A target enrichment high throughput sequencing system for characterization of BLV whole genome sequence, integration sites, clonality and host SNP

Abstract

Bovine leukemia virus (BLV) is an oncogenic retrovirus which induces malignant lymphoma termed enzootic bovine leukosis (EBL) after a long incubation period. Insertion sites of the BLV proviral genome as well as the associations between disease progression and polymorphisms of the virus and host genome are not fully understood. To characterize the biological coherence between virus and host, we developed a DNA-capture-seq approach, in which DNA probes were used to efficiently enrich target sequence reads from the next-generation sequencing (NGS) library. In addition, enriched reads can also be analyzed for detection of proviral integration sites and clonal expansion of infected cells since the reads include chimeric reads of the host and proviral genomes. To validate this DNA-capture-seq approach, a persistently BLV-infected fetal lamb kidney cell line (FLK-BLV), four EBL tumor samples and four non-EBL blood samples were analyzed to identify BLV integration sites. The results showed efficient enrichment of target sequence reads and oligoclonal integrations of the BLV proviral genome in the FLK-BLV cell line. Moreover, three out of four EBL tumor samples displayed multiple integration sites of the BLV proviral genome, while one sample displayed a single integration site. In this study, we found the evidence for the first time that the integrated provirus defective at the 5′ end was present in the persistent lymphocytosis cattle. The efficient and sensitive identification of BLV variability, integration sites and clonal expansion described in this study provide support for use of this innovative tool for understanding the detailed mechanisms of BLV infection during the course of disease progression.

Comprehensive Comparison of Novel Bovine Leukemia Virus (BLV) Integration Sites between B-Cell Lymphoma Lines BLSC-KU1 and BLSC-KU17 Using the Viral DNA Capture High-Throughput Sequencing Method

“…Evaluation of this method showed efficient enrichment of target sequence reads and integration of the BLV proviral genome in persistently infected FLK-BLV cells. Our visualization of sequences of paired-end reads mapped to the host genome in DNA from FLK-BLV cells showed a pattern similar to that reported in a previous publication [ 29 ]. Secondly, we found evidence for the first-time regarding two BLV integration sites in BLSC-KU1 and BLSC-KU17 cells using our newly developed proviral DNA-capture-seq.…”

“…We analyzed the BLV integration sites in FLK-BLV cells. FLK-BLV cells have often been used as controls in studies related to BLV; previous studies have showed multiple copies of the BLV genome integrated into the genome of the FLK-BLV cells by either Southern blotting analysis [ 2 , 33 ] or target enrichment next-generation sequencing (NGS) [ 29 ]. The five biotinylated probes used in this study efficiently and specifically captured the BLV provirus and proviral–host chimeric DNA fragments in the DNA library of FLK-BLV cells.…”

“…Most effort has been devoted to the analysis of proviral integration sites in other retroviruses, such as human immunodeficiency virus type-1 (HIV-1) [ 18 , 19 , 20 , 21 ] and human T-cell leukemia virus type-1 (HTLV-1) [ 10 , 11 , 22 ], and the results indicate that these viruses are preferentially integrated into genes with active transcription [ 10 , 23 , 24 ]; further, both HIV-1- and HTLV-1-infected cells can undergo clonal expansion, which is enhanced by the integration of its provirus in actively transcribed areas of the genome [ 10 , 21 , 25 , 26 , 27 , 28 ]. However, the distribution of BLV proviral integration sites and quantification of clonal expansion in infected cells remains unexplored [ 29 ]. Thus, the impact of BLV integration sites and clonal expansion on lymphoma development remains to be studied in detail.…”

“…Previous studies have shown the applicability of retroviral sequences enrichment using proviral-specific biotinylated probes in the integration-site analysis of retroviruses [ 29 , 30 , 31 , 32 ]. Randomly fragmented DNA containing the junction of provirus–host DNA can be obtained and enriched using virus-specific probes through viral DNA-capture enhancement, and then subjected to analysis of the proviral integration site through high-throughput sequencing.…”

CELLULAR AND EXTRACELLULAR LEVELS OF RETROVIRUS—HOST INTERACTIONS ON THE EXAMPLE OF THE BOVINE LEUKOSE VI-RUS. 2. CRITICAL STAGES — MULTIPLICITY AND VERSATILITY (review)

“…Лимфома, индуцированная BVL, развивается в результате взаимодействия элементов генома вируса и продуктов генома хозяина в дополнение к BoLA-DRB3. Например, интеграция провируса BLV в районе генов хозяина, вовлекаемых в процесс онкогенеза, влияет на их экспрессию (52)(53)(54)(55). В ряде работ отмечается, что достаточно часто интеграция провируса BLV выявляется в районах локализации ретровирусов (56)(57)(58)(59)(60).…”