Inter-comparison of marine microbiome sampling protocols.

Research on marine microbial communities is growing, but studies are hard to compare because of variation in seawater sampling protocols. To help researchers in the inter-comparison of studies that use different seawater sampling methodologies, as well as to help them design future sampling campaigns, we developed the EuroMarine Open Science Exploration initiative (EMOSE). Within the EMOSE framework, we sampled thousands of liters of seawater from a single station in the NW Mediterranean Sea (Service d'Observation du Laboratoire Arago [SOLA], Banyuls-sur-Mer), during one single day. The resulting dataset includes multiple seawater processing approaches, encompassing different material-type kinds of filters (cartridge membrane and flat membrane), three different size fractionations (>0.22 µm, 0.22-3 µm, 3-20 µm and >20 µm), and a number of different seawater volumes ranging from 1 L up to 1000 L. We show that the volume of seawater that is filtered does not have a significant effect on prokaryotic and protist diversity, independently of the sequencing strategy. However, there was a clear difference in alpha and beta diversity between size fractions and between these and "whole water" (with no pre-fractionation). Overall, we recommend care when merging data from datasets that use filters of different pore size, but we consider that the type of filter and volume should not act as confounding variables for the tested sequencing strategies. To the best of our knowledge, this is the first time a publicly available dataset effectively allows for the clarification of the impact of marine microbiome methodological options across a wide range of protocols, including large-scale variations in sampled volume.

The Impact of MOSE (Experimental Electromechanical Module) Flood Barriers on Microphytobenthic Community of the Venice Lagoon.

MOSE is a system of mobile gates engineered to temporarily isolate the Venice Lagoon from the Adriatic Sea and to protect the city from flooding during extreme high tides. Within the framework of the Venezia2021 program, we conducted two enclosure experiments in July 2019 (over 48 h) and October 2020 (over 28 h) by means of 18 mesocosms, in order to simulate the structural alterations that microphytobenthos (MPB) assemblages might encounter when the MOSE system is operational. The reduced hydrodynamics inside the mesocosms favored the deposition of organic matter and the sinking of cells from the water column towards the sediment. Consequently, MPB abundances increased over the course of both experiments and significant changes in the taxonomic composition of the community were recorded. Species richness increased in summer while it slightly decreased in autumn, this latter due to the increase in relative abundances of taxa favored by high organic loads and fine grain size. By coupling classical taxonomy with 18S rRNA gene metabarcoding we were able to obtain a comprehensive view of the whole community potential, highlighting the complementarity of these two approaches in ecological studies. Changes in the structure of MPB could affect sediment biostabilization, water turbidity and lagoon primary production.

Large-scale sequencing and comparative analysis of oenological Saccharomyces cerevisiae strains supported by nanopore refinement of key genomes.

Saccharomyces cerevisiae has long been part of human activities related to the production of food and wine. The industrial demand for fermented beverages with well-defined and stable characteristics boosted the isolation and selection of strains conferring a distinctive aroma profile to the final product. To uncover variants characterizing oenological strains, the sequencing of 65 new S. cerevisiae isolates, and the comparison with other 503 publicly available genomes were performed. A hybrid approach based on short Illumina and long Oxford Nanopore reads allowed the in-depth investigation of eleven genomes and the identification of putative laterally transferred regions and structural variants. A comparative analysis between clusters of strains belonging to different datasets allowed the identification of novel relevant genetic features including single nucleotide polymorphisms, insertions and structural variants. Detection of oenological single nucleotide variants shed light on the existence of different levels of modulation for the mevalonate pathway relevant for the biosynthesis of aromatic compounds.

Prokaryotic Response to Phytodetritus-Derived Organic Material in Epi- and Mesopelagic Antarctic Waters.

Particulate organic matter (POM) export represents the underlying principle of the biological carbon pump, driving the carbon flux from the sunlit to the dark ocean. The efficiency of this process is tightly linked to the prokaryotic community, as >70% of POM respiration is carried out by particle-associated prokaryotes. In the Ross Sea, one of the most productive areas of the Southern Ocean, up to 50% of the surface primary production is exported to the mesopelagic ocean as POM. Recent evidence suggests that a significant fraction of the POM in this area is composed of intact phytoplankton cells. During austral summer 2017, we set up bottle enrichment experiments in which we amended free-living surface and deep prokaryotic communities with organic matter pools generated from native microplankton, mimicking the particle export that may derive from mild (1 µg of Chlorophyll a L-1) and intense (10 µg of Chlorophyll a L-1) phytoplankton bloom. Over a course of 4 days, we followed free-living and particle-attached prokaryotes' abundance, the degradation rates of polysaccharides, proteins and lipids, heterotrophic production as well as inorganic carbon utilization and prokaryotic community structure dynamics. Our results showed that several rare or undetected taxa in the initial community became dominant during the time course of the incubations and that different phytodetritus-derived organic matter sources induced specific changes in microbial communities, selecting for peculiar degradation and utilization processes spectra. Moreover, the features of the supplied detritus (in terms of microplankton taxa composition) determined different colonization dynamics and organic matter processing modes. Our study provides insights into the mechanisms underlying the prokaryotic utilization of phytodetritus, a significant pool of organic matter in the dark ocean.

Setup and Validation of a Targeted Next-Generation Sequencing Approach for the Diagnosis of Lysosomal Storage Disorders.

Lysosomal storage disorders (LSDs) are monogenic diseases, due to accumulation of specific undegraded substrates into lysosomes. LSD diagnosis could take several years because of both poor knowledge of these diseases and shared clinical features. The diagnostic approach includes clinical evaluations, biochemical tests, and genetic analysis of the suspected gene. In this study, we evaluated an LSD targeted sequencing panel as a tool capable to potentially reverse this classic diagnostic route. The panel includes 50 LSD genes and 230 intronic sequences conserved among 33 placental mammals. For the validation phase, 56 positive controls, 13 biochemically diagnosed patients, and nine undiagnosed patients were analyzed. Disease-causing variants were identified in 66% of the positive control alleles and in 62% of the biochemically diagnosed patients. Three undiagnosed patients were diagnosed. Eight patients undiagnosed by the panel were analyzed by whole exome sequencing: for two of them, the disease-causing variants were identified. Five patients, undiagnosed by both panel and exome analyses, were investigated through array comparative genomic hybridization: one of them was diagnosed. Conserved intronic fragment analysis, performed in cases unresolved by the first-level analysis, evidenced no candidate intronic variants. Targeted sequencing has low sequencing costs and short sequencing time. However, a coverage >60× to 80× must be ensured and/or Sanger validation should be performed. Moreover, it must be supported by a thorough clinical phenotyping.

Metabolomic Alterations Do Not Induce Metabolic Burden in the Industrial Yeast M2n[pBKD2-Pccbgl1]-C1 Engineered by Multiple δ-Integration of a Fungal ß-Glucosidase Gene.

In the lignocellulosic yeast development, metabolic burden relates to redirection of resources from regular cellular activities toward the needs created by recombinant protein production. As a result, growth parameters may be greatly affected. Noteworthy, Saccharomyces cerevisiae M2n[pBKD2-Pccbgl1]-C1, previously developed by multiple δ-integration of the ß-glucosidase BGL3, did not show any detectable metabolic burden. This work aims to test the hypothesis that the metabolic burden and the metabolomic perturbation induced by the δ-integration of a yeast strain, could differ significantly. The engineered strain was evaluated in terms of metabolic performances and metabolomic alterations in different conditions typical of the bioethanol industry. Results indicate that the multiple δ-integration did not affect the ability of the engineered strain to grow on different carbon sources and to tolerate increasing concentrations of ethanol and inhibitory compounds. Conversely, metabolomic profiles were significantly altered both under growing and stressing conditions, indicating a large extent of metabolic reshuffling involved in the maintenance of the metabolic homeostasis. Considering that four copies of BGL3 gene have been integrated without affecting any parental genes or promoter sequences, deeper studies are needed to unveil the mechanisms implied in these metabolomic changes, thus supporting the optimization of protein production in engineered strains.

MicroRNA signatures in cardiac biopsies and detection of allograft rejection.

BACKGROUND: Identification of heart transplant (HTx) rejection currently relies on immunohistology and immunohistochemistry. We aimed to identify specific sets of microRNAs (miRNAs) to characterize acute cellular rejection (ACR), antibody-mediated rejection (pAMR), and mixed rejection (MR) in monitoring formalin-fixed paraffin-embedded (FFPE) endomyocardial biopsies (EMBs) in HTx patients. METHODS: In this study we selected 33 adult HTx patients. For each, we chose the first positive EMB for study of each type of rejection. The next-generation sequencing (NGS) IonProton technique and reverse transcript quantitative polymerase chain reaction (RT-qPCR) analysis were performed on FFPE EMBs. Using logistic regression analysis we created unique miRNA signatures as predictive models of each rejection. In situ PCR was carried out on the same EMBs. RESULTS: We obtained >2,257 mature miRNAs from all the EMBs. The 3 types of rejection showed a different miRNA profile for each group. The logistic regression model formed by miRNAs 208a, 126-5p, and 135a-5p identified MR; that formed by miRNAs 27b-3p, 29b-3p, and 199a-3p identified ACR; and that formed by miRNAs 208a, 29b-3p, 135a-5p, and 144-3p identified pAMR. The expression of miRNAs on tissue, through in situ PCR, showed different expressions of the same miRNA in different rejections. miRNA 126-5p was expressed in endothelial cells in ACR but in cardiomyocytes in pAMR. In ACR, miRNA 29b-3p was significantly overexpressed and detected in fibroblasts, whereas in pAMR it was underexpressed and detected only in cardiomyocytes. CONCLUSIONS: miRNA profiling on FFPE EMBs differentiates the 3 types of rejection. Localization of expression of miRNAs on tissue showed different expression of the same miRNA for different cells, suggesting different roles of the same miRNA in different rejections.

High-Throughput Sequencing of microRNAs in Glucocorticoid Sensitive Paediatric Inflammatory Bowel Disease Patients.

The aim of this research was the identification of novel pharmacogenomic biomarkers for better understanding the complex gene regulation mechanisms underpinning glucocorticoid (GC) action in paediatric inflammatory bowel disease (IBD). This goal was achieved by evaluating high-throughput microRNA (miRNA) profiles during GC treatment, integrated with the assessment of expression changes in GC receptor (GR) heterocomplex genes. Furthermore, we tested the hypothesis that differentially expressed miRNAs could be directly regulated by GCs through investigating the presence of GC responsive elements (GREs) in their gene promoters. Ten IBD paediatric patients responding to GCs were enrolled. Peripheral blood was obtained at diagnosis (T0) and after four weeks of steroid treatment (T4). MicroRNA profiles were analyzed using next generation sequencing, and selected significantly differentially expressed miRNAs were validated by quantitative reverse transcription-polymerase chain reaction. In detail, 18 miRNAs were differentially expressed from T0 to T4, 16 of which were upregulated and 2 of which were downregulated. Out of these, three miRNAs (miR-144, miR-142, and miR-96) could putatively recognize the 3’UTR of the GR gene and three miRNAs (miR-363, miR-96, miR-142) contained GREs sequences, thereby potentially enabling direct regulation by the GR. In conclusion, we identified miRNAs differently expressed during GC treatment and miRNAs which could be directly regulated by GCs in blood cells of young IBD patients. These results could represent a first step towards their translation as pharmacogenomic biomarkers.

QueryOR: a comprehensive web platform for genetic variant analysis and prioritization.

BACKGROUND: Whole genome and exome sequencing are contributing to the extraordinary progress in the study of human genetic variants. In this fast developing field, appropriate and easily accessible tools are required to facilitate data analysis. RESULTS: Here we describe QueryOR, a web platform suitable for searching among known candidate genes as well as for finding novel gene-disease associations. QueryOR combines several innovative features that make it comprehensive, flexible and easy to use. Instead of being designed on specific datasets, it works on a general XML schema specifying formats and criteria of each data source. Thanks to this flexibility, new criteria can be easily added for future expansion. Currently, up to 70 user-selectable criteria are available, including a wide range of gene and variant features. Moreover, rather than progressively discarding variants taking one criterion at a time, the prioritization is achieved by a global positive selection process that considers all transcript isoforms, thus producing reliable results. QueryOR is easy to use and its intuitive interface allows to handle different kinds of inheritance as well as features related to sharing variants in different patients. QueryOR is suitable for investigating single patients, families or cohorts. CONCLUSIONS: QueryOR is a comprehensive and flexible web platform eligible for an easy user-driven variant prioritization. It is freely available for academic institutions at http://queryor.cribi.unipd.it/ .

Direct 16S rRNA-seq from bacterial communities: a PCR-independent approach to simultaneously assess microbial diversity and functional activity potential of each taxon.

The analysis of environmental microbial communities has largely relied on a PCR-dependent amplification of genes entailing species identity as 16S rRNA. This approach is susceptible to biases depending on the level of primer matching in different species. Moreover, possible yet-to-discover taxa whose rRNA could differ enough from known ones would not be revealed. DNA-based methods moreover do not provide information on the actual physiological relevance of each taxon within an environment and are affected by the variable number of rRNA operons in different genomes. To overcome these drawbacks we propose an approach of direct sequencing of 16S ribosomal RNA without any primer- or PCR-dependent step. The method was tested on a microbial community developing in an anammox bioreactor sampled at different time-points. A conventional PCR-based amplicon pyrosequencing was run in parallel. The community resulting from direct rRNA sequencing was highly consistent with the known biochemical processes operative in the reactor. As direct rRNA-seq is based not only on taxon abundance but also on physiological activity, no comparison between its results and those from PCR-based approaches can be applied. The novel principle is in this respect proposed not as an alternative but rather as a complementary methodology in microbial community studies.

Why Close a Bacterial Genome? The Plasmid of Alteromonas Macleodii HOT1A3 is a Vector for Inter-Specific Transfer of a Flexible Genomic Island.

Genome sequencing is rapidly becoming a staple technique in environmental and clinical microbiology, yet computational challenges still remain, leading to many draft genomes which are typically fragmented into many contigs. We sequenced and completely assembled the genome of a marine heterotrophic bacterium, Alteromonas macleodii HOT1A3, and compared its full genome to several draft genomes obtained using different reference-based and de novo methods. In general, the de novo assemblies clearly outperformed the reference-based or hybrid ones, covering >99% of the genes and representing essentially all of the gene functions. However, only the fully closed genome (∼4.5 Mbp) allowed us to identify the presence of a large, 148 kbp plasmid, pAM1A3. While HOT1A3 belongs to A. macleodii, typically found in surface waters ("surface ecotype"), this plasmid consists of an almost complete flexible genomic island (fGI), containing many genes involved in metal resistance previously identified in the genomes of Alteromonas mediterranea ("deep ecotype"). Indeed, similar to A. mediterranea, A. macleodii HOT1A3 grows at concentrations of zinc, mercury, and copper that are inhibitory for other A. macleodii strains. The presence of a plasmid encoding almost an entire fGI suggests that wholesale genomic exchange between heterotrophic marine bacteria belonging to related but ecologically different populations is not uncommon.