Catching the Wave: Detecting Strain-Specific SARS-CoV-2 Peptides in Clinical Samples Collected during Infection Waves from Diverse Geographical Locations.

The Coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) resulted in a major health crisis worldwide with its continuously emerging new strains, resulting in new viral variants that drive "waves" of infection. PCR or antigen detection assays have been routinely used to detect clinical infections; however, the emergence of these newer strains has presented challenges in detection. One of the alternatives has been to detect and characterize variant-specific peptide sequences from viral proteins using mass spectrometry (MS)-based methods. MS methods can potentially help in both diagnostics and vaccine development by understanding the dynamic changes in the viral proteome associated with specific strains and infection waves. In this study, we developed an accessible, flexible, and shareable bioinformatics workflow that was implemented in the Galaxy Platform to detect variant-specific peptide sequences from MS data derived from the clinical samples. We demonstrated the utility of the workflow by characterizing published clinical data from across the world during various pandemic waves. Our analysis identified six SARS-CoV-2 variant-specific peptides suitable for confident detection by MS in commonly collected clinical samples.

Impact of high platelet turnover on the platelet transcriptome: Results from platelet RNA-sequencing in patients with sepsis.

BACKGROUND: Sepsis is associated with high platelet turnover and elevated levels of immature platelets. Changes in the platelet transcriptome and the specific impact of immature platelets on the platelet transcriptome remain unclear. Thus, this study sought to address whether and how elevated levels of immature platelets affect the platelet transcriptome in patients with sepsis. METHODS: Blood samples were obtained from patients with sepsis requiring vasopressor therapy (n = 8) and from a control group of patients with stable coronary artery disease and otherwise similar demographic characteristics (n = 8). Immature platelet fraction (IPF) was determined on a Sysmex XE 2100 analyser and platelet function was tested by impedance aggregometry. RNA from leukocyte-depleted platelets was used for transcriptome analysis by Next Generation Sequencing integrating the use of unique molecular identifiers. RESULTS: IPF (median [interquartile range]) was significantly elevated in sepsis patients (6.4 [5.3-8.7] % vs. 3.6 [2.6-4.6] %, p = 0.005). Platelet function testing revealed no differences in adenosine diphosphate- or thrombin receptor activating peptide-induced platelet aggregation between control and sepsis patients. Putative circular RNA transcripts were decreased in platelets from septic patients. Leukocyte contamination defined by CD45 abundance levels in RNA-sequencing was absent in both groups. Principal component analysis of transcripts showed only partial overlap of clustering with IPF levels. RNA sequencing showed up-regulation of 524 and down-regulation of 118 genes in platelets from sepsis patients compared to controls. Upregulated genes were mostly related to catabolic processes and protein translation. Comparison to published platelet transcriptomes showed a large overlap of changes observed in sepsis and COVID-19 but not with reticulated platelets from healthy donors. CONCLUSIONS: Patients with sepsis appear to have a less degraded platelet transcriptome as indicated by increased levels of immature platelets and decreased levels of putative circular RNA transcripts. The present data suggests that increased protein translation is a characteristic mechanism of systemic inflammation.

Fostering accessible online education using Galaxy as an e-learning platform.

The COVID-19 pandemic is shifting teaching to an online setting all over the world. The Galaxy framework facilitates the online learning process and makes it accessible by providing a library of high-quality community-curated training materials, enabling easy access to data and tools, and facilitates sharing achievements and progress between students and instructors. By combining Galaxy with robust communication channels, effective instruction can be designed inclusively, regardless of the students' environments.

A rigorous evaluation of optimal peptide targets for MS-based clinical diagnostics of Coronavirus Disease 2019 (COVID-19).

The Coronavirus Disease 2019 (COVID-19) global pandemic has had a profound, lasting impact on the world's population. A key aspect to providing care for those with COVID-19 and checking its further spread is early and accurate diagnosis of infection, which has been generally done via methods for amplifying and detecting viral RNA molecules. Detection and quantitation of peptides using targeted mass spectrometry-based strategies has been proposed as an alternative diagnostic tool due to direct detection of molecular indicators from non-invasively collected samples as well as the potential for high-throughput analysis in a clinical setting; many studies have revealed the presence of viral peptides within easily accessed patient samples. However, evidence suggests that some viral peptides could serve as better indicators of COVID-19 infection status than others, due to potential misidentification of peptides derived from human host proteins, poor spectral quality, high limits of detection etc. In this study we have compiled a list of 639 peptides identified from Sudden Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) samples, including from in vitro and clinical sources. These datasets were rigorously analyzed using automated, Galaxy-based workflows containing tools such as PepQuery, BLAST-P, and the Multi-omic Visualization Platform as well as the open-source tools MetaTryp and Proteomics Data Viewer (PDV). Using PepQuery for confirming peptide spectrum matches, we were able to narrow down the 639 peptide possibilities to 87 peptides which were most robustly detected and specific to the SARS-CoV-2 virus. The specificity of these sequences to coronavirus taxa was confirmed using Unipept and BLAST-P. Applying stringent statistical scoring thresholds, combined with manual verification of peptide spectrum match quality, 4 peptides derived from the nucleocapsid phosphoprotein and membrane protein were found to be most robustly detected across all cell culture and clinical samples, including those collected non-invasively. We propose that these peptides would be of the most value for clinical proteomics applications seeking to detect COVID-19 from a variety of sample types. We also contend that samples taken from the upper respiratory tract and oral cavity have the highest potential for diagnosis of SARS-CoV-2 infection from easily collected patient samples using mass spectrometry-based proteomics assays.

Decitabine Induces Gene Derepression on Monosomic Chromosomes: In Vitro and In Vivo Effects in Adverse-Risk Cytogenetics AML.

Hypomethylating agents (HMA) have become the backbone of nonintensive acute myeloid leukemia/myelodysplastic syndrome (AML/MDS) treatment, also by virtue of their activity in patients with adverse genetics, for example, monosomal karyotypes, often with losses on chromosome 7, 5, or 17. No comparable activity is observed with cytarabine, a cytidine analogue without DNA-hypomethylating properties. As evidence exists for compounding hypermethylation and gene silencing of hemizygous tumor suppressor genes (TSG), we thus hypothesized that this effect may preferentially be reversed by the HMAs decitabine and azacitidine. An unbiased RNA-sequencing approach was developed to interrogate decitabine-induced transcriptome changes in AML cell lines with or without a deletion of chromosomes 7q, 5q or 17p. HMA treatment preferentially upregulated several hemizygous TSG in this genomic region, significantly derepressing endogenous retrovirus (ERV)3-1, with promoter demethylation, enhanced chromatin accessibility, and increased H3K4me3 levels. Decitabine globally reactivated multiple transposable elements, with activation of the dsRNA sensor RIG-I and interferon regulatory factor (IRF)7. Induction of ERV3-1 and RIG-I mRNA was also observed during decitabine treatment in vivo in serially sorted peripheral blood AML blasts. In patient-derived monosomal karyotype AML murine xenografts, decitabine treatment resulted in superior survival rates compared with cytarabine. Collectively, these data demonstrate preferential gene derepression and ERV reactivation in AML with chromosomal deletions, providing a mechanistic explanation that supports the clinical observation of superiority of HMA over cytarabine in this difficult-to-treat patient group. SIGNIFICANCE: These findings unravel the molecular mechanism underlying the intriguing clinical activity of HMAs in AML/MDS patients with chromosome 7 deletions and other monosomal karyotypes.See related commentary by O'Hagan et al., p. 813.

GLASSgo in Galaxy: high-throughput, reproducible and easy-to-integrate prediction of sRNA homologs.

MOTIVATION: The correct prediction of bacterial sRNA homologs is a prerequisite for many downstream analyses based on comparative genomics, but it is frequently challenging due to the short length and distinct heterogeneity of such homologs. GLobal Automatic Small RNA Search go (GLASSgo) is an efficient tool for the prediction of sRNA homologs from a single input query. To make the algorithm available to a broader community, we offer a Docker container along with a free-access web service. For non-computer scientists, the web service provides a user-friendly interface. However, capabilities were lacking so far for batch processing, version control and direct interaction with compatible software applications as a workflow management system can provide. RESULTS: Here, we present GLASSgo 1.5.2, an updated version that is fully incorporated into the workflow management system Galaxy. The improved version contains a new feature for extracting the upstream regions, allowing the search for conserved promoter elements. Additionally, it supports the use of accession numbers instead of the outdated GI numbers, which widens the applicability of the tool. AVAILABILITY AND IMPLEMENTATION: GLASSgo is available at https://github.com/lotts/GLASSgo/ under the MIT license and is accompanied by instruction and application data. Furthermore, it can be installed into any Galaxy instance using the Galaxy ToolShed.

The omics discovery REST interface.

The Omics Discovery Index is an open source platform that can be used to access, discover and disseminate omics datasets. OmicsDI integrates proteomics, genomics, metabolomics, models and transcriptomics datasets. Using an efficient indexing system, OmicsDI integrates different biological entities including genes, transcripts, proteins, metabolites and the corresponding publications from PubMed. In addition, it implements a group of pipelines to estimate the impact of each dataset by tracing the number of citations, reanalysis and biological entities reported by each dataset. Here, we present the OmicsDI REST interface (www.omicsdi.org/ws/) to enable programmatic access to any dataset in OmicsDI or all the datasets for a specific provider (database). Clients can perform queries on the API using different metadata information such as sample details (species, tissues, etc), instrumentation (mass spectrometer, sequencer), keywords and other provided annotations. In addition, we present two different libraries in R and Python to facilitate the development of tools that can programmatically interact with the OmicsDI REST interface.

Galaxy HiCExplorer 3: a web server for reproducible Hi-C, capture Hi-C and single-cell Hi-C data analysis, quality control and visualization.

The Galaxy HiCExplorer provides a web service at https://hicexplorer.usegalaxy.eu. It enables the integrative analysis of chromosome conformation by providing tools and computational resources to pre-process, analyse and visualize Hi-C, Capture Hi-C (cHi-C) and single-cell Hi-C (scHi-C) data. Since the last publication, Galaxy HiCExplorer has been expanded considerably with new tools to facilitate the analysis of cHi-C and to provide an in-depth analysis of Hi-C data. Moreover, it supports the analysis of scHi-C data by offering a broad range of tools. With the help of the standard graphical user interface of Galaxy, presented workflows, extensive documentation and tutorials, novices as well as Hi-C experts are supported in their Hi-C data analysis with Galaxy HiCExplorer.

The ChemicalToolbox: reproducible, user-friendly cheminformatics analysis on the Galaxy platform.

Here, we introduce the ChemicalToolbox, a publicly available web server for performing cheminformatics analysis. The ChemicalToolbox provides an intuitive, graphical interface for common tools for downloading, filtering, visualizing and simulating small molecules and proteins. The ChemicalToolbox is based on Galaxy, an open-source web-based platform which enables accessible and reproducible data analysis. There is already an active Galaxy cheminformatics community using and developing tools. Based on their work, we provide four example workflows which illustrate the capabilities of the ChemicalToolbox, covering assembly of a compound library, hole filling, protein-ligand docking, and construction of a quantitative structure-activity relationship (QSAR) model. These workflows may be modified and combined flexibly, together with the many other tools available, to fit the needs of a particular project. The ChemicalToolbox is hosted on the European Galaxy server and may be accessed via https://cheminformatics.usegalaxy.eu .

Intuitive, reproducible high-throughput molecular dynamics in Galaxy: a tutorial.

This paper is a tutorial developed for the data analysis platform Galaxy. The purpose of Galaxy is to make high-throughput computational data analysis, such as molecular dynamics, a structured, reproducible and transparent process. In this tutorial we focus on 3 questions: How are protein-ligand systems parameterized for molecular dynamics simulation? What kind of analysis can be carried out on molecular trajectories? How can high-throughput MD be used to study multiple ligands? After finishing you will have learned about force-fields and MD parameterization, how to conduct MD simulation and analysis for a protein-ligand system, and understand how different molecular interactions contribute to the binding affinity of ligands to the Hsp90 protein.

Software engineering for scientific big data analysis.

The increasing complexity of data and analysis methods has created an environment where scientists, who may not have formal training, are finding themselves playing the impromptu role of software engineer. While several resources are available for introducing scientists to the basics of programming, researchers have been left with little guidance on approaches needed to advance to the next level for the development of robust, large-scale data analysis tools that are amenable to integration into workflow management systems, tools, and frameworks. The integration into such workflow systems necessitates additional requirements on computational tools, such as adherence to standard conventions for robustness, data input, output, logging, and flow control. Here we provide a set of 10 guidelines to steer the creation of command-line computational tools that are usable, reliable, extensible, and in line with standards of modern coding practices.

Uncontrolled Diabetes Mellitus Has No Major Influence on the Platelet Transcriptome.

BACKGROUND: Diabetes mellitus (DM) has been associated with increased platelet reactivity as well as increased levels of platelet RNAs in plasma. Here, we sought to evaluate whether the platelet transcriptome is altered in the presence of uncontrolled DM. METHODS: Next-generation sequencing (NGS) was performed on platelet RNA for 5 patients with uncontrolled DM (HbA1c 9.0%) and 5 control patients (HbA1c 5.5%) with otherwise similar clinical characteristics. RNA was isolated from leucocyte-depleted platelet-rich plasma. Libraries of platelet RNAs were created separately for long RNAs after ribosomal depletion and for small RNAs from total RNA, followed by next-generation sequencing. RESULTS: Platelets in both groups demonstrated RNA expression profiles characterized by absence of leukocyte-specific transcripts, high expression of well-known platelet transcripts, and in total 6,343 consistently detectable transcripts. Extensive statistical bioinformatic analysis yielded 12 genes with consistently differential expression at a lenient FDR < 0.1, thereof 8 protein-coding genes and 2 genes with known expression in platelets (MACF1 and ITGB3BP). Three of the four differentially expressed noncoding genes were YRNAs (RNY1, RNY3, and RNY4) which were all downregulated in DM. 23 miRNAs were differentially expressed between the two groups. Of the 13 miRNAs with decreased expression in the diabetic group, 8 belonged to the DLK1-DIO3 gene region on chromosome 14q32.2. CONCLUSIONS: In this study, uncontrolled DM had a remote impact on different components of the platelet transcriptome. Increased expression of MACF1, together with supporting predicted mRNA-miRNA interactions as well as reduced expression of RNYs in platelets, may reflect subclinical platelet activation in uncontrolled DM.

Galaxy HiCExplorer: a web server for reproducible Hi-C data analysis, quality control and visualization.

Galaxy HiCExplorer is a web server that facilitates the study of the 3D conformation of chromatin by allowing Hi-C data processing, analysis and visualization. With the Galaxy HiCExplorer web server, users with little bioinformatic background can perform every step of the analysis in one workflow: mapping of the raw sequence data, creation of Hi-C contact matrices, quality assessment, correction of contact matrices and identification of topological associated domains (TADs) and A/B compartments. Users can create publication ready plots of the contact matrix, A/B compartments, and TADs on a selected genomic locus, along with additional information like gene tracks or ChIP-seq signals. Galaxy HiCExplorer is freely usable at: https://hicexplorer.usegalaxy.eu and is available as a Docker container: https://github.com/deeptools/docker-galaxy-hicexplorer.

The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update.

Galaxy (homepage: https://galaxyproject.org, main public server: https://usegalaxy.org) is a web-based scientific analysis platform used by tens of thousands of scientists across the world to analyze large biomedical datasets such as those found in genomics, proteomics, metabolomics and imaging. Started in 2005, Galaxy continues to focus on three key challenges of data-driven biomedical science: making analyses accessible to all researchers, ensuring analyses are completely reproducible, and making it simple to communicate analyses so that they can be reused and extended. During the last two years, the Galaxy team and the open-source community around Galaxy have made substantial improvements to Galaxy's core framework, user interface, tools, and training materials. Framework and user interface improvements now enable Galaxy to be used for analyzing tens of thousands of datasets, and >5500 tools are now available from the Galaxy ToolShed. The Galaxy community has led an effort to create numerous high-quality tutorials focused on common types of genomic analyses. The Galaxy developer and user communities continue to grow and be integral to Galaxy's development. The number of Galaxy public servers, developers contributing to the Galaxy framework and its tools, and users of the main Galaxy server have all increased substantially.

Distinct epigenetic programs regulate cardiac myocyte development and disease in the human heart in vivo.

Epigenetic mechanisms and transcription factor networks essential for differentiation of cardiac myocytes have been uncovered. However, reshaping of the epigenome of these terminally differentiated cells during fetal development, postnatal maturation, and in disease remains unknown. Here, we investigate the dynamics of the cardiac myocyte epigenome during development and in chronic heart failure. We find that prenatal development and postnatal maturation are characterized by a cooperation of active CpG methylation and histone marks at cis-regulatory and genic regions to shape the cardiac myocyte transcriptome. In contrast, pathological gene expression in terminal heart failure is accompanied by changes in active histone marks without major alterations in CpG methylation and repressive chromatin marks. Notably, cis-regulatory regions in cardiac myocytes are significantly enriched for cardiovascular disease-associated variants. This study uncovers distinct layers of epigenetic regulation not only during prenatal development and postnatal maturation but also in diseased human cardiac myocytes.

High-resolution TADs reveal DNA sequences underlying genome organization in flies.

Despite an abundance of new studies about topologically associating domains (TADs), the role of genetic information in TAD formation is still not fully understood. Here we use our software, HiCExplorer (hicexplorer.readthedocs.io) to annotate >2800 high-resolution (570 bp) TAD boundaries in Drosophila melanogaster. We identify eight DNA motifs enriched at boundaries, including a motif bound by the M1BP protein, and two new boundary motifs. In contrast to mammals, the CTCF motif is only enriched on a small fraction of boundaries flanking inactive chromatin while most active boundaries contain the motifs bound by the M1BP or Beaf-32 proteins. We demonstrate that boundaries can be accurately predicted using only the motif sequences at open chromatin sites. We propose that DNA sequence guides the genome architecture by allocation of boundary proteins in the genome. Finally, we present an interactive online database to access and explore the spatial organization of fly, mouse and human genomes, available at http://chorogenome.ie-freiburg.mpg.de .

DNA methylation signatures follow preformed chromatin compartments in cardiac myocytes.

Storage of chromatin in restricted nuclear space requires dense packing while ensuring DNA accessibility. Thus, different layers of chromatin organization and epigenetic control mechanisms exist. Genome-wide chromatin interaction maps revealed large interaction domains (TADs) and higher order A and B compartments, reflecting active and inactive chromatin, respectively. The mutual dependencies between chromatin organization and patterns of epigenetic marks, including DNA methylation, remain poorly understood. Here, we demonstrate that establishment of A/B compartments precedes and defines DNA methylation signatures during differentiation and maturation of cardiac myocytes. Remarkably, dynamic CpG and non-CpG methylation in cardiac myocytes is confined to A compartments. Furthermore, genetic ablation or reduction of DNA methylation in embryonic stem cells or cardiac myocytes, respectively, does not alter genome-wide chromatin organization. Thus, DNA methylation appears to be established in preformed chromatin compartments and may be dispensable for the formation of higher order chromatin organization.

The RNA workbench: best practices for RNA and high-throughput sequencing bioinformatics in Galaxy.

RNA-based regulation has become a major research topic in molecular biology. The analysis of epigenetic and expression data is therefore incomplete if RNA-based regulation is not taken into account. Thus, it is increasingly important but not yet standard to combine RNA-centric data and analysis tools with other types of experimental data such as RNA-seq or ChIP-seq. Here, we present the RNA workbench, a comprehensive set of analysis tools and consolidated workflows that enable the researcher to combine these two worlds. Based on the Galaxy framework the workbench guarantees simple access, easy extension, flexible adaption to personal and security needs, and sophisticated analyses that are independent of command-line knowledge. Currently, it includes more than 50 bioinformatics tools that are dedicated to different research areas of RNA biology including RNA structure analysis, RNA alignment, RNA annotation, RNA-protein interaction, ribosome profiling, RNA-seq analysis and RNA target prediction. The workbench is developed and maintained by experts in RNA bioinformatics and the Galaxy framework. Together with the growing community evolving around this workbench, we are committed to keep the workbench up-to-date for future standards and needs, providing researchers with a reliable and robust framework for RNA data analysis. AVAILABILITY: The RNA workbench is available at https://github.com/bgruening/galaxy-rna-workbench.

Jupyter and Galaxy: Easing entry barriers into complex data analyses for biomedical researchers.

What does it take to convert a heap of sequencing data into a publishable result? First, common tools are employed to reduce primary data (sequencing reads) to a form suitable for further analyses (i.e., the list of variable sites). The subsequent exploratory stage is much more ad hoc and requires the development of custom scripts and pipelines, making it problematic for biomedical researchers. Here, we describe a hybrid platform combining common analysis pathways with the ability to explore data interactively. It aims to fully encompass and simplify the "raw data-to-publication" pathway and make it reproducible.

BioContainers: an open-source and community-driven framework for software standardization.

MOTIVATION: BioContainers (biocontainers.pro) is an open-source and community-driven framework which provides platform independent executable environments for bioinformatics software. BioContainers allows labs of all sizes to easily install bioinformatics software, maintain multiple versions of the same software and combine tools into powerful analysis pipelines. BioContainers is based on popular open-source projects Docker and rkt frameworks, that allow software to be installed and executed under an isolated and controlled environment. Also, it provides infrastructure and basic guidelines to create, manage and distribute bioinformatics containers with a special focus on omics technologies. These containers can be integrated into more comprehensive bioinformatics pipelines and different architectures (local desktop, cloud environments or HPC clusters). AVAILABILITY AND IMPLEMENTATION: The software is freely available at github.com/BioContainers/. CONTACT: yperez@ebi.ac.uk.