SAMMate: a GUI tool for processing short read alignments in SAM/BAM format

Next-generation sequencing is a valuable tool in our growing understanding of the genetic diversity of viral populations. Using this technology, we have investigated the RNA content of a purified nonenveloped single-stranded RNA virus, flock house virus (FHV). We have also investigated the RNA content of virus-like particles (VLPs) of FHV and the related Nudaurelia capensis omega virus. VLPs predominantly package ribosomal RNA and transcripts of their baculoviral expression vectors. In addition, we find that 5.3% of the packaged RNAs are transposable elements derived from the Sf21 genome. This observation may be important when considering the therapeutic use of VLPs. We find that authentic FHV virions also package a variety of host RNAs, accounting for 1% of the packaged nucleic acid. Significant quantities of host messenger RNAs, ribosomal RNA, noncoding RNAs, and transposable elements are readily detected. The packaging of these host RNAs elicits the possibility of horizontal gene transfer between eukaryotic hosts that share a viral pathogen. We conclude that the genetic content of nonenveloped RNA viruses is variable, not just by genome mutation, but also in the diversity of RNA transcripts that are packaged.deep-sequencing | virus evolution | virus assembly N ext-generation sequencing (NGS) is a powerful tool in the investigation of viral diversity from many different perspectives. NGS has been applied in directed approaches to investigate the prevalence of polymorphism or quasispecies present in a population of known viruses within a host, e.g., foot-and-mouth disease virus (1) or to follow the evolution of a viral genome and the frequency of known polymorphisms during the course of an infection, e.g., human rhinovirus (2) and HIV (3). The total genetic content of biological or clinical samples have been sequenced to uncover viruses present even at very low titers, for example, the presence of porcine circovirus-1 in Rotarix samples (4) and the identification of influenza virus subtypes and norovirus from nasopharyngeal and fecal samples collected during outbreaks (5).NGS has also been applied in unbiased approaches aimed at the discovery of new viral species without any advance genetic information. For example, the Merkel cell polyomavirus was discovered to be the causative agent of its eponymous carcinoma by deep sequencing the transcriptome of tumor cells and analyzing nonhost transcripts (6). Similarly, it has been possible to uncover the history of viral pathogens that have plagued an organism through the de novo assembly of the sequenced siRNAs that would have been generated to tackle viral infections. This approach has revealed the viral genomes of familiar miscreants as well as of new viruses, for example, in fruit flies, mosquitoes, and nematode worms (7).In this report, we employ NGS to investigate the total RNA encapsidated by authentic flock house virus (FHV) virions and by virus-like particles (VLPs) of FHV and of the related tetravirus, Nudaurelia capensis omega virus (NωV). Our analysis emplo...

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“…were determined using SAMMate (44). Reads that did not align to the reference genomes were assembled into contigs using the Velvet package (20).…”

Section: Methodsmentioning

confidence: 99%

Host RNAs, including transposons, are encapsidated by a eukaryotic single-stranded RNA virus

Routh

Domitrovic

Johnson

2012

Proc. Natl. Acad. Sci. U.S.A.

106

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“…Splice junctions were identified using TopHat (17) run on a human plus EBV genome Bowtie index. RPKM calculations (i.e., reads per kilobase of exon per million mapped reads) and genome coverage files (wiggle files) were generated using SAMMate (19). The Integrative Genomics Viewer (IGV) (14) was used to visualize sequence alignments, genomic annotations, and splice junctions.…”

Section: Methodsmentioning

confidence: 99%

Identification of New Viral Genes and Transcript Isoforms during Epstein-Barr Virus Reactivation using RNA-Seq

Concha

Wang

Cao

et al. 2012

J Virol

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Using an enhanced RNA-Seq pipeline to analyze Epstein-Barr virus (EBV) transcriptomes, we investigated viral and cellular gene expression in the Akata cell line following B-cell-receptor-mediated reactivation. Robust induction of EBV gene expression was observed, with most viral genes induced >200-fold and with EBV transcripts accounting for 7% of all mapped reads within the cell. After induction, hundreds of candidate splicing events were detected using the junction mapper TopHat, including a novel nonproductive splicing event at the gp350/gp220 locus and several alternative splicing events at the LMP2 locus. A more detailed analysis of lytic LMP2 transcripts showed an overall lack of the prototypical type III latency splicing events. Analysis of nuclear versus cytoplasmic RNA-Seq data showed that the lytic forms of LMP2, EBNA-2, EBNA-LP, and EBNA-3A, -3B, and -3C have higher nuclear-to-cytoplasmic accumulation ratios than most lytic genes, including classic late genes. These data raise the possibility that at least some lytic transcripts derived from these latency gene loci may have unique, noncoding nuclear functions during reactivation. Our analysis also identified two previously unknown genes, BCLT1 and BCRT2, that map to the BamHI C-region of the EBV genome. Pathway analysis of cellular gene expression changes following B-cell receptor activation identified an inflammatory response as the top predicted function and ILK and TREM1 as the top predicted canonical pathways. E pstein-Barr virus (EBV) is a human pathogen that causes malignancies including Burkitt's lymphoma, Hodgkin's disease, and nasopharyngeal carcinoma (13). EBV has a complex infection cycle involving a number of different viral gene expression programs. These individual programs facilitate distinct tasks that are required for specific infection stages. Like all herpesviruses, EBV utilizes both latent gene expression programs, in which only limited numbers of viral genes are expressed, and a replicative gene expression program, in which the bulk of EBV genes are expressed to produce infectious virus.Efficient and synchronous virus reactivation can be modeled by activating the B-cell receptor (BCR) in the EBV-positive Burkitt's lymphoma cell line Akata (15). In this system, reactivation leads to an ordered induction of immediate-early (e.g., BZLF1 and BRLF1), early (e.g., BMRF1), and late genes, with immediate-early genes peaking at approximately 2 to 6 h and late genes peaking at approximately 6 to 24 h postinduction (6,15,20). Interestingly, Yuan et al. (20) found that EBV latency genes were induced following reactivation, suggesting a role for these genes in the lytic cycle (20).Second-generation RNA-Seq technology allows the simultaneous interrogation of gene expression and transcript structure at a high level of accuracy and at a single-nucleotide resolution. We have recently shown the application of RNA-Seq to the interrogation of EBV transcriptomes in two type I latency cell lines, Akata and Mutu I (11). Here we have improved our RNA-Se...

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“…Expression analysis was performed using our previously reported analysis software, SAMMate (24). Data were displayed using the Integrated Genome Viewer (IGV) (16).…”

Section: Methodsmentioning

confidence: 99%

Detection of Murine Leukemia Virus in the Epstein-Barr Virus-Positive Human B-Cell Line JY, Using a Computational RNA-Seq-Based Exogenous Agent Detection Pipeline, PARSES

Lin

Puetter

Coco

et al. 2012

J Virol

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bMany 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 highthroughput 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 mRNAediting 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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SAMMate: a GUI tool for processing short read alignments in SAM/BAM format

Cited by 55 publications

References 19 publications

Host RNAs, including transposons, are encapsidated by a eukaryotic single-stranded RNA virus

Host RNAs, including transposons, are encapsidated by a eukaryotic single-stranded RNA virus

Identification of New Viral Genes and Transcript Isoforms during Epstein-Barr Virus Reactivation using RNA-Seq

Detection of Murine Leukemia Virus in the Epstein-Barr Virus-Positive Human B-Cell Line JY, Using a Computational RNA-Seq-Based Exogenous Agent Detection Pipeline, PARSES

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