Establishment of a new method for directed sequencing of cDNA-libraries in next-generation sequencing

Um DNA mittels 454-Technologie sequenzieren zu können, muss sie von Adaptersequenzen flankiert sein. Diese werden entweder über Random-Ligation eingeführt, oder mittels PCR heranamplifiziert (454-Amplicon-Library). Letzteres ermöglicht als einziges eine gerichtete Sequenzierung Target-spezifischer Bereiche. Die Herstellung einer 454-Amplicon Library nach bestehenden Protokollen hat einige Mängel: (i) die Adaptersequenzen werden als bis zu 40 Basenpaar lange Overhangs an den 5‘-Enden der Primer eingeführt; (ii) die Primer können Längen von über 60 Basenpaare haben; (iii) die Adaptersequenzen selber können inkompatibel mit Target-spezifischen Sequenzen sein. In dieser Arbeit wurden neue Protokolle zur gerichteten Sequenzierung entwickelt, die auch dann erfolgreich anwendbar sind, wenn bestehende Protokolle scheitern. Die entwickelte Methodik beruht auf gerichteter Ligation von 454-Adaptersequenzen über die Verwendung von unterschiedlichen SfiI-Restriktions-Schnittstellen. Diese können während der cDNA-Synthese in das Template eingeführt, oder unabhängig davon mittels PCR als kurze Overhangs heranamplifiziert werden. Template- und 454-Library-Produktion wurden entkoppelt, wodurch man unabhängig von Template-Sequenzen und verwendeten Primern ist. Die Gestaltung von Multiplex-Runs ist flexibler und die MID-Wahl kann zeitnah durchgeführt werden. Die Sequenzierrichtung kann frei gewählt werden, womit eine optimale Anpassung an biologische Fragestellungen und effektive Ausnutzung der PTP ermöglicht wird. Das Protokoll ist leicht adaptierbar und funktioniert prinzipiell mit jeglichem Template. Aufreinigungs- und Größenselektionsschritte garantieren eine hohe Qualität. Die benötigten Template-Mengen konnten gemessen an den Standard-Protokollen auf weniger als ein Zehntel reduziert werden; 10 ng DNA sind ausreichend. Es wurden erfolgreiche 454-Sequenzier-Läufe von cDNAs und einer Illumina-Library durchgeführt, außerdem wurde ein Deepsequencing von IgH-cDNAs ermöglicht.To be able to sequence DNA with 454-technology, the DNA has to be flanked by adapter-sequences. Those can be introduced by random ligation, or added by PCR (454-amplicon library). Only the latter enables directed sequencing of target-specific areas. 454-amplicon library-generation following Roche’s protocol has some flaws: (i) the adapter-sequences are added as up to 40 base-pair long overhangs at the 5’-ends of primers; (ii) the primers have lengths of over 60 base-pairs overall and (iii) the adaptor-sequences themselves may be incompatible with the target-specific sequences. The outcome of this work are novel protocols for 454-sequencing, which can be successfully applied to templates where sequencing utilizing existing 454-protocols failed. The method developed is based on directed ligation of 454 adapter-sequences by using different SfiI-restriction-sites of the template-DNA. These can be introduced into the template during cDNA-synthesis, or independently as short overhangs by PCR. Template- and 454-library-production-steps are uncoupled and 454-library-production becomes independent of template-sequences and –primers. The design of multiplex-runs becomes more flexible and assignment of MIDs may be done contemporary. Orientation of sequencing direction can be chosen freely, offering optimal adaptation to any biological question and an effective exploitation of PTP-space. The protocol is easily adaptable and works with any template in principle. Purification- and size-selection-steps are performed to guarantee high quality of the library-DNA. The amount of template needed could be reduced to less than a tenth compared to standard protocols; 10 ng of DNA is sufficient.Successful 454-sequencing-runs of cDNA-libraries and an Illumina-library were performed, and also deepsequencing of IgH-cDNAs was made possible

A Novel System of Polymorphic and Diverse NK Cell Receptors in Primates

There are two main classes of natural killer (NK) cell receptors in mammals, the killer cell immunoglobulin-like receptors (KIR) and the structurally unrelated killer cell lectin-like receptors (KLR). While KIR represent the most diverse group of NK receptors in all primates studied to date, including humans, apes, and Old and New World monkeys, KLR represent the functional equivalent in rodents. Here, we report a first digression from this rule in lemurs, where the KLR (CD94/NKG2) rather than KIR constitute the most diverse group of NK cell receptors. We demonstrate that natural selection contributed to such diversification in lemurs and particularly targeted KLR residues interacting with the peptide presented by MHC class I ligands. We further show that lemurs lack a strict ortholog or functional equivalent of MHC-E, the ligands of non-polymorphic KLR in “higher” primates. Our data support the existence of a hitherto unknown system of polymorphic and diverse NK cell receptors in primates and of combinatorial diversity as a novel mechanism to increase NK cell receptor repertoire

Uncertainty Modelling in Metamodels for Fire Risk Analysis

In risk-related research of fire safety engineering, metamodels are often applied to approximate the results of complex fire and evacuation simulations. This approximation may cause epistemic uncertainties, and the inherent uncertainties of evacuation simulations may lead to aleatory uncertainties. However, neither the epistemic ‘metamodel uncertainty’ nor the aleatory ‘inherent uncertainty’ have been included in the results of the metamodels for fire safety engineering. For this reason, this paper presents a metamodel that includes metamodel uncertainty and inherent uncertainty in the results of a risk analysis. This metamodel is based on moving least squares; the metamodel uncertainty is derived from the prediction interval. The inherent uncertainty is modelled with an original approach, directly using all replications of evacuation scenarios without the assumption of a specific probability distribution. This generic metamodel was applied on a case study risk analysis of a road tunnel and showed high accuracy. It was found that metamodel uncertainty and inherent uncertainty have clear effects on the results of the risk analysis, which makes their consideration important

Risk Analysis for Road Tunnels – A Metamodel to Efficiently Integrate Complex Fire Scenarios

Fires in road tunnels constitute complex scenarios with interactions between the fire, tunnel users and safety measures. More and more methodologies for risk analysis quantify the consequences of these scenarios with complex models. Examples for complex models are the computational fluid dynamics model Fire Dynamics Simulator (FDS) and the microscopic evacuation model FDS+Evac. However, the high computational effort of complex models often limits the number of scenarios in practice. To balance this drawback, the scenarios are often simplified. Accordingly, there is a challenge to consider complex scenarios in risk analysis.To face this challenge, we improved the metamodel used in the methodology for risk analysis presented on ISTSS 2016. In general, a metamodel quickly interpolates the consequences of few scenarios simulated with the complex models to a large number of arbitrary scenarios used in risk analysis. Now, our metamodel consists of the projection array-based design, the moving least squares method, and the prediction interval to quantify the metamodel uncertainty. Additionally, we adapted the projection array-based design in two ways: the focus of the sequential refinement on regions with high metamodel uncertainties; and the combination of two experimental designs for FDS and FDS+Evac.To scrutinise the metamodel, we analysed the effects of three sequential refinement steps on the metamodel itself and on the results of risk analysis. We observed convergence in both after the second step (ten scenarios in FDS, 192 scenarios in FDS+Evac). In comparison to ISTSS 2016, we then ran 20 scenarios in FDS and 800 scenarios in FDS+Evac. Thus, we reduced the number of scenarios remarkably with the improved metamodel. In conclusion, we can now efficiently integrate complex scenarios in risk analysis. We further emphasise that the metamodel is broadly applicable on various experimental or modelling issues in fire safety engineering

Genome comparison of the epiphytic bacteria <it>Erwinia billingiae </it>and <it>E. tasmaniensis </it>with the pear pathogen <it>E. pyrifoliae</it>

<p>Abstract</p> <p>Background</p> <p>The genus <it>Erwinia </it>includes plant-associated pathogenic and non-pathogenic <it>Enterobacteria</it>. Important pathogens such as <it>Erwinia amylovora</it>, the causative agent of fire blight and <it>E. pyrifoliae </it>causing bacterial shoot blight of pear in Asia belong to this genus. The species <it>E. tasmaniensis </it>and <it>E. billingiae </it>are epiphytic bacteria and may represent antagonists for biocontrol of fire blight. The presence of genes that are putatively involved in virulence in <it>E. amylovora </it>and <it>E. pyrifoliae </it>is of special interest for these species in consequence.</p> <p>Results</p> <p>Here we provide the complete genome sequences of the pathogenic <it>E. pyrifoliae </it>strain Ep1/96 with a size of 4.1 Mb and of the non-pathogenic species <it>E. billingiae </it>strain Eb661 with a size of 5.4 Mb, <it>de novo </it>determined by conventional Sanger sequencing and next generation sequencing techniques. Genome comparison reveals large inversions resulting from homologous recombination events. Furthermore, comparison of deduced proteins highlights a relation of <it>E. billingiae </it>strain Eb661 to <it>E. tasmaniensis </it>strain Et1/99 and a distance to <it>E. pyrifoliae </it>for the overall gene content as well as for the presence of encoded proteins representing virulence factors for the pathogenic species. Pathogenicity of <it>E. pyrifoliae </it>is supposed to have evolved by accumulation of potential virulence factors. <it>E. pyrifoliae </it>carries factors for type III secretion and cell invasion. Other genes described as virulence factors for <it>E. amylovora </it>are involved in the production of exopolysaccharides, the utilization of plant metabolites such as sorbitol and sucrose. Some virulence-associated genes of the pathogenic species are present in <it>E. tasmaniensis </it>but mostly absent in <it>E. billingiae</it>.</p> <p>Conclusion</p> <p>The data of the genome analyses correspond to the pathogenic lifestyle of <it>E. pyrifoliae </it>and underlines the epiphytic localization of <it>E. tasmaniensis </it>and <it>E. billingiae </it>as a saprophyte<it/>.</p

Onset of Immune Senescence Defined by Unbiased Pyrosequencing of Human Immunoglobulin mRNA Repertoires

<div><p>The immune system protects us from foreign substances or pathogens by generating specific antibodies. The variety of immunoglobulin (Ig) paratopes for antigen recognition is a result of the V(D)J rearrangement mechanism, while a fast and efficient immune response is mediated by specific immunoglobulin isotypes obtained through class switch recombination (CSR). To get a better understanding on how antibody-based immune protection works and how it changes with age, the interdependency between these two parameters need to be addressed. Here, we have performed an in depth analysis of antibody repertoires of 14 healthy donors representing different gender and age groups. For this task, we developed a unique pyrosequencing approach, which is able to monitor the expression levels of all immunoglobulin V(D)J recombinations of all isotypes including subtypes in an unbiased and quantitative manner. Our results show that donors have individual immunoglobulin repertoires and cannot be clustered according to V(D)J recombination patterns, neither by age nor gender. However, after incorporating isotype-specific analysis and considering CSR information into hierarchical clustering the situation changes. For the first time the donors cluster according to age and separate into young adults and elderly donors (>50). As a direct consequence, this clustering defines the onset of immune senescence at the age of fifty and beyond. The observed age-dependent reduction of CSR ability proposes a feasible explanation why reduced efficacy of vaccination is seen in the elderly and implies that novel vaccine strategies for the elderly should include the “Golden Agers”.</p> </div