Env gp120 C2-V5 Region Sequencing

Viral RNA was isolated from cryopreserved plasma samples and purified using the QIAamp Viral RNA Extraction Kit (Qiagen) followed by reverse transcription using the SuperScript III Reverse Transcriptase System (Invitrogen). The Env gp120 C2-V5 (approximately 799 bp, HXB2 [accession number: K03455] positions 6883–7681) was amplified by nested touchdown PCR (primers listed in Table S1). Amplified DNA was purified using the MinElute Gel Extraction Kit (Qiagen), and ligated into a pCR4-TOPO sequencing vector using the TOPO TA Cloning Kit for Sequencing (Invitrogen). Chemically competent One Shot MAX Efficiency DH5α E. coli (Invitrogen) were transformed with the prepared plasmids, and cultured overnight at 37 °C. Eighteen colonies were selected for colony PCR (M13F and M13R primers, Table S1), and the resulting products were purified using ExoSAP-IT (Affymetrix) and sequenced to generate forward and reverse reads (Source BioScience). Contigs were assembled and controlled manually using Geneious v9.0.5^{61 (link)} (HXB2 gp120 positions 661–1455, accession number K03455). Sequences were multiple aligned using MUSCLE^{62 (link)}, and then manually edited in MEGA v6.06^{63 (link)}. Sequences were controlled for intra-patient clustering by maximum-likelihood phylogenetics.