Interactome-Seq: A Protocol for Domainome Library Construction, Validation and Selection by Phage Display and Next Generation Sequencing

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to poorly folding proteins or random open reading frames. We have developed a strategy where, by using TEM-1 \u3b2-lactamase (the enzyme conferring ampicillin resistance) on a genomic scale, we can select collections of correctly folded protein domains from the coding portion of the DNA of any intronless genome. The protein fragments obtained by this approach, the so called "domainome", will be well expressed and soluble, making them suitable for structural/functional studies. By cloning and displaying the "domainome" directly in a phage display system, we have showed that it is possible to select specific protein domains with the desired binding properties (e.g., to other proteins or to antibodies), thus providing essential experimental information for gene annotation or antigen identification. The identification of the most enriched clones in a selected polyclonal population can be achieved by using novel next-generation sequencing technologies (NGS). For these reasons, we introduce deep sequencing analysis of the library itself and the selection outputs to provide complete information on diversity, abundance and precise mapping of each of the selected fragment. The protocols presented here show the key steps for library construction, characterization, and validation

ITSoneDB & ITSoneWB: supporting the metabarcoding investigation of eukaryotic communities

The amplicon-based Metagenomics approach here referred as DNA Metabarcoding, is the method of choice to quickly infer the global composition of both bacterial and eukaryotic communities in a variety of environments, including the human body. In this respect, whereas 16S rRNA is the de facto standard locus for bacteria, the Internal Transcribed Spacer 1 (ITS1) of ribosomal RNA gene cluster is gaining popularity for its high potential in discriminating eukaryotes at the deepest taxonomic levels. In this context the availability of well-curated reference databases, interfaced with established analysis tools, is mandatory to obtain reliable taxonomic profiles. This is not a trivial issue given the paucity of ITS1 targeted databases to date, structured to feed a number of popular tools and services for their use in taxonomic assignment workflows. In order to provide the first curated resource of ITS1 sequences covering all the Eukaryotic domains and well suited for eukaryotic DNA metabarcoding surveys and enhance its connectivity with established analysis pipelines we deployed ITSoneWB (ITS1 workbench). Its virtual instance has been deployed on the INFN-Bari ReCaS cloud facility running OpenStack and includes three taxonomic analysis pipelines pointing to ITSoneDB release 1.138. The current ITSoneDB version (available at itsonedb.cloud.ba.infn.it) hosts 1,174,761 ITS1 sequences spanning 157,531 species, a tool for the barcoding gap computation and for the design of taxon targeted amplification primers and, recently, a system enabling to identify ITS1 regions in shotgun environmental data. ITSoneWB has been designed and developed in the mainframe of the ELIXIR 2017 Implementation Studies for integration of Italian node and the EXCELERATE marine Metagenomics activities. ITSoneWB is available at itsonewb.cloud.ba.infn.it/galaxy and its design allows to scale the underlying resources in order to support large project and/or to include new tools

Analysis of Faecal Microbiota and Small ncRNAs in Autism: Detection of miRNAs and piRNAs with Possible Implications in Host–Gut Microbiota Cross-Talk

Intestinal microorganisms impact health by maintaining gut homeostasis and shaping the host immunity, while gut dysbiosis associates with many conditions, including autism, a complex neurodevelopmental disorder with multifactorial aetiology. In autism, gut dysbiosis correlates with symptom severity and is characterised by a reduced bacterial variability and a diminished beneficial commensal relationship. Microbiota can influence the expression of host microRNAs that, in turn, regulate the growth of intestinal bacteria by means of bidirectional host-gut microbiota cross-talk. We investigated possible interactions among intestinal microbes and between them and host transcriptional modulators in autism. To this purpose, we analysed, by “omics” technologies, faecal microbiome, mycobiome, and small non-coding-RNAs (particularly miRNAs and piRNAs) of children with autism and neurotypical development. Patients displayed gut dysbiosis related to a reduction of healthy gut micro- and mycobiota as well as up-regulated transcriptional modulators. The targets of dysregulated non-coding-RNAs are involved in intestinal permeability, inflammation, and autism. Furthermore, microbial families, underrepresented in patients, participate in the production of human essential metabolites negatively influencing the health condition. Here, we propose a novel approach to analyse faeces as a whole, and for the first time, we detected miRNAs and piRNAs in faecal samples of patients with autism

Analysis of Faecal Microbiota and Small ncRNAs in Autism: Detection of miRNAs and piRNAs with Possible Implications in Host–Gut Microbiota Cross-Talk

Intestinal microorganisms impact health by maintaining gut homeostasis and shaping the host immunity, while gut dysbiosis associates with many conditions, including autism, a complex neurodevelopmental disorder with multifactorial aetiology. In autism, gut dysbiosis correlates with symptom severity and is characterised by a reduced bacterial variability and a diminished beneficial commensal relationship. Microbiota can influence the expression of host microRNAs that, in turn, regulate the growth of intestinal bacteria by means of bidirectional host-gut microbiota cross-talk. We investigated possible interactions among intestinal microbes and between them and host transcriptional modulators in autism. To this purpose, we analysed, by “omics” technologies, faecal microbiome, mycobiome, and small non-coding-RNAs (particularly miRNAs and piRNAs) of children with autism and neurotypical development. Patients displayed gut dysbiosis related to a reduction of healthy gut micro- and mycobiota as well as up-regulated transcriptional modulators. The targets of dysregulated non-coding-RNAs are involved in intestinal permeability, inflammation, and autism. Furthermore, microbial families, underrepresented in patients, participate in the production of human essential metabolites negatively influencing the health condition. Here, we propose a novel approach to analyse faeces as a whole, and for the first time, we detected miRNAs and piRNAs in faecal samples of patients with autism

ITSoneWB: profiling global taxonomic diversity of eukaryotic communities on Galaxy

ITSoneWB (ITSone WorkBench) is a Galaxy-based bioinformatic environment where comprehensive and high-quality reference data are connected with established pipelines and new tools in an automated and easy-to-use service targeted at global taxonomic analysis of eukaryotic communities based on Internal Transcribed Spacer 1 variants high-throughput sequencing

ITSoneWB: profiling global taxonomic diversity of eukaryotic communities on Galaxy

ITSoneWB (ITSone WorkBench) is a Galaxy-based bioinformatic environment where comprehensive and high-quality reference data are connected with established pipelines and new tools in an automated and easy-to-use service targeted at global taxonomic analysis of eukaryotic communities based on Internal Transcribed Spacer 1 variants high-throughput sequencing

Using community events to increase quality and adoption of standards: the case of Bioschemas

We present how a workshop for the local Italian ELIXIR community s teered an improvement of the quality and adoption of Bioschemas, a series of semantic annotation templates for tools, data and samples developed by the ELIXIR Interoperability platform. Gathering a small number of different end-users and having them focus on applying Bioschemas specification to their tools and data resulted in recruitment of early adopters, eight annotated resources, Bioschemas examples for future users and more than ten suggestions for specification improvement. This approach could be applied to other open specifications, promoting a wider adoption and the integration of suggestions in a bottom-up fashion