Many cell lines commonly used for biological studies have been found to harbor exogenous agents such as the human tumor viruses Epstein-Barr virus (EBV) and human papillomavirus. Nevertheless, broad-based, unbiased approaches to globally assess the presence of ectopic organisms within cell model systems have not previously been available. We reasoned that high-throughput sequencing should provide unparalleled insights into the microbiomes of tissue culture cell systems. Here we have used our RNA-seq analysis pipeline, PARSES (Pipeline for Analysis of RNA-Seq Exogenous Sequences), to investigate the presence of ectopic organisms within two EBV-positive B-cell lines commonly used by EBV researchers. Sequencing data sets from both the Akata and JY B-cell lines were found to contain reads for EBV, and the JY data set was found to also contain reads from the murine leukemia virus (MuLV). Further investigation revealed that MuLV transcription in JY cells is highly active. We also identified a number of MuLV alternative splicing events, and we uncovered evidence of APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G)-dependent DNA editing. Finally, reverse transcription-PCR analysis showed the presence of MuLV in three other human B-cell lines (DG75, Ramos, and P3HR1 Cl.13) commonly used by investigators in the Epstein-Barr virus field. We believe that a thorough examination of tissue culture microbiomes using RNA-seq/PARSES-like approaches is critical for the appropriate utilization of these systems in biological studies.
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"The process makes it possible to detect contamination by viral, bacterial or fungal organisms in a variety of samples. While several command line tools have been developed to detect pathogens in sequencing data (e.g., –), the pathogen detection plugin is tightly integrated with GobyWeb and makes it possible to routinely screen samples for pathogen contamination. To measure performance, we analyzed the 72 RNA-Seq Pickrell samples with the pathogen detection plugin (searching viral genomes, using the Minia assembler and stripping Illumina adapters from the reads prior to assembly). "
[Show abstract][Hide abstract]ABSTRACT: We present GobyWeb, a web-based system that facilitates the management and analysis of high-throughput sequencing (HTS) projects. The software provides integrated support for a broad set of HTS analyses and offers a simple plugin extension mechanism. Analyses currently supported include quantification of gene expression for messenger and small RNA sequencing, estimation of DNA methylation (i.e., reduced bisulfite sequencing and whole genome methyl-seq), or the detection of pathogens in sequenced data. In contrast to previous analysis pipelines developed for analysis of HTS data, GobyWeb requires significantly less storage space, runs analyses efficiently on a parallel grid, scales gracefully to process tens or hundreds of multi-gigabyte samples, yet can be used effectively by researchers who are comfortable using a web browser. We conducted performance evaluations of the software and found it to either outperform or have similar performance to analysis programs developed for specialized analyses of HTS data. We found that most biologists who took a one-hour GobyWeb training session were readily able to analyze RNA-Seq data with state of the art analysis tools. GobyWeb can be obtained at http://gobyweb.campagnelab.org and is freely available for non-commercial use. GobyWeb plugins are distributed in source code and licensed under the open source LGPL3 license to facilitate code inspection, reuse and independent extensions http://github.com/CampagneLaboratory/gobyweb2-plugins.
"There are few tools available to groups with less experience in software development for high throughput sequence analysis. PARSES (Pipeline for Analysis of RNA-Seq Exogenous Sequences) is a system that uses BLAST+ for rapid filtering of human reads followed by MEGAN for visualization of metagenomic data. PARSES is designed to work on a 64-bit desktop computer, though with limited memory the time required for analysis of a single dataset can require multiple days. "
[Show abstract][Hide abstract]ABSTRACT: Metagenomics, the study of microbial genomes within diverse environments, is a rapidly developing field. The identification of microbial sequences within a host organism enables the study of human intestinal, respiratory, and skin microbiota, and has allowed the identification of novel viruses in diseases such as Merkel cell carcinoma. There are few publicly available tools for metagenomic high throughput sequence analysis. We present Integrated Metagenomic Sequence Analysis (IMSA), a flexible, fast, and robust computational analysis pipeline that is available for public use. IMSA takes input sequence from high throughput datasets and uses a user-defined host database to filter out host sequence. IMSA then aligns the filtered reads to a user-defined universal database to characterize exogenous reads within the host background. IMSA assigns a score to each node of the taxonomy based on read frequency, and can output this as a taxonomy report suitable for cluster analysis or as a taxonomy map (TaxMap). IMSA also outputs the specific sequence reads assigned to a taxon of interest for downstream analysis. We demonstrate the use of IMSA to detect pathogens and normal flora within sequence data from a primary human cervical cancer carrying HPV16, a primary human cutaneous squamous cell carcinoma carrying HPV 16, the CaSki cell line carrying HPV16, and the HeLa cell line carrying HPV18.
[Show abstract][Hide abstract]ABSTRACT: KSHV inflammatory cytokine syndrome (KICS) is a newly described condition characterized by systemic illness as a result of systemic, lytic KSHV infection. We used Illumina sequencing to establish the DNA vironome of blood from such a patient. It identified concurrent high-level viremia of human herpesvirus (HHV) 8 and 6a. The HHV8 plasma viral load was 5,300,000 copies/ml, which is the highest reported to date; this despite less than five skin lesions and no HHV8 associated lymphoma. This is the first report of systemic HHV6a/KSHV co-infection in a patient. It is the first whole genome KSHV sequence to be determined directly from patient plasma rather than cultured or biopsied tumor material. This case supports KICS as a new clinical entity associated with KSHV.