Identifying viral integration sites using SeqMap 2.0

Hawkins, Troy; Dantzer, Jessica; Peters, Brandon; Dinauer, Mary C.; Mockaitis, Keithanne; Mooney, Sean D.; Cornetta, Kenneth

doi:10.1093/bioinformatics/btq722

Cited by 33 publications

(27 citation statements)

References 20 publications

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“…Since the virus can integrate into a variety of positions in the human genome, these fusion transcripts are specific to each integration site. In recent years, following the introduction of high-throughput sequencing techniques, multiple softwares for detecting pathogen sequences in host sequence data have become available [38, 57–63]. Here, to identify the viral-human fusion transcripts expressed in our epithelia, we used the ViralFusionSeq (VFS) software [61, 64].…”

Section: Resultsmentioning

confidence: 99%

Functional variants of human papillomavirus type 16 demonstrate host genome integration and transcriptional alterations corresponding to their unique cancer epidemiology

et al. 2016

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BackgroundHuman papillomaviruses (HPVs) are a worldwide burden as they are a widespread group of tumour viruses in humans. Having a tropism for mucosal tissues, high-risk HPVs are detected in nearly all cervical cancers. HPV16 is the most common high-risk type but not all women infected with high-risk HPV develop a malignant tumour. Likely relevant, HPV genomes are polymorphic and some HPV16 single nucleotide polymorphisms (SNPs) are under evolutionary constraint instigating variable oncogenicity and immunogenicity in the infected host.ResultsTo investigate the tumourigenicity of two common HPV16 variants, we used our recently developed, three-dimensional organotypic model reminiscent of the natural HPV infectious cycle and conducted various “omics” and bioinformatics approaches. Based on epidemiological studies we chose to examine the HPV16 Asian-American (AA) and HPV16 European Prototype (EP) variants. They differ by three non-synonymous SNPs in the transforming and virus-encoded E6 oncogene where AAE6 is classified as a high- and EPE6 as a low-risk variant. Remarkably, the high-risk AAE6 variant genome integrated into the host DNA, while the low-risk EPE6 variant genome remained episomal as evidenced by highly sensitive Capt-HPV sequencing. RNA-seq experiments showed that the truncated form of AAE6, integrated in chromosome 5q32, produced a local gene over-expression and a large variety of viral-human fusion transcripts, including long distance spliced transcripts. In addition, differential enrichment of host cell pathways was observed between both HPV16 E6 variant-containing epithelia. Finally, in the high-risk variant, we detected a molecular signature of host chromosomal instability, a common property of cancer cells.ConclusionsWe show how naturally occurring SNPs in the HPV16 E6 oncogene cause significant changes in the outcome of HPV infections and subsequent viral and host transcriptome alterations prone to drive carcinogenesis. Host genome instability is closely linked to viral integration into the host genome of HPV-infected cells, which is a key phenomenon for malignant cellular transformation and the reason for uncontrolled E6 oncogene expression. In particular, the finding of variant-specific integration potential represents a new paradigm in HPV variant biology.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3203-3) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Functional variants of human papillomavirus type 16 demonstrate host genome integration and transcriptional alterations corresponding to their unique cancer epidemiology

et al. 2016

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“…QuickMap (Appelt et al, 2009) and SeqMap 2.0 (Hawkins et al, 2011) are also publicly available. Although the general strategy of IS detection is similar for all pipelines, they differ in the individual steps performed during analyses, parameter optimization, use of underlying bioinformatic tools, and biological annotation resources.…”

Section: Discussionmentioning

confidence: 99%

Bioinformatic Clonality Analysis of Next-Generation Sequencing-Derived Viral Vector Integration Sites

Arens

Appelt

Bartholomae

et al. 2012

Human Gene Therapy Methods

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Clonality analysis of viral vector-transduced cell populations represents a convincing approach to dissect the physiology of tissue and organ regeneration, to monitor the fate of individual gene-corrected cells in vivo, and to assess vector biosafety. With the decoding of mammalian genomes and the introduction of next-generation sequencing technologies, the demand for automated bioinformatic analysis tools that can rapidly process and annotate vector integration sites is rising. Here, we provide a publicly accessible, graphical user interface-guided automated bioinformatic high-throughput integration site analysis pipeline. Its performance and key features are illustrated on pyrosequenced linear amplification-mediated PCR products derived from one patient previously enrolled in the first lentiviral vector clinical gene therapy study. Analysis includes trimming of vector genome junctions, alignment of genomic sequence fragments to the host genome for the identification of integration sites, and the annotation of nearby genomic elements. Most importantly, clinically relevant features comprise the determination of identical integration sites with respect to different time points or cell lineages, as well as the retrieval of the most prominent cell clones and common integration sites. The resulting output is summarized in tables within a convenient spreadsheet and can be further processed by researchers without profound bioinformatic knowledge.

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“…The Illumina platform has the advantages of allowing paired-end sequencing and providing larger data volumes. Several reports have described methods for analysis of these data 9, 29, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48. However, to date, none have taken full advantage of all types of paired reads, dealt comprehensively with integration in repeated sequences, or provided a statistical framework for quantitative inference of cell abundances based on integration site data.…”

Section: Introductionmentioning

confidence: 99%

INSPIIRED: A Pipeline for Quantitative Analysis of Sites of New DNA Integration in Cellular Genomes

Sherman

Nobles

Berry

et al. 2017

Molecular Therapy - Methods & Clinical Development

120

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Integration of new DNA into cellular genomes mediates replication of retroviruses and transposons; integration reactions have also been adapted for use in human gene therapy. Tracking the distributions of integration sites is important to characterize populations of transduced cells and to monitor potential outgrow of pathogenic cell clones. Here, we describe a pipeline for quantitative analysis of integration site distributions named INSPIIRED (integration site pipeline for paired-end reads). We describe optimized biochemical steps for site isolation using Illumina paired-end sequencing, including new technology for suppressing recovery of unwanted contaminants, then software for alignment, quality control, and management of integration site sequences. During library preparation, DNAs are broken by sonication, so that after ligation-mediated PCR the number of ligation junction sites can be used to infer abundance of gene-modified cells. We generated integration sites of known positions in silico, and we describe optimization of sample processing parameters refined by comparison to truth. We also present a novel graph-theory-based method for quantifying integration sites in repeated sequences, and we characterize the consequences using synthetic and experimental data. In an accompanying paper, we describe an additional set of statistical tools for data analysis and visualization. Software is available at https://github.com/BushmanLab/INSPIIRED.

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Identifying viral integration sites using SeqMap 2.0

Cited by 33 publications

References 20 publications

Functional variants of human papillomavirus type 16 demonstrate host genome integration and transcriptional alterations corresponding to their unique cancer epidemiology

Functional variants of human papillomavirus type 16 demonstrate host genome integration and transcriptional alterations corresponding to their unique cancer epidemiology

Bioinformatic Clonality Analysis of Next-Generation Sequencing-Derived Viral Vector Integration Sites

INSPIIRED: A Pipeline for Quantitative Analysis of Sites of New DNA Integration in Cellular Genomes

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