Robust and scalable barcoding for massively parallel long‑read sequencing

Nucleic-acid barcoding is an enabling technique for many applications, but its use remains limited in emerging long-read sequencing technologies with intrinsically low raw accuracy. Here, we apply so-called NS-watermark barcodes, whose error correction capability was previously validated in silico, in a proof of concept where we synthesize 3840 NS-watermark barcodes and use them to asymmetrically tag and simultaneously sequence amplicons from two evolutionarily distant species (namely Bordetella pertussis and Drosophila mojavensis) on the ONT MinION platform. To our knowledge, this is the largest number of distinct, non-random tags ever sequenced in parallel and the frst report of microarray-based synthesis as a source for large oligonucleotide pools for barcoding. We recovered the identity of more than 86% of the barcodes, with a crosstalk rate of 0.17% (i.e., one misassignment every 584 reads). This falls in the range of the index hopping rate of established, highaccuracy Illumina sequencing, despite the increased number of tags and the relatively low accuracy of both microarray-based synthesis and long-read sequencing. The robustness of NS-watermark barcodes, together with their scalable design and compatibility with low-cost massive synthesis, makes them promising for present and future sequencing applications requiring massive labeling, such as long-read single-cell RNA-Seq.Fil: Ezpeleta, Joaquín. Centro Internacional Franco Argentino de Ciencias de la Información y de Sistemas; Argentina.Fil: Labari, Ignacio Garcia. Centro Internacional Franco Argentino de Ciencias de la Información y de Sistemas; Argentina.Fil: Bulacio, Pilar. Centro Internacional Franco Argentino de Ciencias de la Información y de Sistemas; Argentina.Fil: Tapia, Elizabeth. Centro Internacional Franco Argentino de Ciencias de la Información y de Sistemas; Argentina.Fil: Ezpeleta, Joaquín. Universidad Nacional de Rosario. Facultad de Ciencias Exactas, Ingeniería y Agrimensura; Argentina.Fil: Bulacio, Pilar. Universidad Nacional de Rosario. Facultad de Ciencias Exactas, Ingeniería y Agrimensura; Argentina.Fil: Tapia, Elizabeth. Universidad Nacional de Rosario. Facultad de Ciencias Exactas, Ingeniería y Agrimensura; Argentina.Fil: Villanova, Gabriela Vanina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina.Fil: Lavista Llanos, Sofía. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina.Fil: Villanova, Gabriela Vanina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio Mixto de Biotecnología Acuática. Centro Científico Tecnológico y Educativo Acuario del Río Paraná; Argentina.Fil: Posner, Victoria María. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio Mixto de Biotecnología Acuática. Centro Científico Tecnológico y Educativo Acuario del Río Paraná; Argentina.Fil: Arranz, Silvia Eda. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio Mixto de Biotecnología Acuática. Centro Científico Tecnológico y Educativo Acuario del Río Paraná; Argentina.Fil: Krsticevic, Flavia. The Hebrew University of Jerusalem. Robert H Smith Faculty of Agriculture, Food and Environment; Israel

Automation-aided high-throughput technologies for synthetic biology

Synthetic biology is a research discipline which harnesses technological progress in de novo DNA synthesis as well as combining expertise of biological sciences and engineering research fields to facilitate construction of novel artificial biological systems. Since the past two decades, application of its methodologies has led to significant advances in metabolic engineering, providing alternative biochemical routes for the production of therapeutic products, cosmetics and biofuels. However, several challenges remain to be addressed to support development of synthetic biology applications, notably the demand for faster, cheaper and more reliable DNA manufacturing as well as efficient methods for genome-scale engineering of living organisms. This doctoral thesis proposes new interdisciplinary approaches to these problems, taking advantage of the latest laboratory automation technologies to improve efficiency of modern DNA assembly and genome editing methods. The first results chapter proposes application of a robotic platform for an acoustic liquid transfer for miniaturisation of DNA fabrication. This research, published in 2016, demonstrates the possibility to cost-efficiently assemble DNA in sub-microlitre assembly reactions. The second results chapter presents efforts to develop a method for genome-scale engineering of a model eukaryote, the budding yeast. This work capitalises on the recent progress in on-chip DNA synthesis and the next-generation sequencing (NGS) technology. Finally, the last results chapter demonstrates computational studies to predict and accelerate turnaround times of a commercial DNA supply chain using probabilistic simulations. The developed software is used to estimate sequence-specific DNA manufacturing turnaround times in order to help plan DNA manufacturing and guide decisions regarding further automation of different experimental procedures

Engineering the central dogma using emulsion based directed evolution

The central dogma of molecular biology forms the most basic (and fundamental) paradigm of how life operates. Despite its elegant simplicity, scientists are still uncovering enigmas of the central dogma - which has been shaped throughout billions of years of the Darwinian process. Even though the core concepts of the central dogma have largely been untouched by evolution (the universality of the genetic code, amino acid utilization, DNA/RNA base identity) scientific advances have shown that these fundamental properties can be altered dramatically. This implies the architectures of life are pliable and likely the result of extreme optimization and fine tuning of semi-random events that took place soon after the origin of life. Reengineering the parameters of life offers a unique way of testing evolutionary processes and perceived optimality of its components. Naturally, coaxing proteins and nucleic acids to function in an unnatural fashion is difficult. Development of techniques to enable these changes has relied heavily on the exploitation of water-in-oil emulsions (or, in vitro compartmentalization), which allows directed evolution at the single cell or even single molecule level. In particular, compartmentalized partnered replication (CPR) is a dual mode selection technique, coupling the in vivo functionality of a gene with the in vitro amplification via emulsion PCR. The CPR technique has enabled the development of synthetic promoter recognition by T7 RNA polymerase, unnatural amino acid incorporation by aminoacyl tRNA synthetase engineering, genetic code reassignment through tRNA evolution, and transcriptional regulation using repressors with novel allosteric effector molecules and operator binding sites. Using a similar technique, the template recognition of an Archaeal DNA polymerase was altered such that the polymerase utilizes both DNA and RNA templates with similar efficiencies. This resulted in a reverse transcriptase that can functionally proofread on RNA templates. These technologies will continue to play a pivotal role in the future development of particular aspects of the central dogma. As certain steps in this process are tweaked to have alternative functionalities and combined together, the gap between natural life and synthetically modified life widens and gives the Darwinian process of evolution new areas to explore.Cellular and Molecular Biolog

The Cryptococcus gattii species complex in koalas: host-pathogen-environment interactions and molecular epidemiology

The Cryptococcus gattii species complex comprises some of the aetiological agents of cryptococcosis, a severe fungal disease that affects a wide variety of hosts and is acquired from the environment by inhalation. Koalas (Phascolarctos cinereus) appear to be particularly susceptible to cryptococcosis. In Australia, eucalypt tree hollows are the classic ecological niche for C. gattii molecular type VGI and therefore are also a potential source of infection. Aspects of the tree hollow microenvironment that may allow for the growth and dispersal of C. gattii VGI remain poorly understood. The C. gattii species complex has been associated with outbreaks and case clusters, and animals are often considered useful sentinels for the disease in these scenarios. The prevalence of cryptococcosis in Australian wildlife remains unknown. Given the koala’s propensity towards developing cryptococcosis, and its regular contact with a common ecological niche for the C. gattii species complex (eucalypts), it is an ideal sentinel species. The host-pathogen-environment interactions of cryptococcosis caused by the C. gattii species complex, particularly progression from exposure to colonisation of the respiratory mucosa to eventual tissue invasion, remain poorly understood. This thesis uses amplicon-based next generation sequencing to characterise the fungal microbiome of Australian tree hollows, focusing on the role that the C. gattii species complex may play in this microenvironment. The prevalence of cryptococcosis in a population of free-ranging koalas is systematically characterised, while the pathogenesis, treatment and diagnosis of the disease in this host species are also explored. Finally, fine-scale molecular epidemiology tools (multi-locus sequence typing and whole genome sequencing) are used to determine sources of infection and examine disease caused by the C. gattii species complex in Australia, using primarily the koala as a model for naturally-occurring cryptococcosis

Continuing professional development - challenge for professional organization

Professions, as one of key sectors of social systems, bear a leading role in the existing social work organization. Free professions take up a special place and significance, all the way from Roman artes liberales to our times. Pharmaceutical profession, as one of the oldest, led by ethical principles, is regulated by postulates accepted by the profession members, and in modern times established through legislations. Typical determinants of the regulated professions, which also refer to pharmacists, as chamber members, are as follows: following ethical principles, specific skills and knowledge, professional development, autonomy at work, continuing improvement, competencies development, professional associations, licensing