High-Resolution Description of Antibody Heavy-Chain Repertoires in Humans

Antibodies' protective, pathological, and therapeutic properties result from their considerable diversity. This diversity is almost limitless in potential, but actual diversity is still poorly understood. Here we use deep sequencing to characterize the diversity of the heavy-chain CDR3 region, the most important contributor to antibody binding specificity, and the constituent V, D, and J segments that comprise it. We find that, during the stepwise D-J and then V-DJ recombination events, the choice of D and J segments exert some bias on each other; however, we find the choice of the V segment is essentially independent of both. V, D, and J segments are utilized with different frequencies, resulting in a highly skewed representation of VDJ combinations in the repertoire. Nevertheless, the pattern of segment usage was almost identical between two different individuals. The pattern of V, D, and J segment usage and recombination was insufficient to explain overlap that was observed between the two individuals' CDR3 repertoires. Finally, we find that while there are a near-infinite number of heavy-chain CDR3s in principle, there are about 3–9 million in the blood of an adult human being

Structural sizing of rotorcraft composite airframe using the open-source based design environment PANDORA

During the last years, the design environment PANDORA has been developed at DLR in order to assess airand rotorcraft airframes. As part of this design environment structural sizing routines have been implemented. The airframe models are numerically solved using commercial Finite Elements solvers and the results are passed to the sizing algorithm using in-house developed converters. This paper introduces the sizing routine that was implemented into PANDORA to assess orthotropic structures using a two-step approach: Gradientbased optimization is conducted using lamination parameters and subsequently an evolutionary algorithm is executed to get a discrete ply lay-up. The theory behind the implemented methods is described and the evaluation of the implemented strength and stability criteria is illustrated. Exemplary sizing runs of rotorcraft main frames are shown and isotropic and orthotropic main frames designs are compared

Rotorcraft Fuselage Sizing Methods in the Open-Source Framework PANDORA

Rural depopulation resulting in altered hospital coverage, new challenges for medical evacuation during military operations, and increased off-shore activities of energy suppliers, lead to changed requirements of helicopter emergency medical services (HEMS). Recently, interest has significantly increased to overcome the traditional physical limitation of flight speed by providing helicopters with auxiliary propulsive devices, so-called compound rotorcraft. In order to assess these novel rotorcraft concepts, an integrated, multidisciplinary, and automated design procedure has been established at the German Aerospace Center (DLR) using the data model CPACS (Common Parametric Aircraft Configuration Schema). The design processes of rotary- and fixed-wing aircraft highly resemble each other: In the first stage of a typical aircraft design process, the conceptual stage, basic characteristics are established that typically consist of e.g. outer dimensions (i.e. its aerodynamic shape), flight performance, mass breakdown, etc. At this stage of the design process mostly fast, analytical, and statistical methods are applied featuring many simplifications. In the subsequent preliminary design phase the detail level increases. The continuously growing computational power has enabled design engineers to integrate higher fidelity methods at this design stage. At the DLR Institute of Structures and Design, tools have been developed in the last couple of years that use finite element (FE) methods to size aeronautical fuselage structures according to static load cases to allow a more precise prediction of the structural mass, and thus in turn to a different maximum take-off mass which is considered as a major design parameter. Although based on the same framework approach, these FE based tools developed for preliminary sizing of rotary- and fixed-wing fuselages diverged over the years due to different project requirements, such as specific modeling aspects, different syntax for the involved FE solvers, or different design emphases. These issues resulted in different tools to generate FE meshes and to conduct analyses with some inconsistencies between the individual tools. In order to unify the tools, the development of the software framework PANDORA (Parametric Numerical Design and Optimization Routines for Aircraft) has been started at DLR in 2016 from scratch using the Python programming language. The key idea behind PANDORA is to generate one common software framework to model, analyze, and size both fixed- and rotary-wing fuselage structures. Particular focus in the development of PANDORA lies in the use of dedicated open-source packages and the interchangeability of different commercial and open-source FE solvers. This paper first shows the approach of the PANDORA toolbox for fixed-wing aircraft. Then, the process of adapting respectively integrating specific modeling and analysis methods for rotorcraft fuselages into the new framework is shown. Concluding this article an outlook of new enhancements into PANDORA is given highlighting its benefits in the context of preliminary structural analysis of novel rotorcraft concepts

Adeno-associated Virus Genome Population Sequencing Achieves Full Vector Genome Resolution and Reveals Human-Vector Chimeras

Recombinant adeno-associated virus (rAAV)-based gene therapy has entered a phase of clinical translation and commercialization. Despite this progress, vector integrity following production is often overlooked. Compromised vectors may negatively impact therapeutic efficacy and safety. Using single molecule, real-time (SMRT) sequencing, we can comprehensively profile packaged genomes as a single intact molecule and directly assess vector integrity without extensive preparation. We have exploited this methodology to profile all heterogeneic populations of self-complementary AAV genomes via bioinformatics pipelines and have coined this approach AAV-genome population sequencing (AAV-GPseq). The approach can reveal the relative distribution of truncated genomes versus full-length genomes in vector preparations. Preparations that seemingly show high genome homogeneity by gel electrophoresis are revealed to consist of less than 50% full-length species. With AAV-GPseq, we can also detect many reverse-packaged genomes that encompass sequences originating from plasmid backbone, as well as sequences from packaging and helper plasmids. Finally, we detect host-cell genomic sequences that are chimeric with inverted terminal repeat (ITR)-containing vector sequences. We show that vector populations can contain between 1.3% and 2.3% of this type of undesirable genome. These discoveries redefine quality control standards for viral vector preparations and highlight the degree of foreign products in rAAV-based therapeutic vectors. Keywords: AAV-GPseq, recombinant adeno-associated virus, single molecule real-time sequencing, rAAV-ITR, gene therapy vector Q

V, D, and J segment use in mouse is also highly uneven.

<p>Lines indicate cumulative distributions (measured on right-hand side y-axis) (a)–(c). Out of space considerations only V segments present at ≥1% are shown. Insets show cumulative distributions. VDJ combinations also appear unevenly, with the ∼200 most frequent VDJ combinations responsible for 50 percent of all recombination events (d).</p

V, D, and J segment use in human is highly uneven.

<p>Lines indicate cumulative distributions (measured on right-hand side y-axis) (a)–(c). Not all V segments are labeled on the x-axis. V, D, and J segments appear at similar frequencies in two different human subjects (d)–(f). Each point corresponds to a single segment. Insets show cumulative distributions. VDJ combinations also appear unevenly, with the 100 most frequent VDJ combinations responsible for 50 percent of all recombination events (g).</p

Number of Reads and Clusters.

<p>Number of Reads and Clusters.</p

Analysis of High-Throughput Sequencing and Annotation Strategies for Phage Genomes

Background: Bacterial viruses (phages) play a critical role in shaping microbial populations as they influence both host mortality and horizontal gene transfer. As such, they have a significant impact on local and global ecosystem function and human health. Despite their importance, little is known about the genomic diversity harbored in phages, as methods to capture complete phage genomes have been hampered by the lack of knowledge about the target genomes, and difficulties in generating sufficient quantities of genomic DNA for sequencing. Of the approximately 550 phage genomes currently available in the public domain, fewer than 5% are marine phage. Methodology/Principal Findings: To advance the study of phage biology through comparative genomic approaches we used marine cyanophage as a model system. We compared DNA preparation methodologies (DNA extraction directly from either phage lysates or CsCl purified phage particles), and sequencing strategies that utilize either Sanger sequencing of a linker amplification shotgun library (LASL) or of a whole genome shotgun library (WGSL), or 454 pyrosequencing methods. We demonstrate that genomic DNA sample preparation directly from a phage lysate, combined with 454 pyrosequencing, is best suited for phage genome sequencing at scale, as this method is capable of capturing complete continuous genomes with high accuracy. In addition, we describe an automated annotation informatics pipeline that delivers high-quality annotation and yields few false positives and negatives in ORF calling. Conclusions/Significance: These DNA preparation, sequencing and annotation strategies enable a high-throughput approach to the burgeoning field of phage genomics