The combined application of linear amplification-mediated PCR (LAM-PCR) protocols with next-generation sequencing (NGS) has had a large impact on our understanding of retroviral pathogenesis. Previously, considerable effort has been expended to optimize NGS methods to explore the genome-wide distribution of proviral integration sites and the clonal architecture of clinically important retroviruses like human T-cell leukemia virus type-1 (HTLV-1). Once sequencing data are generated, the application of rigorous bioinformatics analysis is central to the biological interpretation of the data. To better exploit the potential information available through these methods, we developed an optimized bioinformatics pipeline to analyze NGS clonality datasets. We found that short-read aligners, specifically designed to manage NGS datasets, provide increased speed, significantly reducing processing time and decreasing the computational burden. This is achieved while also accounting for sequencing base quality. We demonstrate the utility of an additional trimming step in the workflow, which adjusts for the number of reads supporting each insertion site. In addition, we developed a recall procedure to reduce bias associated with proviral integration within low complexity regions of the genome, providing a more accurate estimation of clone abundance. Finally, we recommend the application of a "clean-and-recover" step to clonality datasets generated from large cohorts and longitudinal studies. In summary, we report an optimized bioinformatics workflow for NGS clonality analysis and describe a new set of steps to guide the computational process. We demonstrate that the application of this protocol to the analysis of HTLV-1 and bovine leukemia virus (BLV) clonality datasets improves the quality of data processing and provides a more accurate definition of the clonal landscape in infected individuals. The optimized workflow and analysis recommendations can be implemented in the majority of bioinformatics pipelines developed to analyze LAM-PCR-based NGS clonality datasets.

Background Human immunodeficiency virus (HIV) increases the risk of acquiring human T-cell lymphotropic virus (HTLV) and subsequently HTLV's progression to tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM). Existing data have exclusively reported generalized rates of HIV and HTLV-1 chronic viral infections in the Dominican Republic. To our knowledge, no published studies have focused on the rates of HTLV-1/2 in transactional sex workers and drug users, both higher risk groups, in the Dominican Republic. Methods From December 2012 to April 2013 we conducted a study to estimate the seroprevalence of HTLV-1/2 immunoglobulin G (IgG) and HIV antibodies among transactional sex workers and intravenous drug users in Santo Domingo, Dominican Republic. Serological status was analysed with behaviour and demographic data. Results We collected and analysed plasma from 200 participants with a mean age of 27.4 y in men and 25.2 y in women. The overall weighted seroprevalence of HTLV-1/2 IgG antibodies was 13.91% (95% CI 7.59 to 20.23) in men and 10.59% (95% CI 4.05 to 17.13) in women. The overall weighted seroprevalence of HIV-1 was 13.91% (95% CI 7.59 to 20.23%) in men and 17.65% (95% CI 9.55 to 25.75) in women. Male intravenous drug users had an exceptionally high rate of HTLV-positive HIV co-infection, at 75% (95% CI 44.99 to 105.01). Although there an association has been found between HTLV/HIV co-infections and sex work, the adjusted odds revealed a confounding role of HIV infection. Conclusions The results highlight the urgent need for enhanced public health preventive strategies among high-risk populations in the Dominican Republic and other resource-constrained Caribbean settings, as well as global adoption of routine screening for HTLV-associated infections, particularly in these high-risk, underserved populations.