A Simple Data-Adaptive Probabilistic Variant Calling Model

Background: Several sources of noise obfuscate the identification of single nucleotide variation (SNV) in next generation sequencing data. For instance, errors may be introduced during library construction and sequencing steps. In addition, the reference genome and the algorithms used for the alignment of the reads are further critical factors determining the efficacy of variant calling methods. It is crucial to account for these factors in individual sequencing experiments. Results: We introduce a simple data-adaptive model for variant calling. This model automatically adjusts to specific factors such as alignment errors. To achieve this, several characteristics are sampled from sites with low mismatch rates, and these are used to estimate empirical log-likelihoods. These likelihoods are then combined to a score that typically gives rise to a mixture distribution. From these we determine a decision threshold to separate potentially variant sites from the noisy background. Conclusions: In simulations we show that our simple proposed model is competitive with frequently used much more complex SNV calling algorithms in terms of sensitivity and specificity. It performs specifically well in cases with low allele frequencies. The application to next-generation sequencing data reveals stark differences of the score distributions indicating a strong influence of data specific sources of noise. The proposed model is specifically designed to adjust to these differences.Comment: 19 pages, 6 figure

Influence of the sebaceous gland density on the stratum corneum lipidome

The skin surface lipids (SSL) result from the blending of sebaceous and epidermal lipids, which derive from the sebaceous gland (SG) secretion and the permeability barrier of the stratum corneum (SC), respectively. In humans, the composition of the SSL is distinctive of the anatomical distribution of the SG. Thus, the abundance of sebum biomarkers is consistent with the density of the SG. Limited evidence on the influence that the SG exerts on the SC lipidome is available. We explored the differential amounts of sebaceous and epidermal lipids in areas at different SG density with lipidomics approaches. SC was sampled with adhesive patches from forearm, chest, and forehead of 10 healthy adults (8F, 2M) after mechanical removal of sebum with absorbing paper. Lipid extracts of SC were analysed by HPLC/(-)ESI-TOF-MS. In the untargeted approach, the naïve molecular features extraction algorithm was used to extract meaningful entities. Aligned and normalized data were evaluated by univariate and multivariate statistics. Quantitative analysis of free fatty acids (FFA) and cholesterol sulfate (CHS) was performed by targeted HPLC/(-)ESI-TOF-MS, whereas cholesterol and squalene were quantified by GC-MS. Untargeted approaches demonstrated that the relative abundance of numerous lipid species was distinctive of SC depending upon the different SG density. The discriminating species included FFA, CHS, and ceramides. Targeted analyses confirmed that sebaceous FFA and epidermal FFA were increased and decreased, respectively, in areas at high SG density. CHS and squalene, which are biomarkers of epidermal and sebaceous lipid matrices, respectively, were both significantly higher in areas at elevated SG density. Overall, results indicated that the SG secretion intervenes in shaping the lipid composition of the epidermal permeability barrier. © 2018, The Author(s)

Marine life in the North Pacific: the known, unknown, and unknowable

Special Publication 2 On-line version On-line version includes links to the following files (these files are not included into publication): Bacterioplankton [pdf] Phytoplankton [pdf] Zooplankton [pdf] Non-exploited fish and invertebrates [pdf] Commercially-important fish and invertebrates [pdf] Marine birds [pdf] Mammals [pdf] Supplemental table of Unknowns [html

Phylogenetic and Functional Metagenomic Profiling for Assessing Microbial Biodiversity in Environmental Monitoring

Environmental management decisions have to base on holistic understanding of biodiversity and functional capability in ecosystems. Environmental metagenomics is an emerging and powerful approach allowing rapidly and reliably determine and compare microbial biodiversity and functional profiles. Advances in next generation sequencing technologies and bioinformatic tools allow set up analysis pipelines which are useful for well designed and targeted monitoring exercises already today. In the paper we demonstrate how direct sequencing of the total community DNA and analysis of the data are applicable to assess anthropogenic impact on the coastal marine ecosystems.JRC.H.1-Water Resource

Characterization of the newly isolated lytic bacteriophages KTN6 and KT28 and their efficacy against Pseudomonas aeruginosa biofilm

We here describe two novel lytic phages, KT28 and KTN6, infecting Pseudomonas aeruginosa, isolated from a sewage sample from an irrigated field near Wroclaw, in Poland. Both viruses show characteristic features of Pbunalikevirus genus within the Myoviridae family with respect to shape and size of head/tail, as well as LPS host receptor recognition. Genome analysis confirmed the similarity to other PB1-related phages, ranging between 48 and 96%. Pseudomonas phage KT28 has a genome size of 66,381 bp and KTN6 of 65,994 bp. The latent period, burst size, stability and host range was determined for both viruses under standard laboratory conditions. Biofilm eradication efficacy was tested on peg-lid plate assay and PET membrane surface. Significant reduction of colony forming units was observed (70-90%) in 24 h to 72 h old Pseudomonas aeruginosa PAO1 biofilm cultures for both phages. Furthermore, a pyocyanin and pyoverdin reduction tests reveal that tested phages lowers the amount of both secreted dyes in 48-72 h old biofilms. Diffusion and goniometry experiments revealed the increase of diffusion rate through the biofilm matrix after phage application. These characteristics indicate these phages could be used to prevent Pseudomonas aeruginosa infections and biofilm formation. It was also shown, that PB1-related phage treatment of biofilm caused the emergence of stable phage-resistant mutants growing as small colony variants

The evolutionary signal in metagenome phyletic profiles predicts many gene functions

Background. The function of many genes is still not known even in model organisms. An increasing availability of microbiome DNA sequencing data provides an opportunity to infer gene function in a systematic manner. Results. We evaluated if the evolutionary signal contained in metagenome phyletic profiles (MPP) is predictive of a broad array of gene functions. The MPPs are an encoding of environmental DNA sequencing data that consists of relative abundances of gene families across metagenomes. We find that such MPPs can accurately predict 826 Gene Ontology functional categories, while drawing on human gut microbiomes, ocean metagenomes, and DNA sequences from various other engineered and natural environments. Overall, in this task, the MPPs are highly accurate, and moreover they provide coverage for a set of Gene Ontology terms largely complementary to standard phylogenetic profiles, derived from fully sequenced genomes. We also find that metagenomes approximated from taxon relative abundance obtained via 16S rRNA gene sequencing may provide surprisingly useful predictive models. Crucially, the MPPs derived from different types of environments can infer distinct, non-overlapping sets of gene functions and therefore complement each other. Consistently, simulations on > 5000 metagenomes indicate that the amount of data is not in itself critical for maximizing predictive accuracy, while the diversity of sampled environments appears to be the critical factor for obtaining robust models. Conclusions. In past work, metagenomics has provided invaluable insight into ecology of various habitats, into diversity of microbial life and also into human health and disease mechanisms. We propose that environmental DNA sequencing additionally constitutes a useful tool to predict biological roles of genes, yielding inferences out of reach for existing comparative genomics approaches

16S rRNA Gene Metabarcoding Indicates Species-Characteristic Microbiomes in Deep-Sea Benthic Foraminifera

Foraminifera are unicellular eukaryotes that are an integral part of benthic fauna in many marine ecosystems, including the deep sea, with direct impacts on benthic biogeochemical cycles. In these systems, different foraminiferal species are known to have a distinct vertical distribution, i.e., microhabitat preference, which is tightly linked to the physico-chemical zonation of the sediment. Hence, foraminifera are well-adapted to thrive in various conditions, even under anoxia. However, despite the ecological and biogeochemical significance of foraminifera, their ecology remains poorly understood. This is especially true in terms of the composition and diversity of their microbiome, although foraminifera are known to harbor diverse endobionts, which may have a significant meaning to each species' survival strategy. In this study, we used 16S rRNA gene metabarcoding to investigate the microbiomes of five different deep-sea benthic foraminiferal species representing differing microhabitat preferences. The microbiomes of these species were compared intra- and inter-specifically, as well as with the surrounding sediment bacterial community. Our analysis indicated that each species was characterized with a distinct, statistically different microbiome that also differed from the surrounding sediment community in terms of diversity and dominant bacterial groups. We were also able to distinguish specific bacterial groups that seemed to be strongly associated with particular foraminiferal species, such as the family Marinilabiliaceae for Chilostomella ovoidea and the family Hyphomicrobiaceae for Bulimina subornata and Bulimina striata. The presence of bacterial groups that are tightly associated to a certain foraminiferal species implies that there may exist unique, potentially symbiotic relationships between foraminifera and bacteria that have been previously overlooked. Furthermore, the foraminifera contained chloroplast reads originating from different sources, likely reflecting trophic preferences and ecological characteristics of the different species. This study demonstrates the potential of 16S rRNA gene metabarcoding in resolving the microbiome composition and diversity of eukaryotic unicellular organisms, providing unique in situ insights into enigmatic deep-sea ecosystems.Peer reviewe

Physiological ecology of Trichodesmium and its microbiome in the oligotrophic ocean

The colonial, N2 fixing cyanobacterium Trichodesmium is a keystone species in oligotrophic ocean ecosystems. Trichodesmium is responsible for approximately 50% of the total biologically fixed N2 in the ocean, and this “new” nitrogen fuels primary productivity and the amount of carbon sequestered by the ocean. Trichodesmium does not exist in isolation. Colonies occur ubiquitously with an assemblage of epibiotic microorganisms that are distinct from planktonic microbes and modulated across environments, yet the implications of this relationship have not been explored. In this thesis, the ecology, physiology, and potential geochemical impact of interactions within the Trichodesmium host-microbiome system were examined across three different oligotrophic ocean environments. First, to establish the metabolic diversity contributed by the microbiome to Trichodesmium consortia, a whole community metagenomic sequencing approach was used across a transect the western North Atlantic. This study demonstrated that the microbiome contributes a large amount of unique functional potential and is modulated across a geochemical gradient. In the following study, metatranscriptomics was used to show that such metabolic potential in Trichodesmium and the microbiome was expressed and modulated across the environment. Colonies were sampled in the western tropical South Pacific and gene expression dynamics indicated co-limitation by iron and phosphorus, and revealed a mechanism for phosphate reduction by Trichodesmium and subsequent utilization by the microbiome. These activities were verified with phosphate reduction rate measurements and indicated cryptic phosphorus cycling within colonies. Next, the suite of potential physiological interactions between host and microbiome was assessed with metatranscriptome sequencing on high frequency samples of Trichodesmium colonies from the North Pacific subtropical gyre. Synchronized day-night gene expression periodicity between consortia members indicated tightly linked metabolisms. The functional annotations of these synchronous genes indicated intra-consortia cycling of nitrogen, phosphorus and iron, as well as a microbiome dependence on Trichodesmium-derived cobalamin—interactions that could alter the transfer of these resources to the surrounding water column. In the final study, the effect of the microbiome on Trichodesmium N2 fixation was assessed. Using colonies obtained from the North Atlantic, activity in the microbiome was selectively modified using quorum sensing acyl homoserine lactone cell-cell signaling, a mechanism that Trichodesmium itself does not possess. These experiments indicated that the microbiome has the potential to increase or decrease Trichodesmium N2 fixation to a degree that rivals the effects of alterations in nutrient concentration, but at a more rapid rate. In all, the research presented in this thesis demonstrates the integral importance of the microbiome to Trichodesmium physiology and ecology, highlighting the importance of an unexplored facet of marine microbial systems that likely influences the biogeochemistry of the planet

The first steps of adaptation of Escherichia coli to the gut are dominated by soft sweeps

The accumulation of adaptive mutations is essential for survival in novel environments. However, in clonal populations with a high mutational supply, the power of natural selection is expected to be limited. This is due to clonal interference - the competition of clones carrying different beneficial mutations - which leads to the loss of many small effect mutations and fixation of large effect ones. If interference is abundant, then mechanisms for horizontal transfer of genes, which allow the immediate combination of beneficial alleles in a single background, are expected to evolve. However, the relevance of interference in natural complex environments, such as the gut, is poorly known. To address this issue, we studied the invasion of beneficial mutations responsible for Escherichia coli's adaptation to the mouse gut and demonstrate the pervasiveness of clonal interference. The observed dynamics of change in frequency of beneficial mutations are consistent with soft sweeps, where a similar adaptive mutation arises repeatedly on different haplotypes without reaching fixation. The genetic basis of the adaptive mutations revealed a striking parallelism in independently evolving populations. This was mainly characterized by the insertion of transposable elements in both coding and regulatory regions of a few genes. Interestingly in most populations, we observed a complete phenotypic sweep without loss of genetic variation. The intense clonal interference during adaptation to the gut environment, here demonstrated, may be important for our understanding of the levels of strain diversity of E. coli inhabiting the human gut microbiota and of its recombination rate.Howard Hughes Medical Institute (HHMI-55007436), LAO/ITQB, FCT Grants: FRH/BD/80257/2011 and SFRH/BPD/14299/2003