title: 1HC3_Day0_V3loop_PrimerID (2 biological replicates)
design: A Primer-ID deep-sequencing protocol of a ~500 bp region encompassing the base of the V3 loop (HxB2 positions 6854 – 7356) of HIV-1 env was designed. Five subjects were chosen for analysis based on higher viral load and different times of rebound. Primers were designed to bind all 5 subjects’ env based on previously obtained SGS sequences. All primers were ordered from IDT using standard desalting as purification and the cDNA-primer was ordered using hand-mixing of all 10 random nucleotides. RNA was extracted from plasma using the Qiagen MinElute Virus Spin Kit. cDNA synthesis was performed using Superscript III and primer 5’-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNN-NNNNNCAGTTTTACAGTAGAAAAATTCCCCTCCACA-3’. cDNA was purified using Agencourt SPRIselect beads with a bead to reaction ratio of 0.6. All purified cDNA was amplified using primers V3-Forward 5’GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAGNNNNTGAGCCAATTCCCATA-CATTATTGTGC and ADPT_2a 5’-GTGACTGGAGTTCAGACGTGTGCTC-3’. PCR was performed using Phusion Polymerase and a cycling protocol of initial denaturation at 98°C for 30 s, 25 cycles of 98°C for 10 s and 72°C for 1 min, and then a final extension at 72°C for 5 min. First-round PCR products were again purified using Agencourt SPRIselect and eluted in a 50 µl volume. 2 µl of the first-round PCR product was then used in a second PCR using primers Universal Adapter 5’-AATGATACGGCGACCACCGAGATCTACACGCCTCCCTCGCGCCATCAGAGATGTG-3’ and and Indexed adapter 5’-CAAGCAGAAGACGGCATACGAGATNNNNNNGTG-ACTGGAGTTCAGACGTGTGCTC-3’ that carried a six-nucleotide fixed barcode that allowed for the identification of specific samples. Second-round PCR was performed using KAPA HiFi Polymerase 2x Ready Mix and cycling conditions of initial denaturation at 95°C for 2 min, followed by 25 cycles of 98°C 20s, 63°C 15s, 72°C 30s, and final elongation of 3 min at 72°C. Second-round PCR product was again purified with Agencourt SPRIselect beads, visualized on a 2% agarose gel, and gel purified using the Macherey-Nagel Gel and PCR Purification Kit. Final products were analyzed using an Agilent Tapestation and Qubit fluorophotomer. Final products were multiplexed and sequenced paired-end on Illumina MiSeq using 2x 300 bp v3 chemistry.