conventional management systems. For our third question, high tunnels did not affect bacterial or fungal diversity. Use of plastic mulch for a tomato crop in open field plots did not affect bacterial richness, but decreased fungal richness compared to open field plots without plastic mulch. High-throughput sequencing provides a new perspective on the structure and dynamics of these communities. Information from this approach will ultimately improve our ability to manage soil for sustainable productivity by promoting beneficial microorganisms and suppressing pathogenic ones.

Bacteria have been the causative agents of major infectious disease pandemics throughout human history. Over the past 4 decades, a combination of changing medical practices, industrialization, and globalisation have led to a number of emergences and re-emergences of bacterial pathogens. The design of rational control programs and bespoke therapies will require an enhanced understanding of the dynamics underpinning the emergence and transmission of pathogenic clones. The recent development of new technologies for sequencing bacterial genomes rapidly and economically has led to a greatly enhanced understanding of the diversity of bacterial populations. This thesis describes the application of whole genome sequencing of 2 bacterial pathogens, Staphylococcus aureus and Legionella pneumophila, in order to understand the dynamics of bacterial infections on different temporal and spatial scales. The first study involves the examination of S. aureus evolution during a chronic infection of a single patient over a period of 26 months revealing differences in antibiotic resistance profiles and virulence factor expression over time. The genetic variation identified correlated with differences in growth rate, haemolytic activity, and antibiotic sensitivity, implying a profound effect on the ecology of S. aureus. Importantly, polymorphisms were identified in global regulators of virulence, with a high frequency of polymorphisms within the SigB locus identified, suggesting this region may be under selection in this patient. The identification of genes under diversifying selection during long-term infection may inform the design of novel therapeutics for the control of refractory chronic infections. Secondly, the emergence and transmission of 3 pandemic lineages derived from S. aureus clonal complex 30 (CC30) were investigated. Independent origins for each pandemic lineage were identified, with striking molecular correlates of hospital- or community-associated pandemics represented by mobile genetic elements, such as bacteriophage and Staphylococcal pathogenicity islands, and non-synonymous mutations affecting antibiotic resistance and virulence. Hospitals in large cities were identified as hubs for the transmission of MRSA to regional health care centres. In addition, comparison of whole genome sequences revealed that at least 3 independent acquisitions of TSST-1 have occurred in CC30, but a single distinct clade of diverse community-associated CC30 strains was responsible for the TSS epidemic of the late 1970s, and for subsequent cases of TSS in the UK and USA. Finally, whole genome sequencing was used as a tool for investigating a recent outbreak of legionellosis in Edinburgh. An unexpectedly high level of genomic diversity was identified among the outbreak strains, with respect to core genome polymorphisms, and accessory genome content. The data indicate that affected individuals may be infected with heterogeneous strains. The findings highlight the complexities in identifying environmental sources and suggest possible differences in pathogenic potential among isolates from a single outbreak. Taken together, the findings demonstrate applications of bacterial genome sequencing leading to enhanced understanding of bacterial pathogen evolution, emergence, and transmission, which may ultimately inform appropriate infection control measures.

High-throughput sequencing is a powerful tool to study diverse aspects of biology and applies to genome, transcriptome, and small RNA profiling. Ever increasing sequencing throughput and more specialized sequencing assays demand more sophisticated bioinformatics approaches. In this thesis, I present 4 studies for which I developed computational methods to handle high-throughput sequencing data to gain insights into biology. The first study describes the genome of High Five (Hi5) cells, originally derived from Trichoplusia ni eggs. The chromosome-level assembly (scaffold N50 = 14.2 Mb) contains 14,037 predicted protein-coding genes. Examination and curation of multiple gene families, pathways, and small RNA-producing loci reveal species- and order-specific features. The availability of the genome sequence, together with genome editing and single-cell cloning protocols, enables Hi5 cells as a new tool for studying small RNAs. The second study focuses on just one type of piRNAs that are produced at the pachytene stage of mammalian spermatogenesis. Despite their abundance, pachytene piRNAs are poorly understood. I find that pachytene piRNAs cleave transcripts of protein-coding genes and further target transcripts from other pachytene piRNA loci. Subsequently, systematic investigation of piRNA targeting by integrating different types of sequencing data uncovers the piRNA targeting rule. The third study describes computational procedures to map splicing branchpoints using high-throughput sequencing data. Screening >1.2 trillion RNA-seq reads determines >140,000 BPs for both human and mouse. Such branchpoints are compiled into BPDB (BranchPoint DataBase) to provide a comprehensive branchpoint catalog. The final study combines novel experimental and computational procedures to handle PCR duplicates that are prevalent in high-throughput sequencing data. Incorporation of unique molecular identifiers (UMIs) to tag each read enables unambiguous identification of PCR duplicates. Both simulated and experimental datasets demonstrate that UMI incorporation increases the reproducibility of RNA-seq and small RNA-seq. Surveying 7 common variables in high-throughput sequencing reveals that the amount of starting material and sequencing depth, but not the number of PCR cycles, determine the PCR duplicate frequency. Finally, I show that removing PCR duplicates without UMIs leads to substantial bias into data analysis. / 2020-12-11T00:00:00Z

Messenger RNAs (mRNAs) have to undergo a series of post-transcriptional processing steps before translation. One of the post-transcriptional steps - 3' end processing, which consists of cleavage and polyadenylation, is critical for delimiting the 3' end of mRNA and determining regulatory elements for downstream post-transcriptional/translational regulation. Like another well-characterized mRNA processing step - splicing, 3' end processing is very flexible due to the diversity of trans-acting factors and cis-acting elements in the 3' end of mRNA. In recent years, the differential usage of alternative polyA sites (APA) of the same gene, which leads to mRNA isoforms of different 3' UTR, has been increasingly revealed by both experimental and computational studies. More significantly, the global changes of 3' UTR length have been observed in multiple clinical settings, particularly in the cancer cells. However, the depiction of APA phenomenon does not synchronize the efforts to study the mechanism underlying APA biogenesis. In this thesis, we first describe general principle and pipeline to identify APA in different biological or clinical conditions using various high throughput sequencing techniques. After that, we present the work about the global impacts of two RNA binding proteins (ESRP/aCP) and one core 3' end processing factor (CstF64 and its paralog CstF64τ) on the regulation of APA. The APA identification analyses and motif analyses suggest a wide range of APA associated with the expression change of those proteins in different cell lines. In addition, for each protein, we have collect substantial evidence about the mechanism underlying the APA induction. Our findings could provide significant insights into the APA regulation mechanisms. In addition, we also conducted a research on the induction of APA in JEG-3 cells as a response to the change of oxygen supply (Hypoxia and Normoxia). Using a robustness protocol for specifically sequencing 3' end of mRNA, we identified more than 500 APA events and revealed a global shortening pattern of 3' UTR length as a result of hypoxia. The work on APA in this thesis largely increases the understanding of APA regulation by various proteins and provided new evidence for the APA in clinical condition.

The advent of high-throughput DNA and RNA sequencing has made possible the assay of millions of nucleic acid molecules in parallel. This allows functional genomic elements to be identified from background in single-tube experiments. This dissertation discusses the development of two such functional screens as well as work implementing a third that was previously developed in my thesis laboratory. Restriction-Associated DNA sequencing (RAD-Seq) is a complexity reduction sequencing method that allows the same subset of genomic sequence to be read across multiple samples. Differences in sample collection and data analysis allow manifold applications of RAD-Seq. Here we use RAD-Seq to identify mutant genes responsible for altered phenotypes in Caenorhabditis elegans and to identify hyper-invasive alleles in trout population admixtures. Apart from acquiring genomic sequence data, massively-parallel sequencing can be used for counting applications that quantify activity across a large number of test molecules. This dissertation describes the development of a technique for simultaneously quantifying the activity of a restriction enzyme across all possible DNA substrates by linking digest of a sequenced genome to Illumina-sequencing in an unbiased fashion. Finally, a powerful approach to analyze transcriptional activation is described. This method quantifies output from millions of potential DNA transcriptional enhancers via RNA amplicon sequencing of covalently-linked randomer tags and is used in conjunction with RNA-Seq to provide a mechanistic view of hypoxic gene regulation in Drosophila. This dissertation includes previously published, co-authored material

Master of Science / Department of Biology / Stephen K. Chapes / Immunoglobulins are receptors expressed on the outside of a B cell that can specifically bind pathogens and toxic substances within a host. These receptors are heterodimers of two chains: heavy and light, which are encoded at separate loci. Enzymatic splicing of gene segments at heavy and light chain loci within the genomic DNA in every B cell results in a highly diversified and specific repertoire of immunoglobulins in a single host. Spaceflight is known to affect reduce splenic B cell populations and B cell progenitors within the bone marrow, potentially restricting the diversity of the immunoglobulin repertoire (Ig-Rep). The objective of this thesis project was to characterize the impact of spaceflight on the kappa light-chain Ig-Rep of the C57BL/6 mouse. High-throughput sequencing (HTS) technologies have enabled the rapid characterization of Ig-Reps, however, standard Ig-Rep workflows often rely the amplification of immunoglobulin sequences to ensure the capture immunoglobulin sequences from rare B cell clones. Additionally, the Ig-Rep is often assessed in sorted B cell populations. Opportunities for spaceflight experiments are limited and costly, and the exclusive amplification of immunoglobulin sequences prior to HTS results in a dataset that cannot be mined for additional information. Furthermore, due to the difficulties of tissue collection in spaceflight, HTS of sorted B cell populations is not feasible. We optimized a protocol in which the Ig-Rep was assessed from unamplified whole tissue immunoglobulin transcripts. The Ig-Rep was characterized by gene segment usage, gene segment combinations and the region in which gene segments are joined. HTS datasets of ground control animals and animals flown aboard the International Space Station were compared to explore the impact of spaceflight on the unimmunized murine Ig-Rep.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19769号 / 農博第2165号 / 新制||農||1040(附属図書館) / 学位論文||H28||N4985(農学部図書室) / 32805 / 京都大学大学院農学研究科森林科学専攻 / (主査)教授 井鷺 裕司, 教授 北山 兼弘, 教授 神﨑 護 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Forensic scientists and ancient DNA researchers face similar challenges with respect to genetic information acquisition and analysis. However, these communities differ in one critical aspect: while forensic science is regulated by the strict guidelines of the judicial community, ancient DNA is a research-based academic field free to explore emerging technologies as they arise. This thesis investigates the application of two methodologies, developed in ancient DNA research, to challenging extracts, in hopes of modernizing forensic models while maintaining compatibility with current standards. The first chapter focuses on blunt-end sequencing library preparation protocols previously optimized for ancient DNA specimens. Forensically-relevant extracts were converted into libraries and typed by short tandem repeats (STR) amplification. When compared to STR profiles from pre-library extracts, a significant decrease in the quality was observed, in the form of allelic drop-out, heterozygous peak imbalance and increased stutter ratios. The second chapter discusses the efficacy of two enzymatic DNA repair methods, “PreCR® Repair” and “Nelson”, on typical ancient DNA specimens. Based on endogenous sample content, fragment length variation and base misincorporation rates, some DNA repair was reported when using PreCR®. However, the use of the Nelson protocol is not recommended for use in its current state. Both sequencing library preparation and enzymatic DNA repair show potential application to forensic evidential material, but require further analyses to confirm hypotheses and observations outlined in this thesis. / Thesis / Master of Science (MSc)