Physico-chemical foundations underpinning microarray and next-generation sequencing experiments

Hybridization of nucleic acids on solid surfaces is a key process involved in high-throughput technologies such as microarrays and, in some cases, next-generation sequencing (NGS). A physical understanding of the hybridization process helps to determine the accuracy of these technologies. The goal of a widespread research program is to develop reliable transformations between the raw signals reported by the technologies and individual molecular concentrations from an ensemble of nucleic acids. This research has inputs from many areas, from bioinformatics and biostatistics, to theoretical and experimental biochemistry and biophysics, to computer simulations. A group of leading researchers met in Ploen Germany in 2011 to discuss present knowledge and limitations of our physico-chemical understanding of high-throughput nucleic acid technologies. This meeting inspired us to write this summary, which provides an overview of the state-of-the-art approaches based on physico-chemical foundation to modeling of the nucleic acids hybridization process on solid surfaces. In addition, practical application of current knowledge is emphasized

Prevalence and diversity of Arcobacter spp. in poultry meat in New Zealand : a thesis presented in the partial fulfillment of the requirements for the degree of Master of Science in Veterinary Microbiology and Public Health at Massey University, Palmerston North, New Zealand

The microaerophilic bacterium Arcobacter has received increased attention in recent years as an emerging foodborne human pathogen. Although phenotypically related, arcobacters differ from campylobacters in their ability to grow aerobically and at lower temperatures. Poultry are considered a significant reservoir of this organism, with an isolation rate of up to 72% in faecal samples, and up to 100% in meat samples. To date, four species; A. butzleri, A. skirrowii, A. cryaerophilus, and A. cibarius have been isolated from poultry. The first three species have also been found to be associated with human and animal illnesses such as diarrhoea, bacteraemia, mastitis and abortions. The organisms are also found in raw meat products as well as in surface and ground water. Since most laboratories still do not use appropriate isolation techniques, the occurrence of this organism in food sources and their role in human illnesses is greatly underestimated. This is the first investigation of the prevalence of arcobacters in poultry meat in New Zealand. The aim of this study was to compare the most commonly used Arcobacter isolation methods. In addition, this study aimed to estimate the prevalence of Arcobacter spp. in retail poultry in New Zealand. Other aims include comparison of genetic diversity of Arcobacter spp. isolated from three different poultry producers, and by different methods, and estimation of overall genetic diversity of arcobacters present in New Zealand. During the period of May to October 2005, a total of 150 fresh, whole, retail poultry carcass produced by three different producers were purchased through two supermarket outlets in Palmerston North, New Zealand. Isolation of Arcobacter was done by seven different techniques. Arcobacter-like organisms were identified presumptively by phenotypic tests; temperature tolerance, aerotolerance, motility , and oxidase production. These presumptive arcobacters were confirmed by a species-specific multiplex PCR (m-PCR) either as A. butzleri, A. cryaerophilus or A. skirrowii. DNA sequencing was done for selected isolates from both species to further confirm the PCR results. The PCR positive isolates were subjected to Pulsed-Field Gel Electrophoresis (PFGE) following restriction digestion with Eagl. It was found that 55.3 % of 150 retail poultry sold in New Zealand were harbouring Arcobacter species. Two species; A. butzleri and A. cryaerophilus were detected by m-PCR which was later confirmed by sequencing. A total of 189 isolates were detected by six methods from 83 retail poultry samples. A. butzleri was the predominant species and was detected in 51.3% of the samples, whereas A. cryaerophilus was detected only in 8% of the samples. A. butzleri and A. cryaerophilus accounted for 92.6% (n=175) and 7.4% (n-14) of the isolates, respectively. A. butzleri was the only Arcobacter species present in 46.6% samples, and A. cryaerophilus only in 3.3% of the samples. Both species were detected simultaneously in 4.6% of the samples. There was a wide variation among the prevalence rate of Arcobacter spp. in retail poultry from different producers varying from 30 to 98%. There was also a wide variation among the isolation rates of different methods varying from 3.3 to 39.3%. The best isolation method was found to be Arcobacter-broth enrichment followed by passive filtration through a sterile filter of 0.45μm, onto blood-agar plates. No single isolation method detected all arcobacters. PFGE of Arcobacter isolates demonstrated the occurrence of multiple genotypes of both A. butzleri and A. cryaerophilus in the retail poultry from the same producers, and even in a single poultry. The possible explanations for the large amount of heterogeneity include multiple sources of contamination, the occurrence of multiple parent genotypes for both species in a single poultry carcass, and a high degree of genomic recombination among the progeny of historical parent genotypes. This study highlights the high prevalence of Arcobacter spp. in poultry meat in New Zealand. It also indicates prevalence of arcobacters in poultry carcass varies greatly with the choice of isolation method and none of the currently available methods are appropriate for the detection of all species of arcobacters in New Zealand. Therefore, two or more methods should be used in parallel. The level of contamination of poultry carcass may vary with the processing practices of a slaughterhouse. To eliminate or reduce arcobacters in retail poultry, maintenance of slaughter hygiene is of utmost importance. This may be achieved by regular microbiological monitoring of carcasses according to the HACCP principles. Further studies comparing the fingerprinting pattern of Arcobacter spp. isolates obtained from retails poultry with human isolates are necessary to test the hypothesis that poultry meal is an important source for Arcobacter infection in human

Statistical modeling of RNA structure profiling experiments enables parsimonious reconstruction of structure landscapes.

RNA plays key regulatory roles in diverse cellular processes, where its functionality often derives from folding into and converting between structures. Many RNAs further rely on co-existence of alternative structures, which govern their response to cellular signals. However, characterizing heterogeneous landscapes is difficult, both experimentally and computationally. Recently, structure profiling experiments have emerged as powerful and affordable structure characterization methods, which improve computational structure prediction. To date, efforts have centered on predicting one optimal structure, with much less progress made on multiple-structure prediction. Here, we report a probabilistic modeling approach that predicts a parsimonious set of co-existing structures and estimates their abundances from structure profiling data. We demonstrate robust landscape reconstruction and quantitative insights into structural dynamics by analyzing numerous data sets. This work establishes a framework for data-directed characterization of structure landscapes to aid experimentalists in performing structure-function studies

Insights into RNA design from novel molecular tools

RNA, previously recognized merely as a messenger of genetic information, has been recently rediscovered as a versatile molecule with a central role in cellular regulation. These regulatory functions are enabled by its specific chemical makeup that allows it to fold into intricate and flexible structures. In stark contrast with DNA, RNA forms a variety of structural motifs that serve as efficient points of contact in molecular recognition. It is therefore clear, that dynamic RNA structures dictate the binding availability of interfaces that play important roles in molecular regulation inside living cells. As such, the need for tools that can accurately capture and predict RNA structure in vivo continues to be essential to understand RNA function. To this end, my dissertation focuses on the development of molecular tools to predict and characterize accessible RNA interfaces in their native environment. First, I established the usefulness of a fluorescence-based in vivo oligonucleotide hybridization approach to identify accessible interfaces by characterizing numerous RNA regions in several biologically relevant molecules in E. coli. I then described these RNA interactions using a biophysical model based on thermodynamic principles and incorporating large sets of data collected using this fluorescence-based system. This approach displayed improved prediction capabilities of RNA accessibility compared to un-optimized versions without incorporation of in vivo data. Finally, I detailed the development and application of a high throughput tool for the large-scale characterization of accessible interfaces within native RNAs in a single experiment. In this approach, in vivo oligonucleotide hybridization was coupled to transcriptional elongation control to allow analysis via next generation sequencing. This tool was used to obtain complete landscapes of functional structure for 72 regulatory molecules in a single experiment (>1000 regions). Altogether the results of this high throughput approach revealed a pattern indicating that RNA-RNA interaction sites are either highly accessible or highly protected, suggesting their binding status (e.g. actively bound or unbound). In addition, within bacterial small RNAs, our approached revealed the role of the global regulator Hfq as universal structural relaxer. The compendium of these tools provides a unique and fundamental perspective in the study of functional RNA structure, namely, the identification of dynamic structures. Furthermore, the information provided by these approaches significantly aids in the design of synthetic RNAs for a variety of purposes, including gene expression control.Chemical Engineerin

Stability and performance of two GSBR operated in alternating anoxic/aerobic or anaerobic/aerobic conditions for nutrient removal

Two granular sludge sequencing batch reactors (GSBR) with alternating anoxic/aerobic (R1) and anaerobic/aerobic (R2) conditions were operated with a 4-carbon-source synthetic influent. The physical properties of the granular sludge were very good (SVI≈20 mL g−1) and high solid concentrations (up to 35 g L−1) were obtained in the bioreactor operated with a pre-anoxic phase with additional nitrate (R1). In contrast, performance and granule settleability were lower in R2 due to the development of filamentous heterotrophic bacteria on the surface of granules. These disturbances were linked to the fact that a fraction of COD remained during the aerobic phase, which was not stored during the anaerobic period. To stabilize a GSBR with a mixture of organic carbon sources, it is thus necessary to maximize the amount of substrate used during the non-aerated, anaerobic or anoxic, phase. Comparable phosphate removal efficiency was observed in both systems; enhanced biological P removal being greater in anaerobic/aerobic conditions, while the contribution of precipitation (Ca–P) was more significant in anoxic/aerobic conditions

Capturing the ‘ome’ : the expanding molecular toolbox for RNA and DNA library construction

All sequencing experiments and most functional genomics screens rely on the generation of libraries to comprehensively capture pools of targeted sequences. In the past decade especially, driven by the progress in the field of massively parallel sequencing, numerous studies have comprehensively assessed the impact of particular manipulations on library complexity and quality, and characterized the activities and specificities of several key enzymes used in library construction. Fortunately, careful protocol design and reagent choice can substantially mitigate many of these biases, and enable reliable representation of sequences in libraries. This review aims to guide the reader through the vast expanse of literature on the subject to promote informed library generation, independent of the application

Deciphering the Hybridisation History Leading to the Lager Lineage Based on the Mosaic Genomes of Saccharomyces bayanus Strains NBRC1948 and CBS380T

Saccharomyces bayanus is a yeast species described as one of the two parents of the hybrid brewing yeast S. pastorianus. Strains CBS380T and NBRC1948 have been retained successively as pure-line representatives of S. bayanus. In the present study, sequence analyses confirmed and upgraded our previous finding: S. bayanus type strain CBS380T harbours a mosaic genome. The genome of strain NBRC1948 was also revealed to be mosaic. Both genomes were characterized by amplification and sequencing of different markers, including genes involved in maltotriose utilization or genes detected by array-CGH mapping. Sequence comparisons with public Saccharomyces spp. nucleotide sequences revealed that the CBS380T and NBRC1948 genomes are composed of: a predominant non-cerevisiae genetic background belonging to S. uvarum, a second unidentified species provisionally named S. lagerae, and several introgressed S. cerevisiae fragments. The largest cerevisiae-introgressed DNA common to both genomes totals 70kb in length and is distributed in three contigs, cA, cB and cC. These vary in terms of length and presence of MAL31 or MTY1 (maltotriose-transporter gene). In NBRC1948, two additional cerevisiae-contigs, cD and cE, totaling 12kb in length, as well as several smaller cerevisiae fragments were identified. All of these contigs were partially detected in the genomes of S. pastorianus lager strains CBS1503 (S. monacensis) and CBS1513 (S. carlsbergensis) explaining the noticeable common ability of S. bayanus and S. pastorianus to metabolize maltotriose. NBRC1948 was shown to be inter-fertile with S. uvarum CBS7001. The cross involving these two strains produced F1 segregants resembling the strains CBS380T or NRRLY-1551. This demonstrates that these S. bayanus strains were the offspring of a cross between S. uvarum and a strain similar to NBRC1948. Phylogenies established with selected cerevisiae and non-cerevisiae genes allowed us to decipher the complex hybridisation events linking S. lagerae/S. uvarum/S. cerevisiae with their hybrid species, S. bayanus/pastorianus

Prospects for multi-omics in the microbial ecology of water engineering

Advances in high-throughput sequencing technologies and bioinformatics approaches over almost the last three decades have substantially increased our ability to explore microorganisms and their functions – including those that have yet to be cultivated in pure isolation. Genome-resolved metagenomic approaches have enabled linking powerful functional predictions to specific taxonomical groups with increasing fidelity. Additionally, related developments in both whole community gene expression surveys and metabolite profiling have permitted for direct surveys of community-scale functions in specific environmental settings. These advances have allowed for a shift in microbiome science away from descriptive studies and towards mechanistic and predictive frameworks for designing and harnessing microbial communities for desired beneficial outcomes. Water engineers, microbiologists, and microbial ecologists studying activated sludge, anaerobic digestion, and drinking water distribution systems have applied various (meta)omics techniques for connecting microbial community dynamics and physiologies to overall process parameters and system performance. However, the rapid pace at which new omics-based approaches are developed can appear daunting to those looking to apply these state-of-the-art practices for the first time. Here, we review how modern genome-resolved metagenomic approaches have been applied to a variety of water engineering applications from lab-scale bioreactors to full-scale systems. We describe integrated omics analysis across engineered water systems and the foundations for pairing these insights with modeling approaches. Lastly, we summarize emerging omics-based technologies that we believe will be powerful tools for water engineering applications. Overall, we provide a framework for microbial ecologists specializing in water engineering to apply cutting-edge omics approaches to their research questions to achieve novel functional insights. Successful adoption of predictive frameworks in engineered water systems could enable more economically and environmentally sustainable bioprocesses as demand for water and energy resources increases.BT/Industriele Microbiologi