![]() ![]() Previous studies have identified that replication-competent HIV-1 proviruses persist in naïve and resting memory CD4 + T cell subsets (including central, transitional and effector memory T cells), indicating the importance of targeting these cells in future eradication strategies 2, 8, 9.Įarly insights into the distribution, dynamics and maintenance of the latent HIV-1 reservoir were achieved through utilization of single-proviral sequencing (SPS) methods that genetically characterize sub-genomic regions of the HIV-1 genome 10, 11, 12, 13. Two recent studies have found that approximately 5% of HIV-1 proviruses isolated from individuals on long-term ART are genetically intact, and potentially replication-competent, and may contribute to the rapid rebound in HIV-1 plasma levels upon cessation of ART 1, 2, 7. Some mechanisms that generate defective proviral sequences include the error-prone HIV-1 reverse transcriptase enzyme 3, template switching 4, and/or APOBEC-induced hypermutation 5, 6. During the process of reverse transcription, errors are introduced into the integrated proviral sequence. Genetic characterization of the latent HIV-1 reservoir, which persists in individuals on long-term antiretroviral therapy (ART), has been vital to understanding that the majority of integrated proviruses are defective and replication-incompetent 1, 2. FLIPS is a useful tool for understanding the genetic landscape of integrated HIV-1 proviruses, especially within the latent reservoir, however, its utilization can extend to any application in which the genetic composition of integrated HIV-1 is required. FLIPS amplifies and sequences near full-length proviruses enabling replication competency to be determined, and also uses fewer amplification primers, preventing the consequences of primer mismatches. FLIPS provides advantages over traditional methods designed to sequence integrated HIV-1 proviruses, such as single-proviral sequencing. The FLIPS protocol involves: lysis of HIV-1 infected cells, nested PCR of near full-length HIV-1 proviruses (using primers targeted to the HIV-1 5' and 3' LTR), DNA purification and quantification, library preparation for Next-generation Sequencing (NGS), NGS, de novo assembly of proviral contigs, and a simple process of elimination for identifying replication-competent proviruses. The FLIPS assay can be utilized to identify HIV-1 proviruses that lack these defects and are therefore potentially replication-competent. Through identifying defects within HIV-1 proviral sequences that arise during reverse transcription, such as large internal deletions, deleterious stop codons/hypermutation, frameshift mutations, and mutations/deletions in cis acting elements required for virion maturation, FLIPS can identify integrated proviruses incapable of replication. FLIPS allows determination of the genetic composition of integrated HIV-1 within a cell population. First-time users of CLC Gx on our workstation computers must complete the Workstation Request Form.The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near full-length (intact and defective), HIV-1 proviruses.All USC users can freely access the software on our workstation computers. ![]() Equipped with dual-CPU and 512GB RAM, one of our workstation computers is configured specifically to handle large data set and computationally intensive tasks such as de novo genome assembly and sequencing alignment.Wilson Dental Library, the University Park Campus.Norris Medical Library (RM203A), the Health Sciences Campus.The software has been installed on multiple workstation computers:.On workstation computers in the libraries.Mandatory registration is required for installing CLC Gx on your computer. Please submit the Local Installation Request Form.The computer must be connected to the USC network, either via Ethernet cable on campus or via USC VPN when using wireless (applies to both on- and off-campus wireless connections).Minimum hardware requirement for de novo assembly, metagenomics, and raw reads alignment:: 32GB RAM and Intel i7-6700 or faster processor.Minimum hardware requirement for general use: 16GB RAM and Intel i7-2600 or faster processor.The license consists of TWO concurrent user seats. USC has licensed CLC Gx for the free use of USC faculty, students and staff. ![]()
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