We present a Galaxy based web server for processing and visualizing deeply sequenced data. The web server's core functionality consists of a suite of newly developed tools, called deepTools, that enable users with little bioinformatic background to explore the results of their sequencing experiments in a standardized setting. Users can upload pre-processed files with continuous data in standard formats and generate heatmaps and summary plots in a straight-forward, yet highly customizable manner. In addition, we offer several tools for the analysis of files containing aligned reads and enable efficient and reproducible generation of normalized coverage files. As a modular and open-source platform, deepTools can easily be expanded and customized to future demands and developments. The deepTools webserver is freely available at http://deeptools.ie-freiburg.mpg.de and is accompanied by extensive documentation and tutorials aimed at conveying the principles of deep-sequencing data analysis. The web server can be used without registration. deepTools can be installed locally either stand-alone or as part of Galaxy.

SUMMARY Hepatitis C virus (HCV) is a major causative agent of chronic liver disease in humans. To gain insight into host factor requirements for HCV replication we performed a siRNA screen of the human kinome and identified 13 different kinases, including phosphatidylinositol-4 kinase III alpha (PI4KIIIα) as required for HCV replication. Consistent with elevated levels of the PI4KIIIα product phosphatidylinositol-4-phosphate (PI4P) detected in HCV infected cultured hepatocytes and liver tissue from chronic hepatitis C patients, the enzymatic activity of PI4KIIIα was critical for HCV replication. Viral nonstructural protein 5A (NS5A) was found to interact with PI4KIIIα and stimulate its kinase activity. The absence of PI4KIIIα activity induced a dramatic change in the ultrastructural morphology of the membranous HCV replication complex. Our analysis suggests that the direct activation of a lipid kinase by HCV NS5A contributes critically to the integrity of the membranous viral replication complex.

Summary DNA replication is temporally and spatially organized in all eukaryotes, yet the molecular control and biological function of the replication-timing program are poorly understood. A role for three-dimensional chromatin organization has been proposed. Rif1 is required for normal genome-wide regulation of replication timing, but its molecular function is poorly understood. Here we show that in mouse embryonic stem cells Rif1 coats late replicating domains and, together with Lamin B1 identifies the majority of the late replicating genome. Rif1 is an essential determinant of replication timing of non-Lamin B1-bound late domains. We further demonstrate that Rif1 defines and restricts the interactions between replication-timing domains during G1, thereby revealing a novel function of Rif1 as organizer of nuclear architecture. Loss of Rif1 affects both number and replication-timing specificity of the interactions between replication-timing domains. In addition, during S-phase Rif1 ensures temporal coordination of replication of interacting domains. In summary our study identifies Rif1 as the first molecular link between nuclear architecture organization and replication-timing establishment in mammals.

The centromere-specific histone H3 variant CENH3 (also known as CENP-A) is considered to be an epigenetic mark for establishment and propagation of centromere identity. Pulse induction of CENH3 (Drosophila CID) in Schneider S2 cells leads to its incorporation into non-centromeric regions and generates CID islands that resist clearing from chromosome arms for multiple cell generations. We demonstrate that CID islands represent functional ectopic kinetochores, which are non-randomly distributed on the chromosome and show a preferential localization near telomeres and pericentric heterochromatin in transcriptionally silent, intergenic chromatin domains. Although overexpression of heterochromatin protein 1 (HP1) or increasing histone acetylation interferes with CID island formation on a global scale, induction of a locally defined region of synthetic heterochromatin by targeting HP1-LacI fusions to stably integrated Lac operator arrays produces a proximal hotspot for CID deposition. These data indicate that the characteristics of regions bordering heterochromatin promote de novo kinetochore assembly and thereby contribute to centromere identity.

Chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) is a key technique in chromatin research. Although heavily applied, existing ChIP-seq protocols are often highly fine-tuned workflows, optimized for specific experimental requirements. Especially the initial steps of ChIP-seq, particularly chromatin shearing, are deemed to be exceedingly cell-type-specific, thus impeding any protocol standardization efforts. Here we demonstrate that harmonization of ChIP-seq workflows across cell types and conditions is possible when obtaining chromatin from properly isolated nuclei. We established an ultrasound-based nuclei extraction method (NEXSON: Nuclei EXtraction by SONication) that is highly effective across various organisms, cell types and cell numbers. The described method has the potential to replace complex cell-type-specific, but largely ineffective, nuclei isolation protocols. By including NEXSON in ChIP-seq workflows, we completely eliminate the need for extensive optimization and sample-dependent adjustments. Apart from this significant simplification, our approach also provides the basis for a fully standardized ChIP-seq and yields highly reproducible transcription factor and histone modifications maps for a wide range of different cell types. Even small cell numbers (∼10 000 cells per ChIP) can be easily processed without application of modified chromatin or library preparation protocols.

ObjectiveDysarthric speech of persons with Huntington disease (HD) is typically described as hyperkinetic; however, studies suggest that dysarthria can vary and resemble patterns in other neurologic conditions. To test the hypothesis that distinct motor speech subgroups can be identified within a larger cohort of patients with HD, we performed a cluster analysis on speech perceptual characteristics of patient audio recordings.MethodsAudio recordings of 48 patients with mild to moderate dysarthria due to HD were presented to 6 trained raters. Raters provided scores for various speech features (e.g., voice, articulation, prosody) of audio recordings using the classic Mayo Clinic dysarthria rating scale. Scores were submitted to an unsupervised k-means cluster analysis to determine the most salient speech features of subgroups based on motor speech patterns.ResultsFour unique subgroups emerged from the cohort of patients with HD. Subgroup 1 was characterized by an abnormally fast speaking rate among other unique speech features, whereas subgroups 2 and 3 were defined by an abnormally slow speaking rate. Salient speech features for subgroup 2 overlapped with subgroup 3; however, the severity of dysarthria differed. Subgroup 4 was characterized by mild deviations of speech features with typical speech rate. Length of CAG repeats, Unified Huntington’s Disease Rating Scale total motor score, and percent intelligibility were significantly different for pairwise comparisons of subgroups.ConclusionThis study supports the existence of distinct presentations of dysarthria in patients with HD, which may be due to divergent pathologic processes. The findings are discussed in relation to previous literature and clinical implications.

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Background: The amount of published full-text articles has increased dramatically. Text mining tools configure an essential approach to building biological networks, updating databases and providing annotation for new pathways. PESCADOR is an online web server based on LAITOR and NLProt text mining tools, which retrieves protein-protein co-occurrences in a tabular-based format, adding a network schema. Here we present an HPC-oriented version of PESCADOR's native text mining tool, renamed to LAITOR4HPC, aiming to access an unlimited abstract amount in a short time to enrich available networks, build new ones and possibly highlight whether fields of research have been exhaustively studied. Results: By taking advantage of parallel computing HPC infrastructure, the full collection of MEDLINE abstracts available until June 2017 was analyzed in a shorter period (6 days) when compared to the original online implementation (with an estimated 2 years to run the same data). Additionally, three case studies were presented to illustrate LAITOR4HPC usage possibilities. The first case study targeted soybean and was used to retrieve an overview of published co-occurrences in a single organism, retrieving 15,788 proteins in 7894 cooccurrences. In the second case study, a target gene family was searched in many organisms, by analyzing 15 species under biotic stress. Most co-occurrences regarded Arabidopsis thaliana and Zea mays. The third case study concerned the construction and enrichment of an available pathway. Choosing A. thaliana for further analysis, the defensin pathway was enriched, showing additional signaling and regulation molecules, and how they respond to each other in the modulation of this complex plant defense response. Conclusions: LAITOR4HPC can be used for an efficient text mining based construction of biological networks derived from big data sources, such as MEDLINE abstracts. Time consumption and data input limitations will depend on the available resources at the HPC facility. LAITOR4HPC enables enough flexibility for different approaches and data amounts targeted to an organism, a subject, or a specific pathway. Additionally, it can deliver comprehensive results where interactions are classified into four types, according to their reliability.