KNIME4NGS: a comprehensive toolbox for next generation sequencing analysis.

Summary: Analysis of Next Generation Sequencing (NGS) data requires the processing of large datasets by chaining various tools with complex input and output formats. In order to automate data analysis, we propose to standardize NGS tasks into modular workflows. This simplifies reliable handling and processing of NGS data, and corresponding solutions become substantially more reproducible and easier to maintain. Here, we present a documented, linux-based, toolbox of 42 processing modules that are combined to construct workflows facilitating a variety of tasks such as DNAseq and RNAseq analysis. We also describe important technical extensions. The high throughput executor (HTE) helps to increase the reliability and to reduce manual interventions when processing complex datasets. We also provide a dedicated binary manager that assists users in obtaining the modules' executables and keeping them up to date. As basis for this actively developed toolbox we use the workflow management software KNIME. Availability and Implementation: See http://ibisngs.github.io/knime4ngs for nodes and user manual (GPLv3 license). Contact: robert.kueffner@helmholtz-muenchen.de. Supplementary information: Supplementary data are available at Bioinformatics online.

Epstein-Barr virus microRNAs reduce immune surveillance by virus-specific CD8+ T cells.

Infection with Epstein-Barr virus (EBV) affects most humans worldwide and persists life-long in the presence of robust virus-specific T-cell responses. In both immunocompromised and some immunocompetent people, EBV causes several cancers and lymphoproliferative diseases. EBV transforms B cells in vitro and encodes at least 44 microRNAs (miRNAs), most of which are expressed in EBV-transformed B cells, but their functions are largely unknown. Recently, we showed that EBV miRNAs inhibit CD4+ T-cell responses to infected B cells by targeting IL-12, MHC class II, and lysosomal proteases. Here we investigated whether EBV miRNAs also counteract surveillance by CD8+ T cells. We have found that EBV miRNAs strongly inhibit recognition and killing of infected B cells by EBV-specific CD8+ T cells through multiple mechanisms. EBV miRNAs directly target the peptide transporter subunit TAP2 and reduce levels of the TAP1 subunit, MHC class I molecules, and EBNA1, a protein expressed in most forms of EBV latency and a target of EBV-specific CD8+ T cells. Moreover, miRNA-mediated down-regulation of the cytokine IL-12 decreases the recognition of infected cells by EBV-specific CD8+ T cells. Thus, EBV miRNAs use multiple, distinct pathways, allowing the virus to evade surveillance not only by CD4+ but also by antiviral CD8+ T cells.

Epstein-Barr viral miRNAs inhibit antiviral CD4+ T cell responses targeting IL-12 and peptide processing.

Epstein-Barr virus (EBV) is a tumor virus that establishes lifelong infection in most of humanity, despite eliciting strong and stable virus-specific immune responses. EBV encodes at least 44 miRNAs, most of them with unknown function. Here, we show that multiple EBV miRNAs modulate immune recognition of recently infected primary B cells, EBV's natural target cells. EBV miRNAs collectively and specifically suppress release of proinflammatory cytokines such as IL-12, repress differentiation of naive CD4(+) T cells to Th1 cells, interfere with peptide processing and presentation on HLA class II, and thus reduce activation of cytotoxic EBV-specific CD4(+) effector T cells and killing of infected B cells. Our findings identify a previously unknown viral strategy of immune evasion. By rapidly expressing multiple miRNAs, which are themselves nonimmunogenic, EBV counteracts recognition by CD4(+) T cells and establishes a program of reduced immunogenicity in recently infected B cells, allowing the virus to express viral proteins required for establishment of life-long infection.

Genomic factors related to tissue tropism in Chlamydia pneumoniae infection.

BACKGROUND: Chlamydia pneumoniae (Cpn) are obligate intracellular bacteria that cause acute infections of the upper and lower respiratory tract and have been implicated in chronic inflammatory diseases. Although of significant clinical relevance, complete genome sequences of only four clinical Cpn strains have been obtained. All of them were isolated from the respiratory tract and shared more than 99% sequence identity. Here we investigate genetic differences on the whole-genome level that are related to Cpn tissue tropism and pathogenicity. RESULTS: We have sequenced the genomes of 18 clinical isolates from different anatomical sites (e.g. lung, blood, coronary arteries) of diseased patients, and one animal isolate. In total 1,363 SNP loci and 184 InDels have been identified in the genomes of all clinical Cpn isolates. These are distributed throughout the whole chlamydial genome and enriched in highly variable regions. The genomes show clear evidence of recombination in at least one potential region but no phage insertions. The tyrP gene was always encoded as single copy in all vascular isolates. Phylogenetic reconstruction revealed distinct evolutionary lineages containing primarily non-respiratory Cpn isolates. In one of these, clinical isolates from coronary arteries and blood monocytes were closely grouped together. They could be distinguished from all other isolates by characteristic nsSNPs in genes involved in RB to EB transition, inclusion membrane formation, bacterial stress response and metabolism. CONCLUSIONS: This study substantially expands the genomic data of Cpn and elucidates its evolutionary history. The translation of the observed Cpn genetic differences into biological functions and the prediction of novel pathogen-oriented diagnostic strategies have to be further explored.

Phenotypic variation in Acidovorax radicisN35 influences plant growth promotion.

Acidovorax radicis N35, isolated from surface-sterilized wheat roots (Triticum aestivum), showed irreversible phenotypic variation in nutrient broth, resulting in a differing colony morphology. In addition to the wild-type form (rough colony type), a phenotypic variant form (smooth colony type) appeared at a frequency of 3.2 × 10(-3) per cell per generation on NB agar plates. In contrast to the N35 wild type, the variant N35v showed almost no cell aggregation and had lost its flagella and swarming ability. After inoculation, only the wild-type N35 significantly promoted the growth of soil-grown barley plants. After co-inoculation of axenically grown barley seedlings with differentially fluorescently labeled N35 and N35v cells, decreased competitive endophytic root colonization in the phenotypic variant N35v was observed using confocal laser scanning microscopy. In addition, 454 pyrosequencing of both phenotypes revealed almost identical genomic sequences. The only stable difference noted in the sequence of the phenotype variant N35v was a 16-nucleotide deletion identified in a gene encoding the mismatch repair protein MutL. The deletion resulted in a frameshift that revealed a new stop codon resulting in a truncated MutL protein missing a functional MutL C-terminal domain. The mutation was consistent in all investigated phenotype variant cultures and might be responsible for the observed phenotypic variation in A. radicis N35.

Exome sequencing identifies ACAD9 mutations as a cause of complex I deficiency.

An isolated defect of respiratory chain complex I activity is a frequent biochemical abnormality in mitochondrial disorders. Despite intensive investigation in recent years, in most instances, the molecular basis underpinning complex I defects remains unknown. We report whole-exome sequencing of a single individual with severe, isolated complex I deficiency. This analysis, followed by filtering with a prioritization of mitochondrial proteins, led us to identify compound heterozygous mutations in ACAD9, which encodes a poorly understood member of the mitochondrial acyl-CoA dehydrogenase protein family. We demonstrated the pathogenic role of the ACAD9 variants by the correction of the complex I defect on expression of the wildtype ACAD9 protein in fibroblasts derived from affected individuals. ACAD9 screening of 120 additional complex I-defective index cases led us to identify two additional unrelated cases and a total of five pathogenic ACAD9 alleles.

SIMAP--a comprehensive database of pre-calculated protein sequence similarities, domains, annotations and clusters.

The prediction of protein function as well as the reconstruction of evolutionary genesis employing sequence comparison at large is still the most powerful tool in sequence analysis. Due to the exponential growth of the number of known protein sequences and the subsequent quadratic growth of the similarity matrix, the computation of the Similarity Matrix of Proteins (SIMAP) becomes a computational intensive task. The SIMAP database provides a comprehensive and up-to-date pre-calculation of the protein sequence similarity matrix, sequence-based features and sequence clusters. As of September 2009, SIMAP covers 48 million proteins and more than 23 million non-redundant sequences. Novel features of SIMAP include the expansion of the sequence space by including databases such as ENSEMBL as well as the integration of metagenomes based on their consistent processing and annotation. Furthermore, protein function predictions by Blast2GO are pre-calculated for all sequences in SIMAP and the data access and query functions have been improved. SIMAP assists biologists to query the up-to-date sequence space systematically and facilitates large-scale downstream projects in computational biology. Access to SIMAP is freely provided through the web portal for individuals (http://mips.gsf.de/simap/) and for programmatic access through DAS (http://webclu.bio.wzw.tum.de/das/) and Web-Service (http://mips.gsf.de/webservices/services/SimapService2.0?wsdl).