Partial or complete E1/ E2 gene loss may also occur. One study identified the E2 hinge region (3172 to 3659 bp) as the most frequently disrupted site the most prevalent site for E1 was located between 10 bp. Post integration, the HPV E2 gene (often with E1) is frequently disrupted and functionally incapacitated for E6/ E7 repression. Chromosomal fragile sites (loci sensitive to replication stress and breakage) and transcriptionally active regions of open chromatin are reportedly more common. HPV integrates fortuitously but preferentially at vulnerable regions of the host genome. However, during vegetative amplification, HPV E1/E2 loads onto the viral ori and binds directly to host chromatin to initiate, recruit, and exploit cellular DNA damage response for self-replication. Next, the viral genome unwinds bidirectionally at the ori via the HPV E1 helicase, replicates using cellular proteins, and partitions equally into the daughter cells. Specifically, the HPV genome in the form of extrachromosomal plasmids may tether randomly or pairwise to sister chromatids at the viral origin of replication (ori) by HPV E2 binding. During cell division, two models of viral genome replication have been described, i.e., Random Attachment and Faithful Partitioning. “Accidental integration” may result from intimate interactions between the viral and host genomes throughout replication. Throughout the viral life cycle, HPV replicates in three distinct phases, namely, “initial replication” after viral entry, “maintenance replication” during cellular replication, and “vegetative replication” in differentiated cells. The HPV genome is an ~8000 base pair (bp), double-stranded, circular DNA encoding 6 early genes ( E1, E2, E4, E5, E6, and E7) and 2 late genes ( L1 and L2). Applying HPV VIS-mapping to pre- or invasive tumors will advance our understanding of viral oncogenesis and facilitate the discovery of prognostic biomarkers and therapeutic targets. Overall, the VHC and VIS workflows proved to be a rapid and accurate means of localizing viral-host integration site(s) and identifying disrupted and neighboring human genes. Key findings, including disrupted and nearby genes, were summarized in an auto-generated report. The VIS dynamic graphical outputs included read mappings, virus-host genomic breakpoints, and virus-host integration circular plots. The VHC and VIS workflow median runtimes were 19 and 7 min per sample, respectively. The workflows embedded with HPV and human reference genomes were used to analyze a publicly available NGS dataset derived from pre- and cancerous HPV+ cervical cytology of 21 Gabonese women.
#Clc genomics workbench citation software#
Here, we tested two new, automated workflows in CLC Microbial Genomics, i.e., Viral Hybrid Capture (VHC) Data Analysis and Viral Integration Site (VIS) Identification for software performance and efficiency. However, a simple, streamlined bioinformatics workflow has been non-existent until recently. With next-generation sequencing (NGS), identification of viral and host genomic breakpoints and chimeric sequences are now possible. Sequences that did not map to the PA14 reference genome were aligned to the human genome using CLC Genomics Workbench software (Qiagen).Human papillomavirus (HPV) integration within the host genome may contribute to carcinogenesis through various disruptive mechanisms. Sequencing libraries were prepared with the QIAseq smRNA kit (Qiagen) and 75-bp single-end reads were generated using an Illumina MiniSeq. # 217004), including the on-column DNA digestion. Total RNA was isolated with the miRNeasy Mini Kit (Qiagen, Germantown, MD, Cat. The cells were then washed again in PBS, and cytoplasmic RNA was harvested after lysis of bacterial cell wall and inner membrane. To avoid possible carry-over of EVs (and miRNA) attached to the outside of the bacteria, the bacteria were washed with PBS and the bacterial outer membrane was lysed with EDTA to release periplasmic contents and factors associated with the bacterial outer membrane and periplasm (77-79). aeruginosa (1.5*E+07 CFU) were exposed to PBS vehicle control or EVs (5E+09 EV/ml) for 6 h. aeruginosa, sequences that did not map to the PA14 reference genome were aligned to the human genome. To assess transfer of human EV small RNAs to P. Total RNA was isolated, small RNA libraries were prepared with the QIAseq smRNA kit (Qiagen) and 75-bp single-end reads were generated using Illumina MiniSeq. aeruginosa were exposed to extracellular vesicles (EVs) secreted by primary human airway epithelial cells from 3 donors or PBS vehicle control for 6 h. Transfer of small RNA from extracellular vesicles secreted by primary human airway epithelial cells to Pseudomonas aeruginosaĮxpression profiling by high throughput sequencing GEO help: Mouse over screen elements for information.