RSEQREP: RNA-Seq Reports, an open-source cloud-enabled framework for reproducible RNA-Seq data processing, analysis, and result reporting

RNA-Seq is increasingly being used to measure human RNA expression on a genome-wide scale. Expression profiles can be interrogated to identify and functionally characterize treatment-responsive genes. Ultimately, such controlled studies promise to reveal insights into molecular mechanisms of treatment effects, identify biomarkers, and realize personalized medicine. RNA-Seq Reports (RSEQREP) is a new open-source cloud-enabled framework that allows users to execute start-to-end gene-level RNA-Seq analysis on a preconfigured RSEQREP Amazon Virtual Machine Image (AMI) hosted by AWS or on their own Ubuntu Linux machine. The framework works with unstranded, stranded, and paired-end sequence FASTQ files stored locally, on Amazon Simple Storage Service (S3), or at the Sequence Read Archive (SRA). RSEQREP automatically executes a series of customizable steps including reference alignment, CRAM compression, reference alignment QC, data normalization, multivariate data visualization, identification of differentially expressed genes, heatmaps, co-expressed gene clusters, enriched pathways, and a series of custom visualizations. The framework outputs a file collection that includes a dynamically generated PDF report using R, knitr, and LaTeX, as well as publication-ready table and figure files. A user-friendly configuration file handles sample metadata entry, processing, analysis, and reporting options. The configuration supports time series RNA-Seq experimental designs with at least one pre- and one post-treatment sample for each subject, as well as multiple treatment groups and specimen types. All RSEQREP analyses components are built using open-source R code and R/Bioconductor packages allowing for further customization. As a use case, we provide RSEQREP results for a trivalent influenza vaccine (TIV) RNA-Seq study that collected 1 pre-TIV and 10 post-TIV vaccination samples (days 1-10) for 5 subjects and two specimen types (peripheral blood mononuclear cells and B-cells)

RSEQREP: RNA-Seq Reports, an open-source cloud-enabled framework for reproducible RNA-Seq data processing, analysis, and result reporting [version 2; referees: 2 approved]

RNA-Seq is increasingly being used to measure human RNA expression on a genome-wide scale. Expression profiles can be interrogated to identify and functionally characterize treatment-responsive genes. Ultimately, such controlled studies promise to reveal insights into molecular mechanisms of treatment effects, identify biomarkers, and realize personalized medicine. RNA-Seq Reports (RSEQREP) is a new open-source cloud-enabled framework that allows users to execute start-to-end gene-level RNA-Seq analysis on a preconfigured RSEQREP Amazon Virtual Machine Image (AMI) hosted by AWS or on their own Ubuntu Linux machine via a Docker container or installation script. The framework works with unstranded, stranded, and paired-end sequence FASTQ files stored locally, on Amazon Simple Storage Service (S3), or at the Sequence Read Archive (SRA). RSEQREP automatically executes a series of customizable steps including reference alignment, CRAM compression, reference alignment QC, data normalization, multivariate data visualization, identification of differentially expressed genes, heatmaps, co-expressed gene clusters, enriched pathways, and a series of custom visualizations. The framework outputs a file collection that includes a dynamically generated PDF report using R, knitr, and LaTeX, as well as publication-ready table and figure files. A user-friendly configuration file handles sample metadata entry, processing, analysis, and reporting options. The configuration supports time series RNA-Seq experimental designs with at least one pre- and one post-treatment sample for each subject, as well as multiple treatment groups and specimen types. All RSEQREP analyses components are built using open-source R code and R/Bioconductor packages allowing for further customization. As a use case, we provide RSEQREP results for a trivalent influenza vaccine (TIV) RNA-Seq study that collected 1 pre-TIV and 10 post-TIV vaccination samples (days 1-10) for 5 subjects and two specimen types (peripheral blood mononuclear cells and B-cells)

Failure to detect mutations in U2AF1 due to changes in the GRCh38 reference sequence

The U2AF1 gene is a core part of mRNA splicing machinery and frequently contains somatic mutations that contribute to oncogenesis in myelodysplastic syndrome, acute myeloid leukemia, and other cancers. A change introduced in the GRCh38 version of the human reference build prevents detection of mutations in this gene, and others, by variant calling pipelines. This study describes the problem in detail and shows that a modified GRCh38 reference build with unchanged coordinates can be used to ameliorate the issue

A review of clinical trials of cetuximab combined with radiotherapy for non-small cell lung cancer

Treatment of non-small cell lung cancer (NSCLC) is challenging in many ways. One of the problems is disappointing local control rates in larger volume disease. Moreover, the likelihood of both nodal and distant spread increases with primary tumour (T-) stage. Many patients are elderly and have considerable comorbidity. Therefore, aggressive combined modality treatment might be contraindicated or poorly tolerated. In many cases with larger tumour volume, sufficiently high radiation doses can not be administered because the tolerance of surrounding normal tissues must be respected. Under such circumstances, simultaneous administration of radiosensitizing agents, which increase tumour cell kill, might improve the therapeutic ratio. If such agents have a favourable toxicity profile, even elderly patients might tolerate concomitant treatment. Based on sound preclinical evidence, several relatively small studies have examined radiotherapy (RT) with cetuximab in stage III NSCLC. Three different strategies were pursued: 1) RT plus cetuximab (2 studies), 2) induction chemotherapy followed by RT plus cetuximab (2 studies) and 3) concomitant RT and chemotherapy plus cetuximab (2 studies). Radiation doses were limited to 60-70 Gy. As a result of study design, in particular lack of randomised comparison between cetuximab and no cetuximab, the efficacy results are difficult to interpret. However, strategy 1) and 3) appear more promising than induction chemotherapy followed by RT and cetuximab. Toxicity and adverse events were more common when concomitant chemotherapy was given. Nevertheless, combined treatment appears feasible. The role of consolidation cetuximab after RT is uncertain. A large randomised phase III study of combined RT, chemotherapy and cetuximab has been initiated

Ribosome-associated vesicles: A dynamic subcompartment of the endoplasmic reticulum in secretory cells

The endoplasmic reticulum (ER) is a highly dynamic network of membranes. Here, we combine live-cell microscopy with in situ cryo–electron tomography to directly visualize ER dynamics in several secretory cell types including pancreatic β-cells and neurons under near-native conditions. Using these imaging approaches, we identify a novel, mobile form of ER, ribosome-associated vesicles (RAVs), found primarily in the cell periphery, which is conserved across different cell types and species. We show that RAVs exist as distinct, highly dynamic structures separate from the intact ER reticular architecture that interact with mitochondria via direct intermembrane contacts. These findings describe a new ER subcompartment within cells

MWCNTs of different physicochemical properties cause similar inflammatory responses, but differences in transcriptional and histological markers of fibrosis in mouse lungs

Multi-walled carbon nanotubes (MWCNTs) are extensively produced and used in composite materials and electronic applications, thus increasing risk of worker and consumer exposure. MWCNTs are an inhomogeneous group of nanomaterials that come in various lengths, shapes and with different metal contaminations, which makes hazard evaluation difficult. However, several studies suggest that length plays an important role in the toxicity induced by MWCNTs. How the length influences toxicity at the molecular level is yet to be characterized. Female C57BL/6 mice were exposed by single intratracheal instillation to 18, 54 or 162 µg/mouse of a short MWCNT (NRCWE-026, 847±102 nm in length) or long MWCNT (NM-401, 4048±366 nm in length). The two MWCNTs were extensively characterized. Lung tissues were harvested 24 h, 3 d and 28 d after exposure. We employed DNA microarrays, bronchoalveolar lavage fluid analysis, comet assay and dichlorodihydrofluorescein assay in order to profile the pulmonary responses. Bioinformatics tools were then applied to compare and contrast the expression profiles and to build a length dependent property-response matrix for gene-by-gene comparison. The toxicogenomic analysis of the global mRNA changes after exposure to the short, entangled NRCWE-026 or the longer, stiffer NM-401 showed high degree of similarities. The toxicity of both MWCNTs was driven by strong inflammatory and acute phase responses, which peaked at day 3 and was observed both in bronchoalveolar lavage cell influx and in gene expression profiles. The inflammatory response was sustained at post-exposure day 28. Also, at the sub-chronic level, we identified a sub-set of 14 fibrosis related genes that were uniquely differentially regulated after exposure to NM-401. Acellular ROS production occurred almost exclusively with NRCWE-026, however the longer NM-401 induced in vivo DNA strand breaks and differential regulation of genes involved in free radical scavenging more readily than NRCWE-026. Our results indicate that the global mRNA response after exposure to MWCNTs is length independent at the acute time points, but that fibrosis may be length dependent sub-chronic end point.JRC.H.6-Digital Earth and Reference Dat