One step superscript 3 rt platinum taq high fidelity enzyme mix

HIV-1 RNA was extracted from blood plasma samples using the RNeasy Lipid Tissue Mini Kit (QIAGEN) with modifications from the manufacturer’s standard protocol (Esbjörnsson et al. 2010 ). Briefly, 100 µl patient blood plasma was lysed in 1000 μl Qiazol Reagent. Reverse transcription and amplification of partial HIV-1 pol gene were performed using the One-Step Superscript III RT/Platinum Taq High Fidelity Enzyme Mix (ThermoFisher Scientific^TM) with the pol-specific primer pair JA269 and JA272 (Hedskog et al. 2010 ). First-round PCR products were amplified in a nested PCR with DreamTaq Green DNA Polymerase (ThermoFisher Scientific^TM) using pol-specific primers JA271 and JA270 (Hedskog et al. 2010 ). PCR products were sequenced in both directions with the nested PCR primers using the BigDye terminator kit v1.1 (Applied Biosystems). New HIV-1 pol sequences (approximately 1020 nucleotides [nt], HXB2 [K03455] positions 2267–3287) were determined on an ABI PRISM 3130 × 1 Genetic Analyzer (Applied Biosystems).

HIV-1 RNA was extracted from blood plasma samples using the RNeasy Lipid Tissue Mini Kit (QIAGEN) with modifications from the manufacturer’s standard protocol^{50 (link)}. Briefly, 100 µl of blood plasma was efficiently lysed in 1000 µl Qiazol Lysis Reagent (Qiagen). DNA was removed by treating the column with RNase-free DNase 1 (Qiagen) prior to RNA elution in 40 µl nuclease-free water. Reverse transcription and amplification of partial pol gene were performed using the One-Step Superscript III RT/Platinum Taq High Fidelity Enzyme Mix (ThermoFisher Scientific^TM) with the pol-specific primer pair JA269 and JA272^{51 (link)}. First-round PCR products were amplified in a nested PCR with DreamTaq Green DNA Polymerase (ThermoFisher Scientific^TM) using pol-specific primers JA271 and JA270. PCR products were sequenced in both directions with the nested PCR primers using the BigDye terminator kit v1.1 (Applied Biosystems) and the sequences were determined on an ABI PRISM 3130×1 Genetic Analyzer (Applied Biosystems).

The HIV-1 pol sequences were comprised of 1,020 nucleotides, HXB2 [K03455] positions 2267–3287. HIV-1 RNA was purified from patient blood plasma using the RNeasy Lipid Tissue Mini Kit (QIAGEN) as previously described (Esbjörnsson et al., 2010 (link)). Reverse transcription and amplification of partial pol gene were performed using the One-Step Superscript III RT/Platinum Taq High Fidelity Enzyme Mix (Thermo Fisher Scientific™) with the pol-specific primer pair JA269 and JA272 (Hedskog et al., 2010 (link)). First-round PCR products were amplified in a nested PCR with DreamTaq Green DNA Polymerase (Thermo Fisher Scientific™) using pol-specific primers JA271 and JA270 (Hedskog et al., 2010 (link)). PCR products were sequenced in both directions with the nested PCR primers using the BigDye terminator kit v1.1 (Applied Biosystems), and the sequences were determined on an ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems).
Additional Kenyan HIV-1 pol sequences (referred to as published sequences, 2006–2019) were retrieved (October 11th 2021) from the Los Alamos HIV-1 sequence database (Los Alamos National Laboratory, 2019 ). The combined new and published sequences (referred to as the Kenyan dataset) were annotated with information on sampling dates and geographical area of residence during sampling (i.e., province; Coast, Nairobi, and Nyanza).