title: HIV-1 Gag-Pol
design: HIV viral RNA was extracted from plasma using the EZ1 Advanced XL instrument and the EZ1 Virus Mini Kit v2.0 (Qiagen). The extracted RNA was used for amplification of the full-length HIV gag-pol region (~4,58 Kb) through one-step reverse transcription PCR (RT-PCR) with the PrimeScript One Step RT-PCR Kit v2 (Takara) and HIV-specific primers 5'-GCGRCTGGTGAGTACGCC and 5'-CCTAGTGGGATGTGTACTTCTGAAC. The RT-PCR was conducted in three steps: an initial step at 60 C for 1 hour and 94 C for 2 minutes; followed by 60 cycles at 94 C for 1 minute, 61 C for 30 seconds, and 72 C for 5 minutes; and a final extension at 72 C for 10 minutes. The quality of the PCR products was evaluated using agarose gel electrophoresis. The amplified products were then purified and quantified with the Qubit dsDNA Assay Kit (Thermo Fisher). Sequencing library preparation and normalization were performed using the Nextera XT DNA Library Preparation Kit and Nextera XT Index Kit (Illumina). Briefly, 1 ng of purified amplicons underwent enzymatic tagmentation, which fragmented the DNA and added partial adapter sequences to the ends of the amplicons. Indexing PCR was subsequently performed to incorporate index and adapter sequences required for cluster generation on the flow cell during sequencing. The resulting NGS libraries were purified, normalized, and treated with NaOH, followed by equimolar pooling. The libraries were then loaded onto a MiSeq Reagent Nano Kit v2 cartridge (500 cycles), and sequencing was conducted on the Illumina MiSeq instrument according to the manufacturers standard protocol.
identifiers.namespace: SUB15059624
identifiers.id: 3639