Biocrusts and the ecosystem services they provide are becoming more susceptible to fire as exotic annual grass invasions facilitate the spread of desert wildfires. Further, precipitation patterns across the western United States are predicted to change over the next century, and have the potential to dramatically influence fire regimes and the recovery of burned biocrusts. Despite these changes to desert fire and precipitation cycles, our understanding of post-fire biocrust recovery is limited, especially regarding the first two years after fire. To investigate biocrust recovery, we created burn manipulations (i.e., unburned and burned) and tracked crust form and function over two years in one cold and one hot desert ecosystem (UT, USA). We evaluated the entire bacterial community, but focused on Cyanobacteria species that confer soil stability and N fixation capabilities to biocrusts. Specifically, we quantified shifts in biocrust bacterial community composition using target metagenomics of 16S rDNA; monitored biocrust moss and lichen cover; measured N fixation potential; and assessed soil infiltration rates and soil stability. We found little evidence that biocrust form or function recovered from fire within two years. Based on pyrosequencing results, fire altered biocrust community composition in interspace and shrub biocrusts. Cyanobacteria species were almost completely eliminated by fire, constituting 9-21% of unburned plots and less than 0.01% of burned interspace and shrub biocrust communities. Based on cover estimates, no lichen or moss species survived the fire or recovered within two years. N fixation potentials decreased by at least six-fold in burned interspace biocrusts, representing a reduction in soil N inputs into already N-limited desert soils. Soil infiltration rates also drastically declined in burned biocrusts and remained depressed, but only remained depressed for one year. To investigate the interactions between biocrust recovery, fire, and precipitation, we nested precipitation treatments manipulating the amount of monthly rainfall (i.e., ambient, plus 30% and minus 30%) within burn treatments during the second year. Soil NH4+ was the only parameter to be affected by precipitation, and exhibited a positive relationship with precipitation magnitude at the end of one year. Our results demonstrate that fire is a strong destabilizer of the bacterial components of biocrust communities and that the ecosystem services provided by crusts recover at different rates, with N dynamics recovering more slowly than soil ecohydrology.

No description available.

Thema dieser Arbeit war die klinische sowie molekulargenetische Analyse einer Familie mit der Verdachtsdiagnose autosomal dominante lamelläre Ichthyose (ADLI). Mit direkter Sequenzierung des Loricrin-Gens (LOR) wurde die Mutation 730insG identifiziert und die Diagnose Loricrin-Keratoderma gestellt. Durch Analyse weiterer Patienten wurde gezeigt, dass ADLI keine Loricrin-Keratoderma darstellt. Nach eingehender klinischer Untersuchung und Literaturanalyse konnten für die hier beschriebene Entität folgende Merkmale definiert werden: Als Hauptmerkmale eine honigwabenförmige Palmoplantarkeratose sowie eine leichte Ichthyose, als Nebenmerkmale Pseudoainhums, Autoamputationen, Kollodiumbaby, prominente Fingerknöchel sowie Hyperkeratosen an Knien und Ellenbögen. Die genetisch als Loricrin-Keratoderma charakterisierte Verhornungsstörung in der beschriebenen Familie sollte nunmehr klinisch als honigwabenförmige Palmoplantarkeratose mit Ichthyose bezeichnet werden. Zur Standarddiagnostik von Loricrin-Keratoderma wurde die direkte Sequenzierung von LOR auf DNA-Basis etabliert. Die Mutation 730insG resultiert in einer neuen argininreichen Domäne und einer Verlängerung des Proteins um 22 Aminosäuren. Eine Expressionsanalyse mittels Pyrosequenzierung zeigte eine gleichwertige Expression des mutierten und des Wildtyp-Allels. Dies unterstützt die „gain-of-function“-Theorie für das veränderte Loricrin und stützt die Aussage des für Loricrin-Keratoderma existierenden transgenen Mausmodells. / The main focus of this thesis was the clinical and genetic analysis of a family referred to us with the diagnosis of autosomal dominant lamellar ichthyosis (ADLI). Through direct sequencing of the loricrin gene (LOR) the mutation 730insG was identified and the family was diagnosed as having loricrin keratoderma. By sequencing further patients it was shown that ADLI is not a loricrin keratoderma. Based on refined clinical examination and analysis of the literature the following criteria could be defined for the entity seen: Compulsory features are honeycomb-like palmoplantar keratoderma and ichthyosis, optional features are pseudoainhums, autoamputations, collodion baby, prominent knuckle pads as well as hyperkeratotic lesions on knees and elbows. Therefore the disorder of keratinisation of the family described here genetically characterised as loricrin keratoderma should be clinically termed “honeycomb-like palmoplantar keradoderma with ichthyosis”. To molecularly diagnose loricrin keratoderma direct sequencing of LOR with DNA samples was established. The mutation 730insG results in a new arginine rich domain and an elongation of the protein by 22 residues. Expression analysis showed an equal expression of mutant and wild-type allele. This underlined the “gain-of-function” theory of the modified loricrin and supported the findings in the transgenic mouse model for loricrin keratoderma.

Despite the global licensure of two live-attenuated rotavirus vaccines, Rotarix® and RotaTeq®, rotavirus remains the major cause of severe dehydrating diarrhoea in young mammals and the need for further development of additional rotavirus vaccines, especially vaccines effective against regional strains in developing country settings, is increasing. The design and formulation of new effective multivalent rotavirus vaccines is complicated by the wide rotavirus strain diversity. Novel rotavirus strains emerge periodically due to the propensity of rotaviruses to evolve using mechanisms such as point mutation, genome segment reassortment, genome segment recombination and interspecies transmission. Mutations occurring within the primer binding regions targeted by the current commonly employed sequence-dependent genotyping techniques lead to difficulties in genotyping novel mutant rotavirus strains. Therefore, use of sequence-independent techniques coupled with online rotavirus genotyping tools will help to understand the complete epidemiology of the circulating strains which, in turn, is vital for developing intervention measures such as vaccine and anti-viral therapies. In this study, sequence-independent cDNA synthesis that uses a single set of oligonucleotides that do not require prior sequence knowledge of the rotavirus strains, 454® pyrosequencing, and an online rotavirus genotyping tool, RotaC, were used to swiftly characterise the whole genome of rotaviruses. The robustness of this approach was demonstrated in characterising the complete genetic constellations and evolutionary origin of selected human rotavirus strains that emerged in the past two decades worldwide, human rotavirus strains frequently detected in Africa, and the whole genomes of some common strains frequently detected in bovine species. Most of the characterised strains emerged either through intra- or interspecies genome segment reassortment processes. The methods used in this study also allowed determination of the whole consensus genome sequence of multiple rotavirus variants present in a single stool sample and the elucidation of the evolutionary mechanisms that explained their origin. The 454® pyrosequence-generated data revealed evidence of intergenotype rotavirus genome segment recombination between the genome segments 6 (VP6), 8 (NSP2) and 10 (NSP4) of Wa-like and DS-1-like origin. The use of next generation sequencing technology combined with sequence-independent amplification of the rotavirus genomes allowed the determination of the consensus nucleotide sequence for each of the genome segments of the selected study strains directly from stool sample. The consensus nucleotide sequences of the genome segments encoding VP2, VP4, VP6 and VP7 of some of the study strains were codon optimised for insect cell expression and used to generate recombinant baculoviruses. The Bac-to-Bac baculovirus expression system was used to generate chimaeric rotavirus virus-like particles (RV-VLPs). These chimaeric RV-VLPs contained inner capsids (VP2 and VP6) derived from a South African RVA/Humanwt/ ZAF/GR10924/1999/G9P[6] strain, on to which outer capsid layer proteins composed of various combinations of VP4 and VP7 were assembled. The outer capsid proteins were derived from the dsRNA of G2, G8, G9 or G12 strains associated with either P[4], P[6] or P[8] genotypes that were directly extracted from human stool faecal specimens. The structures of these chimaeric RV-VLPs were morphologically evaluated using transmission electron microscopy (TEM). Based on the size and morphology of the particles, doublelayered (dRV-VLPs) and triple-layered RV-VLPs (tRV-VLPs) were produced. Recombinant rotavirus proteins readily assembled into dRV-VLPs, whereas approximately 10 – 30% of the assembled RV-VLPs from insect expressed recombinant VP2/6/7/4 were chimaeric tRVVLPs. These RV-VLPs will be evaluated in future animal studies as potential non-live rotavirus vaccine candidates. The novel approach of producing RV-VLPs introduced in this study, namely by using the consensus nucleotide sequence derived from dsRNA extracted directly from clinical specimens, should speed up vaccine research and development by bypassing the need to adapt the viruses to tissue culture and circumventing some other problems associated with cell culture adaptation as well. Thus, it is now possible to generate RV-VLPs for evaluation as non-live vaccine candidates for any human or animal field rotavirus strain. / Thesis (PhD (Biochemistry))--North-West University, Potchefstroom Campus, 2012

Submitted in complete fulfillment for the degree of Doctor of Philosophy (Biotechnology), Durban University of Technology, Durban, South Africa, 2016. / Seasonal nitrification breakdown is a major problem in wastewater treatment plants which makes it difficult for the plant operators to meet discharge limits. The present study focused on understanding the seasonal impact of environmental and operational parameters on nitrifiers and nitrification, in a biological nutrient removal wastewater treatment works situated in the midlands of KwaZulu Natal. Composite sludge samples (from the aeration tank), influent and effluent water samples were collected twice a month for 237 days. A combination of fluorescent in-situ hybridization, polymerase chain reaction (PCR)-clone library, quantitative polymerase chain reaction (qPCR) were employed for characterizing and quantifying the dominant nitrifiers in the plant. In order to have more insight into the activated sludge community structure, pyrosequencing was used in profiling the amoA locus of ammonia oxidizing bacteria (AOB) community whilst Illumina sequencing was used in characterising the plant’s total bacterial community. The nonlinear effect of operating parameters and environmental conditions on nitrification was also investigated using an adaptive neuro-fuzzy inference system (ANFIS), Pearson’s correlation coefficient and quadratic models. The plant operated with higher MLSS of 6157±783 mg/L during the first phase (winter) whilst it was 4728±1282 mg/L in summer. The temperature recorded in the aeration tanks ranged from 14.2oC to 25.1oC during the period. The average ammonia removal during winter was 60.0±18% whereas it was 83±13% during summer and this was found to correlate with temperature (r = 0.7671; P = 0.0008). A significant correlation was also found between the AOB (amoA gene) copy numbers and temperature in the reactors (α= 0.05; P=0.05), with the lowest AOB abundance recorded during winter. Sanger sequencing analysis indicated that the dominant nitrifiers were Nitrosomonas spp. Nitrobacter spp. and Nitrospira spp. Pyrosequencing revealed significant differences in the AOB population which was 6 times higher during summer compared to winter. The AOB sequences related to uncultured bacterium and uncultured AOB also showed an increase of 133% and 360% respectively when the season changed from winter to summer. This study suggests that vast population of novel, ecologically significant AOB species, which remain unexploited, still inhabit the complex activated sludge communities. Based on ANFIS model, AOB increased during summer season, when temperature was 1.4-fold higher than winter (r 0.517, p 0.048), and HRT decreased by 31% as a result of rainfall (r - 0.741, p 0.002). Food: microorganism ratio (F/M) and HRT formed the optimal combination of two inputs affecting the plant’s specific nitrification (qN), and their quadratic equation showed r2-value of 0.50. This study has significantly contributed towards understanding the complex relationship between the microbial population dynamics, wastewater composition and nitrification performance in a full-scale treatment plant situated in the subtropical region. This is the first study applying ANFIS technique to describe the nitrification performance at a full-scale WWTP, subjected to dynamic operational parameters. The study also demonstrated the successful application of ANFIS for determining and ranking the impact of various operating parameters on plant’s nitrification performance, which could not be achieved by the conventional spearman correlation due to the non-linearity of the interactions during wastewater treatment. Moreover, this study also represents the first-time amoA gene targeted pyrosequencing of AOB in a full-scale activated sludge is being done. / D

L’aulacode (Thryonomys swinderianus) est un rongeur herbivore récemment domestiqué en Afrique pour la production de viande. Quelques études antérieures ont portés sur l’alimentation de cet animal, dans le but d’améliorer la productivité des élevages. A ce jour, nos connaissances sur la digestion et les besoins nutritionnels de cet animal sont encore très parcellaires. Le premier objectif de notre étude était d’améliorer nos connaissances sur la physiologie digestive de l’aulacode en croissance, en particulier en relation avec les apports de fibres alimentaires, avec pour finalité de proposer des recommandations nutritionnelles en fibres pour optimiser la croissance et la santé digestive de cet animal. Notre second objectif était d’analyser les effets d’une diminution du ratio protéines digestibles / énergie digestible parallèlement à une hausse des apports d’amidon, sur la digestion et les performances. La finalité était d’analyser les possibilités de formuler un aliment complet moins onéreux pour les éleveurs et qui respecte les besoins de l’aulacode en croissance. Notre étude a permis de savoir que le caecum est le compartiment digestif le plus important du jeune aulacode entre 1 et 3 mois d’âge, avec plus de 40% du contenu digestif total. L’activité microbienne caecale (100 mM d’acides gras volatils totaux (AGVt) par gramme de contenu frais) est élevée, et similaire à celle des ruminants ou d’autres herbivores monogastriques. Le profil fermentaire est caractérisé par une prédominance de l’acétate (75 % des AGVt) et un ratio propionate / butyrate supérieur à 1. Le pyroséquençage 454 de l’ADN16S bactérien a permis de caractériser le microbiote caecal. Au sevrage, nous observons une prédominance du phylum des Bacteroidetes, avec 51 % d’abondance relative, alors que le phylum des Firmicutes devient majoritaire (50%) à 3 mois d’âge. Le microbiote caecal est caractérisé par la présence de genres souvent identifiés dans d’autres écosystèmes digestifs d’herbivores, tels que : RC9 (2 à 8%), Parabacteroides (1 à 8%), Prevotella (3 à 6%) et Xylanibacter (1%), Erysipelotrichaceae Turicibacter (1 à 7%), Lachnospiraceae Incertae_Sedis (4 à 5%), Ruminococcaceae Incertae_Sedis (1 à 2%) et Ruminococcus (1 à 3%). D’autres genres, absents chez des espèces voisines comme le lapin et le cobaye, semblent plus spécifiques de l’aulacode, tels que Termite_Treponema_cluster (1.7 à 2.2%) et Treponema (7 à 13%), du phylum des Spirochaetes. L’analyse des performances zootechniques indique qu’un taux de fibres compris entre 17 et 21 % d’ADF représenterait un bon compromis entre santé digestive et croissance de l’aulacode après son sevrage. Descendre au dessous de 6 g de protéines digestibles par MJ d’énergie digestible, via une hausse importante des apports d’amidon et une baisse importante du taux de protéines brutes (en dessous de 11 %) et de fibres, est préjudiciable à la croissance des animaux. / The cane rat or grasscutter (Thryonomys swinderianus) is a rodent herbivore recently domesticated in Africa for meat production. Some previous studies focused on the feeding of this animal, in order to improve the productivity of farms. To date, our knowledge of digestion and nutritional requirements of this animal are still very scarce. Our first objective was to improve our knowledge of digestive physiology of the young grasscutter, particularly in relation to dietary fibre supply, in order to improve the recommendations for dietary fibre content of diets to optimize growth and digestive health. Our second objective was to analyze the effects of a decreased digestible protein / digestible energy ratio, along with an increased intake of starch, on digestion and performances. The final aim was to analyze the possibilities to formulate a complete feed, cheaper for farmers and that meets the requirements of the young grasscutter. Our study found that the caecum is the most important digestive compartment of the young grasscutter between 1 and 3 months of age, with more than 40% of the total gut contents. The caecal microbial activity (100 mM of total volatile fatty acids (VFA) per gram of fresh content) is high and similar to that of ruminants or other herbivorous monogastric animals. The fermentation profile is characterized by a predominance of acetate (75% of total VFA) and a propionate / butyrate ratio greater than 1. A pyrosequencing of bacterial 16S-DNA was used to characterize the caecal microbiota. At weaning (one month), we observe a predominance of the Bacteroidetes phylum, with 51% of relative abundance, whereas the Firmicutes phylum becomes predominant (50%) at 3 months of age. Caecal microbiota is characterized by the presence of genera often identified in other digestive ecosystems of herbivores, such as: RC9 (2-8%), Parabacteroides (1-8%), Prevotella (3.6%) and Xylanibacter (1%), Erysipelotrichaceae Turicibacter (1-7%), Lachnospiraceae Incertae_Sedis (4-5%), Ruminococcaceae Incertae_Sedis (1-2%) and Ruminococcus (1-3%). Other genera, absent in related species such as rabbits and guinea pigs, seemed more specific of the grasscutter, such as Termite_Treponema_cluster (1.7-2.2%) and Treponema (7-13%) of the Spirochaetes phylum. The analysis of growth performances indicated that a dietary fibre content between 17% and 21% of ADF represents a good compromise between digestive health and growth of the grasscutter after weaning. Decreasing below 6g of digestible protein / MJ of digestible energy, via a high increase in starch intake and a significant decline in crude protein content (below 11%) and fibre, is detrimental to the growth of animals.

The remediation of persistent chlorinated aromatic compounds has become a priority of great relevance due to the teratogenic, carcinogenic and endocrine-disrupting properties of these xenobiotics. The use of biological methodologies for the clean-up of contaminated sites, collectively referred to as "bioremediation", has been gaining an increasing interest in recent years because it represents an effective, cost-competitive and environmentally friendly alternative to the physico-chemical and thermal treatments. In this respect, "white rot" fungi, an ecological subgroup of filamentous fungi, display features that make them excellent candidates to design an effective remediation technology ("mycoremediation"). In spite of this, fungi have not been widely exploited for their metabolic capabilities and the mechanism by which they are able to degrade the aforementioned pollutants has not been fully elucidated yet. Within this frame, the present Ph.D thesis was aimed at: i) assessing the efficiency of different mycoremediation strategies for the clean-up of a polychlorinated biphenyl (PCBs)-contaminated soil; ii) understanding the fungal degradation pathways of polychlorinated biphenyls and their major metabolites, namely chlorobenzoic acids (CBAs) and hydroxylated polychlorinated biphenyls (OH-PCBs). i)...

No description available.

Au niveau des fonds océaniques, souvent considérés comme des déserts aux conditions de vie extrêmes, des oasis luxuriants de vie existent pourtant. Les sources hydrothermales et les suintements froids, principalement localisés au niveau des dorsales océaniques et des marges continentales, vont permettre le développement de communautés microbiennes et animales très particulières. Le Bassin de Guaymas, situé dans le Golfe de Californie (Mexique) présente la particularité de regrouper à la fois une zone de sources hydrothermales et une zone de suintements froids, situées à une soixantaine de kilomètres l’une de l’autre, et toutes deux recouvertes par une épaisse couche sédimentaire. Ces deux zones sont également colonisées par des étendues de bivalves, des buissons de vers tubicoles ainsi que de tapis microbiens blancs ou colorés comparables. Afin de mieux comprendre le fonctionnement global de ces deux types d’écosystèmes et notamment le rôle structurant des communautés microbiennes sédimentaires sur la répartition des différents assemblages de surface, les travaux entrepris dans cette thèse se proposaient d’étudier les communautés microbiennes sédimentaires associés à la zone de suintements froids (Marge de Sonora) et la zone de sources hydrothermales (Southern Trough) du Bassin de Guaymas. Pour cela, la diversité des communautés microbiennes (Bacteria et Archaea) de différents habitats caractérisés par une faune et des profils géochimiques particuliers, a été étudiée grâce à l’utilisation d’une technique de séquençage haut-débit, le pyroséquençage par la technique du 454, combinée à d’autres techniques comme le FISH ou la PCR quantitative. Cette étude a permis de déterminer que la structure et la diversité des communautés microbiennes dans ces sédiments étaient très spécifiques de ces environnements. Par ailleurs, les colonisateurs présents en surface des sédiments reflétaient des profils géochimiques et des communautés microbiennes très différentes au sein des sédiments. Dans les sédiments colonisés par les tapis microbiens, riches en méthane, les communautés microbiennes dominantes (ANME, Deltaproteobacteria…) utiliseraient notamment les émissions de méthane des couches sédimentaires profondes et produiraient d'importantes concentrations de sulfures, nécessaires à l’installation des communautés microbiennes thiotrophes de surface formant les tapis. Ces fortes concentrations en sulfure excluraient en revanche les communautés animales de ce périmètre. A contrario, dans les sédiments présentant de faibles concentrations en méthane et en sulfure, permettant l'installation d’assemblages faunistiques variés, les communautés microbiennes méthanotrophes anaérobies et productrices de sulfure étaient minoritaires. L'activité et le métabolisme de ces colonisateurs de surface pourraient par ailleurs permettre le développement des lignées microbiennes détectées dans ces habitats (MBG-D, Chloroflexi…). L'analyse des larges jeux de données obtenus au cours de cette étude nous a donc permis de mettre en évidence un système dynamique complexe fonctionnant en équilibre entre les communautés microbiennes sédimentaires, les organismes colonisant la surface du sédiment et la composition géochimique des eaux interstitielles. / Whereas the deep-sea environment is often considered to be a desert, hydrothermal vents andcold seeps provide “oases” of biological activity on the ocean floor. Vent and seep ecosystems support complex food webs based on microbial chemoautotrophic primary production. These hydrothermal vent and cold seeps ecosystems both release hydrocarbon- and sulfide-rich fluids,fueling various surface assemblages such as mat-forming giant bacteria or symbiont-bearinginvertebrates (e.g. bivalves, tubeworms). In the Guaymas Basin, the nearby presence at a few tens of kilometers of cold seeps and hydrothermal vents coupled with comparable sedimentary settings and depths offer a unique opportunity to assess and compare the microbial community composition of these ecosystems. Tobetter understand their overall functioning, we studied sedimentary microbial communities associated with cold seep and hydrothermal vent areas in the Guaymas Basin. The diversity of microbial communities inhabiting sediments was studied using high throughput sequencing (454pyrosequencing), combined with complementary approaches, such as FISH and quantitative PCR. This study reveals that sediments found in the Guaymas Basin were colonized by microbial communities typically found in these types of ecosystems. Our results revealed a high similarity between microbial communities composition associated with the cold seep and hydrothermal vent areas as a probable consequence of the sedimentary context. Nonetheless, thermophilic and hyperthermophilic lineages (e.g.: Thermodesulfobacteria, Desulfurococcales, etc) were exclusively identified in hydrothermally influenced sediments highlighting the strong influence of temperature gradients and other hydrothermally-related factors on microbial community composition. Furthermore, sediments populated by different surface assemblages show distinct porewater geochemistry features and are associated with distinct microbial communities. Indeed, in the sediments underlying microbial mats characterized by high methane porewater concentrations,microbial communities were dominated by anaerobic methane oxidizers (ANME), known to produce sulfide which provides high fluxes of sulfide to the seafloor. In contrast, sediment associated microbial communities underlying faunal assemblages were characterized by a lower biomass and lower methane porewater concentrations in sediments, limiting porewater sulfide concentrations. Without elevated and toxic sulfide concentrations, faunal assemblages can colonize the surface. Together, geochemical and microbial surveys indicate that porewater methane concentrations play an important role in the microbial community structure and subsequently in the establishment of the surface colonizers. Furthermore, presence and activity of the surface colonizers influence the underlying microbial communities probably because of modification of energy source availabilities. Finally, the existence of similar microbial populations between the two ecosystems also raises the question of their dispersal mechanisms. Our results support the hypothesis of a potential continuity among deep-sea ecosystems. In absence of physical borders, environmental conditions (temperature, specific compounds associated withhydrothermal fluids) might select specific and highly adapted microorganisms from the pool of microorganisms dispersed globally across the seafloor.

Advancements in the field of biotechnology are expanding the scientific horizon and a promising era is envisioned with personalized medicine for improved health. The amount of genetic data is growing at an ever-escalating pace due to the availability of novel technologies that allow massively parallel sequencing and whole-genome genotyping, that are supported by the advancements in computer science and information technologies. As the amount of information stored in databases throughout the world is growing and our knowledge deepens, genetic signatures with significant importance are discovered. The surface of such a set in the data mining process may include causative- or marker single nucleotide polymorphisms (SNPs), revealing predisposition to disease, or gene expression signatures, profiling a pathological state. When targeting a reduced set of signatures in a large number of samples for diagnostic- or fine-mapping purposes, efficient interrogation and scoring require appropriate preparations. These needs are met by miniaturized and parallelized platforms that allow a low sample and template consumption. This doctoral thesis describes an attempt to tackle some of these challenges by the design and implementation of a novel assay denoted Trinucleotide Threading (TnT). The method permits multiplex amplification of a medium size set of specific loci and was adapted to genotyping, gene expression profiling and digital allelotyping. Utilizing a reduced number of nucleotides permits specific amplification of targeted loci while preventing the generation of spurious amplification products. This method was applied to genotype 96 individuals for 75 SNPs. In addition, the accuracy of genotyping from minute amounts of genomic DNA was confirmed. This procedure was performed using a robotic workstation running custom-made scripts and a software tool was implemented to facilitate the assay design. Furthermore, a statistical model was derived from the molecular principles of the genotyping assay and an Expectation-Maximization algorithm was chosen to automatically call the generated genotypes. The TnT approach was also adapted to profiling signature gene sets for the Swedish Human Protein Atlas Program. Here 18 protein epitope signature tags (PrESTs) were targeted in eight different cell lines employed in the program and the results demonstrated high concordance rates with real-time PCR approaches. Finally, an assay for digital estimation of allele frequencies in large cohorts was set up by combining the TnT approach with a second-generation sequencing system. Allelotyping was performed by targeting 147 polymorphic loci in a genomic pool of 462 individuals. Subsequent interrogation was carried out on a state-of-the-art massively parallelized Pyrosequencing instrument. The experiment generated more than 200,000 reads and with bioinformatic support, clonally amplified fragments and the corresponding sequence reads were converted to a precise set of allele frequencies. / QC 20100813