Despite combined antiretroviral therapy (cART), HIV+ patients still develop neurological disorders, which may be due to persistent HIV infection and selective evolution in brain tissues. Single-molecule real-time (SMRT) sequencing technology offers an improved opportunity to study the relationship among HIV isolates in the brain and lymphoid tissues because it is capable of generating thousands of long sequence reads in a single run. Here, we used SMRT sequencing to generate ~ 50,000 high-quality full-length HIV envelope sequences (> 2200 bp) from seven autopsy tissues from an HIV+/cART+ subject, including three brain and four non-brain sites. Sanger sequencing was used for comparison with SMRT data and to clone functional pseudoviruses for in vitro tropism assays. Phylogenetic analysis demonstrated that brain-derived HIV was compartmentalized from HIV outside the brain and that the variants from each of the three brain tissues grouped independently. Variants from all peripheral tissues were intermixed on the tree but independent of the brain clades. Due to the large number of sequences, a clustering analysis at three similarity thresholds (99, 99.5, and 99.9%) was also performed. All brain sequences clustered exclusive of any non-brain sequences at all thresholds; however, frontal lobe sequences clustered independently of occipital and parietal lobes. Translated sequences revealed potentially functional differences between brain and non-brain sequences in the location of putative N-linked glycosylation sites (N-sites), V1 length, V3 charge, and the number of V4 N-sites. All brain sequences were predicted to use the CCR5 co-receptor, while most non-brain sequences were predicted to use CXCR4 co-receptor. Tropism results were confirmed by in vitro infection assays. The study is the first to use a SMRT sequencing approach to study HIV compartmentalization in tissues and supports other reports of limited trafficking between brain and non-brain sequences during cART. Due to the long sequence length, we could observe changes along the entire envelope gene, likely caused by differential selective pressure in the brain that may contribute to neurological disease.

The past year has brought substantial progress in the development of functional and structural genomic tools for strawberry. Sequencing of cDNA library clones from the cultivated strawberry Fragaria × ananassa and the diploid model species Fragaria vesca has provided more than 3000 new EST sequences. We have also constructed a large (∼40 kb) insert genomic (fosmid) library from F. vesca. About 33,000 fosmid clones have been picked and spotted onto hybridization filters. Filters have been successfully probed with three single copy gene probes, one gene family probe, and chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) probe sets. The combined cpDNA and mtDNA clone content of the library is about 11%. After correction for organelle insert content, the nuclear genome coverage of the library is about 6×. Complete sequencing of two fosmid clones identified 12 putative protein-encoding genes, four of which were organized in colinearity with the corresponding chromosomal region of Arabidopsis thaliana. We will sequence an additional 50 fosmid clones, and use the resulting sequence data as the basis for developing a novel marker technology, to be described. These genomic tools will provide a basis for connecting specific genes to specific traits in the octoploid, cultivated strawberry, paving the way for implementation of gene-based, marker assisted selection as a tool for strawberry breeders. Opportunity for cross-species comparisons of gene sequence and composition, as well as genome organization and linkage group structure, between Fragaria and other members of the economically important Rosaceae family has been significantly enhanced, thus expanding the relevance of the project results to peach, cherry, apple, rose, brambles, and many other Rosaceous species.

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