ocwieja_hiv_pacbio
Dynamic regulation of HIV-1 mRNA populations analyzed by single molecule enrichment and long read sequencing
PacBio sequencing of HIV transcripts
Alternative RNA splicing affects an estimated 95% of human genes, thereby greatly expanding the number of encoded proteins. Next generation sequencing (NGS) is attractive for studying alternative splicing because of the efficiency and low cost per base, but the short reads typical of NGS only report one or a few splice junctions, thereby failing to specify the full structure for most multiply-spliced RNAs. An extreme example of alternative splicing is provided by the HIV-1 provirus, which is only 9700 bp in length but uses alternative RNA splicing to encode 9 major proteins or polyproteins. Here we report use of single molecule PCR enrichment followed by long-read single molecule sequencing to characterize HIV-1 message populations in primary T cells and transformed human osteosarcoma cells. We find that the clinical isolate HIV-189.6 encodes at least 113 different spliced RNAs after infection of T cells, including a previously unappreciated size class of messages (1kb). Among these are several with new open reading frames (ORFs). HIV-1 message populations differed between cell types, longitudinally during infection, and between T cells from different human donors. These findings open a new window on a little-studied aspect of HIV-1 replication, suggest therapeutic opportunities, and provide a model for the use of single molecule enrichment and next generation sequencing in the study of alternative splicing.