Improving the methodology for high throughput mapping of proviral integration sites

Bovine Leukemia Virus (BLV) and Human T-cell leukemia virus-1 (HTLV-1) are closely related delta-retroviruses provoking a polyclonal expansion of infected B- and T-cells respectively, with monoclonal leukemia/lymphoma developing in about ~5% of infected individuals. To date, the molecular mechanisms leading to cellular transformation remain unclear. Both proviruses are largely transcriptionally silent in tumors and their integration sites in the host genome appear very variable. Mapping proviral insertion sites using high throughput sequencing techniques has provided insights into the evolution of BLV/HTLV-1 infections and the expansion of transformed clones in delta-retrovirus induced leukemia/lymphoma.
The methods currently used have a number of limitations such as the utilisation of custom sequencing primers, relatively high sequencing costs, no examination of the 5’LTR host flanking region and a limited dynamic range for determining clone abundance. We have developed an alternative high-throughput sequencing protocol for tracking proviral integration sites and determining clonal abundance in BLV and HTLV-1 infected individuals.
We implemented the use of off-the-shelf Illumina primers for the addition of adapters and indexes, which facilitates library multiplexing and avoids the need for custom sequencing primers. Our method reduces the amount of sequencing of PCR duplicates by reducing the number of PCR cycles via an enrichment of BLV- and HTLV-1-carrying DNA fragments. Moreover, in addition to the proviral 3’LTR, our approach also assays the 5’LTR, providing additional information on the frequency of 5’-end deletions in proviruses and increasing the dynamic range of the assay. We have tested the approach on over 100 BLV and HTLV-1 samples, representing both tumors and asymptomatic stages.
Our approach allowed for a more accurate determination of clone abundance in tumors and by assaying the provirus 5’ end, identified clones overlooked with previously published methods. Finally, by facilitating greater multiplexing of libraries we have reduced the cost to a level where the technique may be attractive in a clinical setting.