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- W2886315079 abstract "Abstract Purpose/Objectives: Integration of human papillomavirus (HPV) DNA into the human genome occurs in a fraction of viral infections and is a near ubiquitous factor in cervical tumorigenesis. While use of next-generation sequencing (NGS) methods has significantly advanced our understanding of HPV DNA integration and its consequences, short read approaches may yield false positives or misinterpretation of associated genomic structural variants. With the goal of resolving genomic rearrangements, we applied both HPV16 hybridization capture-NGS and long read Nanopore technology to the well-studied CaSki cervical carcinoma cell line that has many sites of HPV16 DNA integration. Methods: CaSki genomic DNA was sheared, ligated to Illumina adaptors, hybridized to HPV16 capture probes (Roche Nimblegen SeqCap EZ System), and sequenced by Illumina HiSeq 2500, paired end, 300 bp mode. Reads were aligned to a custom human (GRCh37/hg19) plus HPV16 reference genome using BWA mem, and junction fragments were computationally identified using Delly and SplazerS. For long read analysis, isolated CaSki DNA was sheared to 20 kb. Genomic libraries were prepared and sequenced on an Oxford Nanopore MinION Mk1b device (ONT) using standard 48 hour scripts. Base calling and fastq file extraction were performed with Albacore v2.0. Reads were aligned using Ngmlr, and structural variants called using Sniffles. Results: For Illumina analysis, 34 million demultiplexed read pairs were obtained with average, HPV genome coverage depth of 2.8*106. 47 previously identified, HPV-human junctions were verified along with 193 novel junctions. Use of PCR has validated 4 of 6 novel junctions tested to date. Multiple junctions were noted to be clustered at some chromosomal locations, with 30 (13.5%) called less than 1kb apart on the human genomic side. For long read analysis, ~150K reads were obtained. Mean read length was 16K with genome and HPV16 coverage of 0.60X and 220X, respectively. Integrations were detected on chromosomes 2, 5, 7, 10, 19, 20, and X, and all consisted of >2 tandem viral genomes. Discussion: Our molecular approaches generated unprecedented long-range analyses of HPV DNA integration sites in the CaSki cervical carcinoma cell line. HPV DNA was often present in tandem arrays. We interpret the clusters of nearby HPV-human junctions as potential evidence of frequent, ongoing, subclonal, genomic rearrangements at these sites. Combining deep-sequencing and long-range methodologies allowed important insights, and we are currently analyzing clinical cancer samples using them. Citation Format: Anne Van Arsdale, Jack Lenz, Nicole Patterson, Elaine Maggi, Dennis Y. Kuo, Cristina Montagna. Elucidation of integrated HPV DNA structure in a cervical carcinoma cell line by combined high resolution and long range sequencing analyses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-334." @default.
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- W2886315079 date "2018-07-01" @default.
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- W2886315079 title "Abstract LB-334: Elucidation of integrated HPV DNA structure in a cervical carcinoma cell line by combined high resolution and long range sequencing analyses" @default.
- W2886315079 doi "https://doi.org/10.1158/1538-7445.am2018-lb-334" @default.
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