<p>Although common knowledge dictates that the lichen thallus is formed solely by a fungus (mycobiont) that develops a symbiotic relationship with an alga and/or cyanobacterium (photobiont), the non-photoautotrophic bacteria found in lichen microbiomes are increasingly regarded as integral components of lichen thalli and significant players in the ecology and physiology of lichens. Despite recent interest in this topic, the phylogeny, ecology, and function of these bacteria remain largely unknown. The experiments presented in this dissertation employ culture-free methods to examine the bacteria housed in these unique environments to ultimately inform an assessment of their status with regard to the lichen symbiosis. Microbiotic surveys of lichen thalli using new oligonucleotide-primers targeting the 16S SSU rRNA gene (developed as part of this study to target Bacteria, but exclude sequences derived from chloroplasts and Cyanobacteria) revealed the identity of diverse bacterial associates, including members of an undescribed lineage in the order Rhizobiales (Lichen-Associated Rhizobiales 1; `LAR1'). It is shown that the LAR1 bacterial lineage, uniquely associated with lichen thalli, is widespread among lichens formed by distantly related lichen-forming fungi and is found in lichens collected from the tropics to the arctic. Through extensive molecular cloning of the 16S rRNA gene and 454 16S amplicon sequencing, ecological trends were inferred based on mycobiont, photobiont, and geography. The implications for using lichens as microcosms to study larger principles of ecology and evolution are discussed. In addition to phylogenetic and ecological studies of lichen-associated bacterial communities, this dissertation provides a first assessment of the functions performed by these bacteria within the lichen microbiome in nature through 454 sequencing of two different lichen metatranscriptomes (one from a chlorolichen, <italic>Cladonia grayi</italic>, and one from a cyanolichen, <italic>Peltigera praetextata</italic>). Non-photobiont bacterial genes for nitrogen fixation were not detected in the <italic>Cladonia</italic> thallus (even though transcripts of cyanobacterial nitrogen fixation genes from two different pathways were detected in the cyanolichen thallus), implying that the role of nitrogen fixation in the maintenance of chlorolichens might have previously been overstated. Additionally, bacterial polyol dehydrogenases were found to be expressed in chlorolichen thalli (along with fungal polyol dehydrogenases and kinases from the mycobiont), suggesting the potential for bacteria to begin the process of breaking down the fixed carbon compounds secreted by the photobiont for easier metabolism by the mycobiont. This first look at the group of functional genes expressed at the level of transcription provides initial insights into the symbiotic network of interacting genes within the lichen microbiome.</p> / Dissertation

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HIV-1 genetic diversity is a major obstacle for developing an effective vaccine. My hypothesis is that HIV-1 genetic diversity can be characterized and that cross-clade immunogens can be predicted at the population level. I systematically investigated positive selection (PS) pressures on HIV-1 Env and Gag proteins based on the analysis of the sequences collected from the Los Alamos Sequence Database. I identified PS sites, investigated PS patterns, correlated PS with the known functional sites of the two proteins, calculated frequencies of HLA alleles targeting CTL epitopes, and compared PS patterns among major subtypes. The results showed that PS pressure was widely dispersed across the entire regions of both HIV-1 Env and Gag proteins, suggesting the conserved regions are under host immune response pressure. The neutralizing antibody, non-neutralizing antibody, and CTL responses were found to be the major forces driving genetic diversity of HIV-1 env and gag genes at population level. However, PS pressures on both Env and Gag proteins remain stable over time, suggesting genetic diversity of HIV-1 driven by host immune responses changed very little over the last 29 years. Furthermore, the results also demonstrated that up to 70% PS sites were shared among the major HIV-1 clades, implying the existence of cross-clade immunogenicity. A number of potential cross-clades immunogens were predicted to elicit CTL or neutralizing antibody responses from Env and Gag proteins. I also detected a significant correlation between HLA allele frequencies and host CTL responses elicited by Accessory/Regulator’s proteins at population level. Moreover, I detected an association between the frequency of HLA-B7 supertype and the number of identified optimal CTL epitopes. The results suggest HLA class I allele frequencies in a population influence the evolution of HIV-1. I also systematically evaluated the utility of ultra-deep pyrosequencing to characterize genetic diversity of HIV-1 gag genes within quasispecies. The results showed that ultra-deep pyrosequencing of amplified HIV genes is a better method than the traditional Sanger-clone-based method in the comprehensive characterization of genetic diversity of HIV-1 quasispecies, especially in detecting low frequency variations. In conclusion, my thesis provides important information for rational design of an effective HIV-1 vaccine.

HIV-1 genetic diversity is a major obstacle for developing an effective vaccine. My hypothesis is that HIV-1 genetic diversity can be characterized and that cross-clade immunogens can be predicted at the population level. I systematically investigated positive selection (PS) pressures on HIV-1 Env and Gag proteins based on the analysis of the sequences collected from the Los Alamos Sequence Database. I identified PS sites, investigated PS patterns, correlated PS with the known functional sites of the two proteins, calculated frequencies of HLA alleles targeting CTL epitopes, and compared PS patterns among major subtypes. The results showed that PS pressure was widely dispersed across the entire regions of both HIV-1 Env and Gag proteins, suggesting the conserved regions are under host immune response pressure. The neutralizing antibody, non-neutralizing antibody, and CTL responses were found to be the major forces driving genetic diversity of HIV-1 env and gag genes at population level. However, PS pressures on both Env and Gag proteins remain stable over time, suggesting genetic diversity of HIV-1 driven by host immune responses changed very little over the last 29 years. Furthermore, the results also demonstrated that up to 70% PS sites were shared among the major HIV-1 clades, implying the existence of cross-clade immunogenicity. A number of potential cross-clades immunogens were predicted to elicit CTL or neutralizing antibody responses from Env and Gag proteins. I also detected a significant correlation between HLA allele frequencies and host CTL responses elicited by Accessory/Regulator’s proteins at population level. Moreover, I detected an association between the frequency of HLA-B7 supertype and the number of identified optimal CTL epitopes. The results suggest HLA class I allele frequencies in a population influence the evolution of HIV-1. I also systematically evaluated the utility of ultra-deep pyrosequencing to characterize genetic diversity of HIV-1 gag genes within quasispecies. The results showed that ultra-deep pyrosequencing of amplified HIV genes is a better method than the traditional Sanger-clone-based method in the comprehensive characterization of genetic diversity of HIV-1 quasispecies, especially in detecting low frequency variations. In conclusion, my thesis provides important information for rational design of an effective HIV-1 vaccine.

Recent proceedings in biotechnology have enabled completely new avenues in life science research to be explored. By allowing increased parallelization an ever-increasing complexity of cell samples or experiments can be investigated in shorter time and at a lower cost. This facilitates for example large-scale efforts to study cell heterogeneity at the single cell level, by analyzing cells in parallel that also can include global genomic analyses. The work presented in this thesis focuses on massively parallel analysis of cells or nucleic acid samples, demonstrating technology developments in the field as well as use of the technology in life sciences. In stem cell research issues such as cell morphology, cell differentiation and effects of reprogramming factors are frequently studied, and to obtain information on cell heterogeneity these experiments are preferably carried out on single cells. In paper I we used a high-density microwell device in silicon and glass for culturing and screening of stem cells. Maintained pluripotency in stem cells from human and mouse was demonstrated in a screening assay by antibody staining and the chip was furthermore used for studying neural differentiation. The chip format allows for low sample volumes and rapid high-throughput analysis of single cells, and is compatible with Fluorescence Activated Cell Sorting (FACS) for precise cell selection. Massively parallel DNA sequencing is revolutionizing genomics research throughout the life sciences by constantly producing increasing amounts of data from one sequencing run. However, the reagent costs and labor requirements in current massively parallel sequencing protocols are still substantial. In paper II-IV we have focused on flow-sorting techniques for improved sample preparation in bead-based massive sequencing platforms, with the aim of increasing the amount of quality data output, as demonstrated on the Roche/454 platform. In paper II we demonstrate a rapid alternative to the existing shotgun sample titration protocol and also use flow-sorting to enrich for beads that carry amplified template DNA after emulsion PCR, thus obtaining pure samples and with no downstream sacrifice of DNA sequencing quality. This should be seen in comparison to the standard 454-enrichment protocol, which gives rise to varying degrees of sample purity, thus affecting the sequence data output of the sequencing run. Massively parallel sequencing is also useful for deep sequencing of specific PCR-amplified targets in parallel. However, unspecific product formation is a common problem in amplicon sequencing and since these shorter products may be difficult to fully remove by standard procedures such as gel purification, and their presence inevitably reduces the number of target sequence reads that can be obtained in each sequencing run. In paper III a gene-specific fluorescent probe was used for target-specific FACS enrichment to specifically enrich for beads with an amplified target gene on the surface. Through this procedure a nearly three-fold increase in fraction of informative sequences was obtained and with no sequence bias introduced. Barcode labeling of different DNA libraries prior to pooling and emulsion PCR is standard procedure to maximize the number of experiments that can be run in one sequencing lane, while also decreasing the impact of technical noise. However, variation between libraries in quality and GC content affects amplification efficiency, which may result in biased fractions of the different libraries in the sequencing data. In paper IV barcode specific labeling and flow-sorting for normalization of beads with different barcodes on the surface was used in order to weigh the proportion of data obtained from different samples, while also removing mixed beads, and beads with no or poorly amplified product on the surface, hence also resulting in an increased sequence quality. In paper V, cell heterogeneity within a human being is being investigated by low-coverage whole genome sequencing of single cell material. By focusing on the most variable portion of the human genome, polyguanine nucleotide repeat regions, variability between different cells is investigated and highly variable polyguanine repeat loci are identified. By selectively amplifying and sequencing polyguanine nucleotide repeats from single cells for which the phylogenetic relationship is known, we demonstrate that massively parallel sequencing can be used to study cell-cell variation in length of these repeats, based on which a phylogenetic tree can be drawn. / QC 20111031

Marine micro-organisms are important components of various biogeochemical cycles, complex food webs and ecological niches. Metagenomic sequencing can provide rapid profile of metabolic activities within the sponge and resident microbes. However, the study of metatranscriptomes from sponges using high throughput sequencing technology has only recently begun. Through this study we isolated, characterized and compared metatranscriptome profiles of Axinella corrugata host and sponge-specific microbial communities using 454 pyrosequencing technology. Four cDNA libraries (two eukaryotic and two prokaryotic) were generated from Axinella corrugata sponge samples collected in December 2009 and May 2010, and were characterized to a) reveal which metabolic genes were actively expressed and b) reveal possible interactions between the sponge and its microbial symbionts. The techniques used for isolation of mRNA and cDNA normalization also helped in optimization of whole-transcriptome amplification. More than 130,000 ESTs were generated for the two seasonal sponge samples and the metagenomic data sets were analyzed using bioinformatics tool, MG-RAST. Several stress-related transcripts were found which can increase our understanding of sensitivity of the sponge to changes in physical parameters in nature. The involvement of the sponge and its microbial consortia is depicted through actively expressed nitrogen and sulfur metabolism genes. Novel genes involved in several functional pathways may be discovered upon further studying hypothetical genes found across all four metagenomic data sets. Metatranscriptomic data sheds light on the functional role of microbes within the sponges and the extent of their involvement in sponge metabolism. 16S rRNA analysis was also carried out using genomic DNA of the same samples, to better elucidate the bacterial taxa abundance in the sponge. This study provides a profile of active mRNA trancripts in Axinella corrugata which include eukaryotic as well as prokaryotic sequences. The data analysis of this research provides new information at the cross-disciplinary interface between molecular biology and computational science.

One in 3,000 people in the US are born with cystic fibrosis (CF), a genetic disorder affecting the reproductive system, pancreas, and lungs. Lung disease caused by chronic bacterial and fungal infections is the leading cause of morbidity and mortality in CF. Identities of the microbes are traditionally determined by culturing followed by phenotypic and biochemical assays. It was first thought that the bacterial infections were caused by a select handful of bacteria such as S. aureus, H. influenzae, B. cenocepacia, and P. aeruginosa. With the advent of PCR and molecular techniques, the polymicrobial nature of the CF lung became evident. The CF lung contains numerous bacteria and the communities are diverse and unique to each patient. The total complexity of the bacterial infections is still being determined. In addition, only a few members of the fungal communities have been identified. Much of the fungal community composition is still a mystery. This dissertation addresses this gap in knowledge. A snap shot of CF sputa bacterial community was obtained using the length heterogeneity-PCR community profiling technique. The profiles show that south Florida CF patients have a unique, diverse, and dynamic bacterial community which changes over time. The identities of the bacteria and fungi present were determined using the state-of-the-art 454 sequencing. Sequencing results show that the CF lung microbiome contains commonly cultured pathogenic bacteria, organisms considered a part of the healthy core biome, and novel organisms. Understanding the dynamic changes of these identified microbes will ultimately lead to better therapeutical interventions. Early detection is key in reducing the lung damage caused by chronic infections. Thus, there is a need for accurate and sensitive diagnostic tests. This issue was addressed by designing a bacterial diagnostic tool targeted towards CF pathogens using SPR. By identifying the organisms associated with the CF lung and understanding their community interactions, patients can receive better treatment and live longer.

In der vorliegenden Dissertation wurde der Einfluss der Baumdiversität auf die Bodenpilzgemeinschaft in Chinesischen subtropischen experimentellen Waldflächen untersucht.

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

Quinoa (Chenopodium quinoa Willd.) is an important food crop for subsistence farmers in the Altiplano (high plains) of Peru, Bolivia, and Argentina. Saponins are part of a diverse family of secondary metabolites that are found in high concentrations in the pericarp of many varieties of quinoa. Due to their bitter taste and anti-nutritive properties, saponins must be removed before the quinoa grain is consumed. There are ‘sweet’ varieties of quinoa that have significantly reduced levels of saponin. Previous research suggests saponin production is controlled by a single locus. The major objective of this research was to elucidate the genetic components in the saponin biosynthesis pathway. Thus, we report the development and annotation of the first large scale expressed sequence tag (EST) collection for quinoa based on Sanger and 454 pyrosequencing of maturing seed tissue expressing saponins. Sanger sequencing produced 18,325 reads with an average read length of 693 nucleotides, while 454 GS-FLX pyrosequencing generated 295,048 reads with an average read length of 202 nucleotides. A hybrid assembly of all sequences generated 39,366 unigenes, consisting of 16,728 contigs and 22,638 singletons. Repeat sequence analysis of the unigene set identified 291 new microsatellite markers. From the unigene set, a custom microarray was developed and used to assay transcriptional changes in developing seeds of saponin-containing and saponin-free quinoa lines. The microarray consisted of 102,834 oligonucleotide probes representing 37,716 sequences of the unigenes set. Three different statistical comparisons, based on comparisons of ‘sweet’ vs. ‘bitter’ seed tissue at two developmental stages, were assayed on the custom array. Using a p-value cutoff threshold of 0.01, we identified a list of 198 significantly differentially expressed candidate genes common to all three comparisons. We also identified a list of candidate genes (p-value ≤ 0.05) that are known to be associated with identified triterpenoid (saponin) biosynthetic pathways that were differentially expressed in all three comparisons. Included in this list are candidate genes that share homology to cytochrome P450s (20), cytochrome P450 monooxygenases (10), and glycosyltransferases (49) suggesting that transcriptional differences in the saponin biosynthesis pathway possibly responsible for the absence or presence of saponin in quinoa are determined after the formation of the β-amyrin skeleton. These candidate genes are suggested for use in future studies in the production of saponin in quinoa.