Unipept Desktop 2.0: Construction of Targeted Reference Protein Databases for Metaproteogenomics Analyses.

Unipept Desktop 2.0 is the most recent iteration of the Unipept Desktop tool that adds support for the analysis of metaproteogenomics datasets. Unipept Desktop now supports the automatic construction of targeted protein reference databases that only contain proteins (originating from the UniProtKB resource) associated with a predetermined list of taxa. This improves both the taxonomic and functional resolution of a metaproteomic analysis and yields several technical advantages. By limiting the proteins present in a reference database, it is also possible to perform (meta)proteogenomics analyses. Since the protein reference database resides on the user's local machine, they have complete control over the database used during an analysis. Data no longer need to be transmitted over the Internet, decreasing the time required for an analysis and better safeguarding privacy-sensitive data. As a proof of concept, we present a case study in which a human gut metaproteome dataset is analyzed with Unipept Desktop 2.0 using different targeted databases based on matched 16S rRNA gene sequencing data.

FragGeneScanRs: faster gene prediction for short reads.

BACKGROUND: FragGeneScan is currently the most accurate and popular tool for gene prediction in short and error-prone reads, but its execution speed is insufficient for use on larger data sets. The parallelization which should have addressed this is inefficient. Its alternative implementation FragGeneScan+ is faster, but introduced a number of bugs related to memory management, race conditions and even output accuracy. RESULTS: This paper introduces FragGeneScanRs, a faster Rust implementation of the FragGeneScan gene prediction model. Its command line interface is backward compatible and adds extra features for more flexible usage. Its output is equivalent to the original FragGeneScan implementation. CONCLUSIONS: Compared to the current C implementation, shotgun metagenomic reads are processed up to 22 times faster using a single thread, with better scaling for multithreaded execution. The Rust code of FragGeneScanRs is freely available from GitHub under the GPL-3.0 license with instructions for installation, usage and other documentation ( https://github.com/unipept/FragGeneScanRs ).

UMGAP: the Unipept MetaGenomics Analysis Pipeline.

BACKGROUND: Shotgun metagenomics yields ever richer and larger data volumes on the complex communities living in diverse environments. Extracting deep insights from the raw reads heavily depends on the availability of fast, accurate and user-friendly biodiversity analysis tools. RESULTS: Because environmental samples may contain strains and species that are not covered in reference databases and because protein sequences are more conserved than the genes encoding them, we explore the alternative route of taxonomic profiling based on protein coding regions translated from the shotgun metagenomics reads, instead of directly processing the DNA reads. We therefore developed the Unipept MetaGenomics Analysis Pipeline (UMGAP), a highly versatile suite of open source tools that are implemented in Rust and support parallelization to achieve optimal performance. Six preconfigured pipelines with different performance trade-offs were carefully selected, and benchmarked against a selection of state-of-the-art shotgun metagenomics taxonomic profiling tools. CONCLUSIONS: UMGAP's protein space detour for taxonomic profiling makes it competitive with state-of-the-art shotgun metagenomics tools. Despite our design choices of an extra protein translation step, a broad spectrum index that can identify both archaea, bacteria, eukaryotes and viruses, and a highly configurable non-monolithic design, UMGAP achieves low runtime, manageable memory footprint and high accuracy. Its interactive visualizations allow for easy exploration and comparison of complex communities.

Unipept Visualizations: an interactive visualization library for biological data.

SUMMARY: The Unipept Visualizations library is a JavaScript package to generate interactive visualizations of both hierarchical and non-hierarchical quantitative data. It provides four different visualizations: a sunburst, a treemap, a treeview and a heatmap. Every visualization is fully configurable, supports TypeScript and uses the excellent D3.js library. AVAILABILITY AND IMPLEMENTATION: The Unipept Visualizations library is available for download on NPM: https://npmjs.com/unipept-visualizations. All source code is freely available from GitHub under the MIT license: https://github.com/unipept/unipept-visualizations.

Unipept Desktop: A Faster, More Powerful Metaproteomics Results Analysis Tool.

Metaproteomics has become an important research tool to study microbial systems, which has resulted in increased metaproteomics data generation. However, efficient tools for processing the acquired data have lagged behind. One widely used tool for metaproteomics data interpretation is Unipept, a web-based tool that provides, among others, interactive and insightful visualizations. Due to its web-based implementation, however, the Unipept web application is limited in the amount of data that can be analyzed. In this manuscript we therefore present Unipept Desktop, a desktop application version of Unipept that is designed to drastically increase the throughput and capacity of metaproteomics data analysis. Moreover, it provides a novel comparative analysis pipeline and improves the organization of experimental data into projects, thus addressing the growing need for more efficient and versatile analysis tools for metaproteomics data.

Unipept CLI 2.0: adding support for visualizations and functional annotations.

SUMMARY: Unipept is an ecosystem of tools developed for fast metaproteomics data-analysis consisting of a web application, a set of web services (application programming interface, API) and a command-line interface (CLI). After the successful introduction of version 4 of the Unipept web application, we here introduce version 2.0 of the API and CLI. Next to the existing taxonomic analysis, version 2.0 of the API and CLI provides access to Unipept's powerful functional analysis for metaproteomics samples. The functional analysis pipeline supports retrieval of Enzyme Commission numbers, Gene Ontology terms and InterPro entries for the individual peptides in a metaproteomics sample. This paves the way for other applications and developers to integrate these new information sources into their data processing pipelines, which greatly increases insight into the functions performed by the organisms in a specific environment. Both the API and CLI have also been expanded with the ability to render interactive visualizations from a list of taxon ids. These visualizations are automatically made available on a dedicated website and can easily be shared by users. AVAILABILITY AND IMPLEMENTATION: The API is available at http://api.unipept.ugent.be. Information regarding the CLI can be found at https://unipept.ugent.be/clidocs. Both interfaces are freely available and open-source under the MIT license. CONTACT: pieter.verschaffelt@ugent.be. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Connecting MetaProteomeAnalyzer and PeptideShaker to Unipept for Seamless End-to-End Metaproteomics Data Analysis.

Although metaproteomics, the study of the collective proteome of microbial communities, has become increasingly powerful and popular over the past few years, the field has lagged behind on the availability of user-friendly, end-to-end pipelines for data analysis. We therefore describe the connection from two commonly used metaproteomics data processing tools in the field, MetaProteomeAnalyzer and PeptideShaker, to Unipept for downstream analysis. Through these connections, direct end-to-end pipelines are built from database searching to taxonomic and functional annotation.

Introducing SPeDE: High-Throughput Dereplication and Accurate Determination of Microbial Diversity from Matrix-Assisted Laser Desorption-Ionization Time of Flight Mass Spectrometry Data.

The isolation of microorganisms from microbial community samples often yields a large number of conspecific isolates. Increasing the diversity covered by an isolate collection entails the implementation of methods and protocols to minimize the number of redundant isolates. Matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry methods are ideally suited to this dereplication problem because of their low cost and high throughput. However, the available software tools are cumbersome and rely either on the prior development of reference databases or on global similarity analyses, which are inconvenient and offer low taxonomic resolution. We introduce SPeDE, a user-friendly spectral data analysis tool for the dereplication of MALDI-TOF mass spectra. Rather than relying on global similarity approaches to classify spectra, SPeDE determines the number of unique spectral features by a mix of global and local peak comparisons. This approach allows the identification of a set of nonredundant spectra linked to operational isolation units. We evaluated SPeDE on a data set of 5,228 spectra representing 167 bacterial strains belonging to 132 genera across six phyla and on a data set of 312 spectra of 78 strains measured before and after lyophilization and subculturing. SPeDE was able to dereplicate with high efficiency by identifying redundant spectra while retrieving reference spectra for all strains in a sample. SPeDE can identify distinguishing features between spectra, and its performance exceeds that of established methods in speed and precision. SPeDE is open source under the MIT license and is available from https://github.com/LM-UGent/SPeDEIMPORTANCE Estimation of the operational isolation units present in a MALDI-TOF mass spectral data set involves an essential dereplication step to identify redundant spectra in a rapid manner and without sacrificing biological resolution. We describe SPeDE, a new algorithm which facilitates culture-dependent clinical or environmental studies. SPeDE enables the rapid analysis and dereplication of isolates, a critical feature when long-term storage of cultures is limited or not feasible. We show that SPeDE can efficiently identify sets of similar spectra at the level of the species or strain, exceeding the taxonomic resolution of other methods. The high-throughput capacity, speed, and low cost of MALDI-TOF mass spectrometry and SPeDE dereplication over traditional gene marker-based sequencing approaches should facilitate adoption of the culturomics approach to bacterial isolation campaigns.

A Method for Comprehensive Proteomic Analysis of Human Faecal Samples to Investigate Gut Dysbiosis in Patients with Cystic Fibrosis.

BACKGROUND: This chapter reports the evaluation of two shotgun metaproteomic workflows. The methods were developed to investigate gut dysbiosis via analysis of the faecal microbiota from patients with cystic fibrosis (CF). We aimed to set up an unbiased and effective method to extract the entire proteome, i.e. to extract sufficient bacterial proteins from the faecal samples in combination with a maximum of host proteins giving information on the disease state. METHODS: Two protocols were compared; the first method involves an enrichment of the bacterial proteins while the second method is a more direct method to generate a whole faecal proteome extract. The different extracts were analysed using denaturing polyacrylamide gel electrophoresis followed by liquid chromatography-tandem mass spectrometry aiming a maximal coverage of the bacterial protein content in faecal samples. RESULTS AND CONCLUSIONS: In all extracts, microbial proteins are detected, and in addition, nonbacterial proteins are detected in all samples providing information about the host status. Our study demonstrates the huge influence of the used protein extraction method on the obtained result and shows the need for a standardised and appropriate sample preparation for metaproteomic analysis. To address questions on the health status of the patients, a whole protein extract is preferred over a method to enrich the bacterial fraction. In addition, the method of the whole protein fraction is faster, which gives the possibility to analyse more biological replicates.

Unipept 4.0: Functional Analysis of Metaproteome Data.

Unipept ( https://unipept.ugent.be ) is a web application for metaproteome data analysis, with an initial focus on tryptic-peptide-based biodiversity analysis of MS/MS samples. Because the true potential of metaproteomics lies in gaining insight into the expressed functions of complex environmental samples, the 4.0 release of Unipept introduces complementary functional analysis based on GO terms and EC numbers. Integration of this new functional analysis with the existing biodiversity analysis is an important asset of the extended pipeline. As a proof of concept, a human faecal metaproteome data set from 15 healthy subjects was reanalyzed with Unipept 4.0, yielding fast, detailed, and straightforward characterization of taxon-specific catalytic functions that is shown to be consistent with previous results from a BLAST-based functional analysis of the same data.

Multifunctional sequence-defined macromolecules for chemical data storage.

Sequence-defined macromolecules consist of a defined chain length (single mass), end-groups, composition and topology and prove promising in application fields such as anti-counterfeiting, biological mimicking and data storage. Here we show the potential use of multifunctional sequence-defined macromolecules as a storage medium. As a proof-of-principle, we describe how short text fragments (human-readable data) and QR codes (machine-readable data) are encoded as a collection of oligomers and how the original data can be reconstructed. The amide-urethane containing oligomers are generated using an automated protecting-group free, two-step iterative protocol based on thiolactone chemistry. Tandem mass spectrometry techniques have been explored to provide detailed analysis of the oligomer sequences. We have developed the generic software tools Chemcoder for encoding/decoding binary data as a collection of multifunctional macromolecules and Chemreader for reconstructing oligomer sequences from mass spectra to automate the process of chemical writing and reading.

High-throughput metaproteomics data analysis with Unipept: A tutorial.

In recent years, shotgun metaproteomics has established itself as an important tool to study the composition of complex ecosystems and microbial communities. Two key steps in metaproteomics data analysis are the inference of proteins from the identified peptides, and the determination of the taxonomic origin and function of these proteins. This tutorial therefore introduces the Unipept command line interface (http://unipept.ugent.be/clidocs) as a platform-independent tool for such metaproteomics data analyses. First, a detailed overview is given of the available Unipept commands and their functions. Next, the power of the Unipept command line interface is illustrated using two case studies that analyze a single tryptic peptide, and a set of peptides retrieved from a shotgun metaproteomics experiment, respectively. Finally, the analysis results obtained using these command line tools are compared with the interactive taxonomic analysis that is available on the Unipept website.

Temporal Dynamics of Bacterial and Fungal Colonization on Plastic Debris in the North Sea.

Despite growing evidence that biofilm formation on plastic debris in the marine environment may be essential for its biodegradation, the underlying processes have yet to be fully understood. Thus, far, bacterial biofilm formation had only been studied after short-term exposure or on floating plastic, yet a prominent share of plastic litter accumulates on the seafloor. In this study, we explored the taxonomic composition of bacterial and fungal communities on polyethylene plastic sheets and dolly ropes during long-term exposure on the seafloor, both at a harbor and an offshore location in the Belgian part of the North Sea. We reconstructed the sequence of events during biofilm formation on plastic in the harbor environment and identified a core bacteriome and subsets of bacterial indicator species for early, intermediate, and late stages of biofilm formation. Additionally, by implementing ITS2 metabarcoding on plastic debris, we identified and characterized for the first time fungal genera on plastic debris. Surprisingly, none of the plastics exposed to offshore conditions displayed the typical signature of a late stage biofilm, suggesting that biofilm formation is severely hampered in the natural environment where most plastic debris accumulates.

The unique peptidome: Taxon-specific tryptic peptides as biomarkers for targeted metaproteomics.

The Unique Peptide Finder (http://unipept.ugent.be/peptidefinder) is an interactive web application to quickly hunt for tryptic peptides that are unique to a particular species, genus, or any other taxon. Biodiversity within the target taxon is represented by a set of proteomes selected from a monthly updated list of complete and nonredundant UniProt proteomes, supplemented with proprietary proteomes loaded into persistent local browser storage. The software computes and visualizes pan and core peptidomes as unions and intersections of tryptic peptides occurring in the selected proteomes. In addition, it also computes and displays unique peptidomes as the set of all tryptic peptides that occur in all selected proteomes but not in any UniProt record not assigned to the target taxon. As a result, the unique peptides can serve as robust biomarkers for the target taxon, for example, in targeted metaproteomics studies. Computations are extremely fast since they are underpinned by the Unipept database, the lowest common ancestor algorithm implemented in Unipept and modern web technologies that facilitate in-browser data storage and parallel processing.

Global alternans instability and its effect on non-linear wave propagation: dynamical Wenckebach block and self terminating spiral waves.

The main mechanism of formation of reentrant cardiac arrhythmias is via formation of waveblocks at heterogeneities of cardiac tissue. We report that heterogeneity and the area of waveblock can extend itself in space and can result formation of new additional sources, or termination of existing sources of arrhythmias. This effect is based on a new form of instability, which we coin as global alternans instability (GAI). GAI is closely related to the so-called (discordant) alternans instability, however its onset is determined by the global properties of the APD-restitution curve and not by its slope. The APD-restitution curve relates the duration of the cardiac pulse (APD) to the time interval between the pulses, and can easily be measured in an experimental or even clinical setting. We formulate the conditions for the onset of GAI, study its manifestation in various 1D and 2D situations and discuss its importance for the onset of cardiac arrhythmias.

Unipept web services for metaproteomics analysis.

UNLABELLED: Unipept is an open source web application that is designed for metaproteomics analysis with a focus on interactive datavisualization. It is underpinned by a fast index built from UniProtKB and the NCBI taxonomy that enables quick retrieval of all UniProt entries in which a given tryptic peptide occurs. Unipept version 2.4 introduced web services that provide programmatic access to the metaproteomics analysis features. This enables integration of Unipept functionality in custom applications and data processing pipelines. AVAILABILITY AND IMPLEMENTATION: The web services are freely available at http://api.unipept.ugent.be and are open sourced under the MIT license. CONTACT: Unipept@ugent.be SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Dynamics in the Strawberry Rhizosphere Microbiome in Response to Biochar and Botrytis cinerea Leaf Infection.

Adding biochar, the solid coproduct of biofuel production, to peat can enhance strawberry growth, and disease resistance against the airborne fungal pathogen Botrytis cinerea. Additionally, biochar can induce shifts in the strawberry rhizosphere microbiome. However, the moment that this biochar-mediated shift occurs in the rhizosphere is not known. Further, the effect of an above-ground infection on the strawberry rhizosphere microbiome is unknown. In the present study we established two experiments in which strawberry transplants (cv. Elsanta) were planted either in peat or in peat amended with 3% biochar. First, we established a time course experiment to measure the effect of biochar on the rhizosphere bacterial and fungal communities over time. In a second experiment, we inoculated the strawberry leaves with B. cinerea, and studied the impact of the infection on the rhizosphere bacterial community. The fungal rhizosphere community was stabilized after 1 week, except for the upcoming Auriculariales, whereas the bacterial community shifted till 6 weeks. An effect of the addition of biochar to the peat on the rhizosphere microbiome was solely measured for the bacterial community from week 6 of plant growth onwards. When scoring the plant development, biochar addition was associated with enhanced root formation, fruit production, and postharvest resistance of the fruits against B. cinerea. We hypothesize that the bacterial rhizosphere microbiome, but also biochar-mediated changes in chemical substrate composition could be involved in these events. Infection of the strawberry leaves with B. cinerea induced shifts in the bacterial rhizosphere community, with an increased bacterial richness. This disease-induced effect was not observed in the rhizospheres of the B. cinerea-infected plants grown in the biochar-amended peat. The results show that an above-ground infection has its effect on the strawberry rhizosphere microbiome, changing the bacterial interactions in the root-substrate interface. This infection effect on the bacterial rhizosphere microbiome seems to be comparable to, but less pronounced than the effect of biochar-addition to the peat. The biological meaning of these observations needs further research, but this study indicates that biochar and an above-ground pathogen attack help the plant to recruit rhizosphere microbes that may aid them in their plant growth and health.

Faecal proteomics: A tool to investigate dysbiosis and inflammation in patients with cystic fibrosis.

BACKGROUND: Several microbial studies reported gut microbiota dysbiosis in patients with cystic fibrosis (CF). The functional consequences of this phenomenon are poorly understood. Faecal metaproteomics allows the quantitative analysis of host and microbial proteins to address functional changes resulting from this dysbiosis. METHODS: We analysed faecal protein extracts from fifteen patients with CF that have pancreatic insufficiency and from their unaffected siblings by shotgun proteomics. Novel computational and statistical tools were introduced to evaluate changes in taxonomic composition and protein abundance. RESULTS: Faecal protein extracts from patients with CF were dominated by host proteins involved in inflammation and mucus formation. Taxonomic analysis of the microbial proteins confirmed the strong reduction of butyrate reducers such as Faecalibacterium prausnitzii and increase of Enterobacteriaceae, Ruminococcus gnavus and Clostridia species. CONCLUSION: Faecal metaproteomics provides insights in intestinal dysbiosis, inflammation in patients with CF and can be used to monitor different disease markers in parallel.

Bacterial Community Profiling of Plastic Litter in the Belgian Part of the North Sea.

Bacterial colonization of marine plastic litter (MPL) is known for over four decades. Still, only a few studies on the plastic colonization process and its influencing factors are reported. In this study, seafloor MPL was sampled at different locations across the Belgian part of the North Sea to study bacterial community structure using 16S metabarcoding. These marine plastic bacterial communities were compared with those of sediment and seawater, and resin pellets sampled on the beach, to investigate the origin and uniqueness of plastic bacterial communities. Plastics display great variation of bacterial community composition, while each showed significant differences from those of sediment and seawater, indicating that plastics represent a distinct environmental niche. Various environmental factors correlate with the diversity of MPL bacterial composition across plastics. In addition, intrinsic plastic-related factors such as pigment content may contribute to the differences in bacterial colonization. Furthermore, the differential abundance of known primary and secondary colonizers across the various plastics may indicate different stages of bacterial colonization, and may confound comparisons of free-floating plastics. Our studies provide insights in the factors that shape plastic bacterial colonization and shed light on the possible role of plastic as transport vehicle for bacteria through the aquatic environment.

A Long Fragment Aligner called ALFALFA.

BACKGROUND: Rapid evolutions in sequencing technology force read mappers into flexible adaptation to longer reads, changing error models, memory barriers and novel applications. RESULTS: ALFALFA achieves a high performance in accurately mapping long single-end and paired-end reads to gigabase-scale reference genomes, while remaining competitive for mapping shorter reads. Its seed-and-extend workflow is underpinned by fast retrieval of super-maximal exact matches from an enhanced sparse suffix array, with flexible parameter tuning to balance performance, memory footprint and accuracy. CONCLUSIONS: ALFALFA is open source and available at http://alfalfa.ugent.be .