<p>Advances in sequencing technologies constantly improves the throughput andaccuracy of sequencing instruments. Together with this development comes newdemands and opportunities to fully take advantage of the massive amounts of dataproduced within a sequence run. One way of doing this is by analyzing a large set ofsamples in parallel by pooling them together prior to sequencing and associating thereads to the corresponding samples using DNA sequence tags. Amplicon sequencingis a common application for this technique, enabling ultra deep sequencing andidentification of rare allelic variants. However, a common problem for ampliconsequencing projects is formation of unspecific PCR products and primer dimersoccupying large portions of the data sets.</p><p>This thesis is based on two papers exploring these new kinds of possibilities andissues. In the first paper, a method for including thousands of samples in the samesequencing run without dramatically increasing the cost or sample handlingcomplexity is presented. The second paper presents how the amount of high qualitydata from an amplicon sequencing run can be maximized.</p><p>The findings from the first paper shows that a two-tagging system, where the first tagis introduced by PCR and the second tag is introduced by ligation, can be used foreffectively sequence a cohort of 3500 samples using the 454 GS FLX Titaniumchemistry. The tagging procedure allows for simple and easy scalable samplehandling during sequence library preparation. The first PCR introduced tags, that arepresent in both ends of the fragments, enables detection of chimeric formation andhence, avoiding false typing in the data set.</p><p>In the second paper, a FACS-machine is used to sort and enrich target DNA covered emPCR beads. This is facilitated by tagging quality beads using hybridization of afluorescently labeled target specific DNA probe prior to sorting. The system wasevaluated by sequencing two amplicon libraries, one FACS sorted and one standardenriched, on the 454 showing a three-fold increase of quality data obtained.</p> / QC20100907

Intratumoural heterogeneity complicates the molecular interpretation of biopsies, as multiple distinct tumour genomes are sampled and analysed at once. Ignoring the presence of these populations can lead to erroneous conclusions, and so a correct analysis must account for the clonal structure of the sample. Several methods to reconstruct tumour clonality from sequencing data have been proposed, spanning methods that either do not consider phylogenetic constraints or posit a perfect phylogeny. Models of the first type are typically latent feature models that can describe the observed data flexibly, but whose results may not be reconcilable with a phylogeny. The second type, instead, generally comprises non-parametric mixture models, with strict assumptions on the tumour’s evolutionary process. The focus of this dissertation is on the development of a phylogenetic latent feature model that can bridge the advantages of these two approaches, allowing deviations from a perfect phylogeny. The work is recounted by three statistical models of increasing complexity. First, I present a non-parametric model based on the Indian Buffet Process prior, and highlight the need for phylogenetic constraints. Second, I develop a finite, phylogenetic extension of the previous model, and show that it can outperform competing methods. Third, I generalise the phylogenetic model to arbitrary copy-number states. Markov chain Monte Carlo algorithms are presented to perform inference. The models are tested on datasets that include synthetic data, controlled biological data, and clinical data. In particular, the copy-number generalisation is applied to longitudinal circulating tumour DNA samples. Liquid biopsies that leverage circulating tumour DNA require sensitive techniques in order to detect mutations at low allele fractions. One method that allows sensitive mutation calling is the amplicon sequencing strategy TAm-Seq. I present bioinformatic tools to improve both the development of TAm-Seq amplicon panels and the analysis of its sequencing data. Finally, an enhancement of this method is presented and shown to detect mutations de novo and in a multiplexed manner at allele fractions less than 0.1%.

Antimicrobial engineered nanomaterials (ENM) are increasingly incorporated into products despite limited understanding of the interactions between ENMs and bacteria that lead to toxic impacts. The hazard posed by increasing environmental release of antimicrobial ENMs is also poorly characterized. The overall objective of this thesis is to inform questions about the types of interactions that lead to an ENM inducing bacterial toxicity. Many antimicrobial ENMs are soluble, and the ion plays an important role in their toxicity. Some believe that, beyond release of ions, ENM toxicity is expected to derive from a nanoparticle (NP)-specific effect. This research compares bacterial responses to ENMs, their metal salts, and/or their transformed species within different experimental settings to improve our understanding of the interactions that enable ENM bacterial toxicity. The first objective is to characterize the potential hazard posed by pristine and transformed antimicrobial ENMs on microbial communities within a complex environmental system. One pair of ENMs (Ag0 and Ag2S) led to differential short-term impacts on surficial sediment microbial communities, while the other did not (CuO and CuS), showing that ENM transformation does not universally lead to distinct impacts. The metal ion (Cu2+) had a more profound microbial community impact than did any of the four ENMs. By 300 days the microbial community structure and composition re-converged, suggesting minimal long-term impacts of high pulse inputs of antimicrobial ENMs on microbial communities within complex environments. The second objective is to identify NP-specific effects of a common antimicrobial ENM on a model bacterium. Analysis of transcriptional responses identified NP-specific induction of a membrane stress responsive gene, providing evidence of a NP-specific effect. Otherwise, our results suggest that CuO NP toxicity triggers the same stress responses as does Cu2+, but at more moderate levels. Two ion treatments with the same total Cu input – one with pulse addition and one with gradual addition that was meant to better represent the slow dissolution of the CuO NP – led to temporally distinct responses. This calls for the use of more representative ion controls for comparison against soluble NP impacts in future nanotoxicity studies. The third objective is to investigate the potential use of CuO ENMs to reduce virulence and growth of an emerging bacterial pathogen. CuO NP exposure led to reduction in relative expression of three Staphylococcus aureus virulence factor genes, especially in methicillinresistant S. aureus (MRSA) clinical isolates. Growth was inhibited at high CuO NP concentrations for all four isolates, too. Comparison across all genes assayed showed isolatespecific transcriptional responses, but with NP- and ion-induced responses showing clear differences for each isolate, too. Altogether, this research contributes novel knowledge that will guide efforts to characterize potential hazard from release of ENMs into the environment and to apply ENMs for effective antibacterial treatment.

The rapid development of DNA sequencing technologies has opened up new avenues of research, including the investigation of population structure within infectious diseases (both within patient and between populations). In order to take advantage of these advances in technologies and the generation of new types of data, novel bioinformatics tools are needed that won’t succumb to artifacts introduced by the data generation, and thus provide accurate and precise results. To achieve this goal I have create several tools. First, SeekDeep, a pipeline for analyzing targeted amplicon sequencing datasets from various technologies, is able to achieve 1-base resolution even at low frequencies and read depths allowing for accurate comparison between samples and the detection of important SNPs. Next, PathWeaver, a local haplotype assembler designed for complex infections and highly variable genomic regions with poor reference mapping. PathWeaver is able to create highly accurate haplotypes without generating chimeric assemblies. PathWeaver was used on the key protein in pregnancy-associated malaria Plasmodium falciparum VAR2CSA which revealed population sub-structuring within the key binding domain of the protein observed to be present globally along with confirming copy number variation. Finally, the program Carmen is able to utilize PathWeaver to augment the results from targeted amplicon approaches by reporting where and when local haplotypes have been found previously. These rigorously tested tools allow the analysis of local haplotype data from various technologies and approaches to provide accurate, precise and easily accessible results.

Advances in sequencing technologies constantly improves the throughput andaccuracy of sequencing instruments. Together with this development comes newdemands and opportunities to fully take advantage of the massive amounts of dataproduced within a sequence run. One way of doing this is by analyzing a large set ofsamples in parallel by pooling them together prior to sequencing and associating thereads to the corresponding samples using DNA sequence tags. Amplicon sequencingis a common application for this technique, enabling ultra deep sequencing andidentification of rare allelic variants. However, a common problem for ampliconsequencing projects is formation of unspecific PCR products and primer dimersoccupying large portions of the data sets. This thesis is based on two papers exploring these new kinds of possibilities andissues. In the first paper, a method for including thousands of samples in the samesequencing run without dramatically increasing the cost or sample handlingcomplexity is presented. The second paper presents how the amount of high qualitydata from an amplicon sequencing run can be maximized. The findings from the first paper shows that a two-tagging system, where the first tagis introduced by PCR and the second tag is introduced by ligation, can be used foreffectively sequence a cohort of 3500 samples using the 454 GS FLX Titaniumchemistry. The tagging procedure allows for simple and easy scalable samplehandling during sequence library preparation. The first PCR introduced tags, that arepresent in both ends of the fragments, enables detection of chimeric formation andhence, avoiding false typing in the data set. In the second paper, a FACS-machine is used to sort and enrich target DNA covered emPCR beads. This is facilitated by tagging quality beads using hybridization of afluorescently labeled target specific DNA probe prior to sorting. The system wasevaluated by sequencing two amplicon libraries, one FACS sorted and one standardenriched, on the 454 showing a three-fold increase of quality data obtained. / QC20100907

Methicillin-resistant Staphylococcus aureus (MRSA) is a variant of the more common Methicillin-Susceptible Staphylococcus aureus (MSSA), an opportunistic pathogen a portion of the human population carries as normal bacterial flora. When an outbreak of MRSA occurs, it is often important to determine if and how these strains are related to each other. In this report two different types of epidemiological methods were combined (namely Multi-Locus Sequence Typing and staphylococcal protein A-typing), in order to reduce workload, costs and time. A panel of resistance and virulence markers was also added to gather as much information about the culture as possible in a single analysis. To test the viability of the method extracted DNA and heat-treated bacterial cultures of both MRSA and MSSA were amplified with a curated panel of primers. These products were later sequenced with Nanopore’s MinION using the Flongle flow-cell. The method showed promise and worked as intended regarding the staphylococcal protein A-typing and the panel of resistance and virulence markers. However, the Multi-Locus Sequence Typing did still require optimization in order to be used clinically. In summary the project can be viewed as a success since it succeeded in being more time, cost and work efficient than many of its predecessors, when the problems with the Multi-Locus Sequence Typing are solved.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22485号 / 農博第2389号 / 新制||農||1075(附属図書館) / 学位論文||R2||N5265(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 吉田 天士, 教授 澤山 茂樹, 教授 菅原 達也 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

The overall aim of this study was to investigate the potential influence of chitosan, a biodegradable and antimicrobial compound, on termite hindgut symbionts. For this purpose, a morphological quantifying technique was conducted on the protist community’s hindgut after feeding termites on chitosan-treated wood. The aim was to characterize the diversity of protist species in the economically important dark southern subterranean termite, Reticulitermes virginicus. A molecular phylogenetic analysis of the V3 and V4 hyper-variable regions of 16S ribosomal RNA (rRNA) gene of the bacterial community in the hindgut of R. virginicus was performed on termites exposed to chitosan treatment. Light microscopy visualization of protist species residing in the hindgut of workers showed presence of ten protist species both in the control sample and in termites fed a low concentration of chitosan. In this study, the coexistence of two species of the genus Trichonympha (T. agilis and T. burlesquei) is reported for the first time in R. virginicus. Monocercomonas sp. and Trichomitus trypanoides were the only two protists found in termites exposed to wood treated with higher chitosan concentration solutions and the absence of wood fragments in their food vacuoles was clear. This feature indicates that these two protists may not be involved in the digestion of the wood fragments impregnated with chitosan. The results of this study indicated that the potential effect of chitosan caused elimination of the protist species in termite hindguts. The genomic DNA of bacterial hindgut community of R. virginicus were profiled using sequences which amplified theV3-V4 sub-regions of 16S rRNA gene. Sequences were analyzed using a taxonomic analysis tool, Quantitative Insights Into Microbial Ecology (OIIME 2), in order to infer the effect of chitosan on the composition of the bacterial fauna in the hindgut. The richness and evenness results indicated that the most diversity was observed in the bacteria from termites not being exposed (UNX) to treatment compared to other treatment groups. On the other hand, the lowest richness and evenness were determined for chitosan-treated wood (CTE) and starved termites (STV). Of 28 bacterial phyla, Bacteroidetes, Firmicutes, Elusimicrobia, and Proteobacteria were the most dominant phyla across all the treatment groups. The results suggest that chitosan treated wood led to the microbial community shifts in R. virginicus. In addition, lack of a nutrition source and other changes in termite’s food affect the termite hindgut bacterial diversity.

Cattle are commonly treated with antibiotics that may survive digestion and promote antibiotic resistance when manure or composted manure is used as a soil amendment for crop production. This study was conducted to determine the effects of antibiotic administration and soil amendment practices on microbial diversity and antibiotic resistance of bacteria recovered from the surfaces of lettuce and radishes grown using recommended application rates. Vegetables were planted in field plots amended with raw manure from antibiotic-treated dairy cows, composted-manure from cows with different histories of antibiotic administration, or a chemical fertilizer control (12 plots, n=3). Culture-based methods, 16SrDNA amplicon sequencing, qPCR and shot-gun metagenomics were utilized to profile bacteria and characterize the different gene markers for antibiotic resistance. Culture-based methodologies revealed that lettuce grown in soils amended with BSAs had significantly larger clindamycin resistant populations compared to control conditions. Growth in BSAs was associated with significant changes to the bacterial community composition of radish and lettuce. Total sul1 copies were 160X more abundant on lettuce grown in manure and total tet(W) copies were 30X more abundant on radishes grown in manure. Analysis of shotgun metagenomic data revealed that lettuce grown in manure-amended soils possessed resistance genes for three additional antibiotic classes compared to other treatments. This study demonstrates that raw, antibiotic-exposed manure may alter microbiota and the antibiotic resistance genes present on vegetables. Proper composting of BSAs as recommended by the U.S. Department of Agriculture and Environmental Protection Agency is recommended to mitigate the spread of resistance to vegetable surfaces. / MSLFS

Eukaryotic Translation Elongation Factor 1 Alpha (EEF1A) exists as two forms with different tissue specificities and encoded by separate loci: eEF1A1 on 6q13 and eEF1A2 on 20q13.3. eEF1A1 is ubiquitously expressed whereas eEF1A2 expression is normally limited to the heart, brain and skeletal muscles. eEF1A proteins are GTP-binding proteins that recruit an amino-acylated tRNA to the ribosome during the elongation phase of protein translation. eEF1A2 mRNA and protein are highly expressed in 50–60% of primary human breast tumors and metastases but not in normal breast epithelium. Since it is also overexpressed in 30% of primary human ovarian tumors, transforms rodent fibroblasts and increases their tumorigenicity in nude mice, eEF1A2 is considered to be a potential human oncogene. The mechanism of eEF1A2 expression is yet to be determined. Studies showed no gene mutation and no correlation between locus amplification or methylation and gene expression. Phosphate Tyrosine Kinase-6 (PTK6) is also located on 20q13.3. It is 48kb upstream of EEF1A2. PTK6 is a non-receptor tyrosine-kinase that is normally expressed in epithelial linings, prostate, skin and oral epithelium but it is not detected in the normal human mammary epithelium. PTK6 has been found to be expressed in many breast cancer cell lines and in approximately 60% of primary human breast tumors but it has not been detected in normal human breast tissue nor in fibroadenomas. Like other tyrosine kinases, PTK6 phosphorylates and activates downstream substrates that would be predicted to lead to increased transcriptional activity and therefore mediates proliferation of breast cancer cells. PTK6 is considered a prognostic marker of metastasis-free survival in breast cancer independent of the classical markers of tumor size, lymph node involvement and HER2 status. The aim of this project was to characterize for the first time the genomic region containing the two mentioned breast cancer oncogenes and understand their various roleswhether they act in tandem or independently in breast tumorigenesis. Immunohistochemistry was performed on tissue microarrays from 300 breast cancer patients to detect the expression levels of eEF1A2 and PTK6. Tumors that showed a high co-expression were analyzed for the genes’ copy number. An increased copy number of PTK6 was detected but not of eEF1A2 nor of adjacent genes on the 20q13.3 amplicon. To understand the effect of EEF1A2 expression on other genes, microarray analysis was performed on NIH-3T3 cells stably transfected with EEF1A2. Many upregulated genes were associated with different types of cancers. This was further confirmed by real-time PCR. However, when the NIH-3T3 cells were transiently transfected with EEF1A2, the genes that were upregulated in the microarray study showed no change in expression. In conclusion, EEF1A2 and PTK6 act independently and each acts through a different mechanism in breast tumorigenesis.