*4.2. Single-Genome Sequencing*

A previously described, single genome sequencing assay [38] was modified by designing new antisense primers at the end of *IN*, and used to sequence full-length *pol* from sequential samples. cDNA synthesis and single genome PCR reactions were carried out as described, but using the antisense primer KVL069 (5--TTCTTCCTGCCATAGGARATGCCT AAG-3-) [39] followed by a nested PCR reaction using either 5095- (5--TAATCCTCATCCTG TCTACYTGCCACAC-3-), KVL084 (5--TCCTGTATGCARACCCCAATATG-3-) [39] or 5222- deg (5--TGTCTATAAAACCATCCTYTAGC-3-) antisense primers.

#### *4.3. Single-Replication Cycle Drug Susceptibility Assay*

To study phenotypic drug susceptibility, we used a previously described three plasmidbased retroviral vector system, utilising luciferase expression as a measure of infectivity [13,40]. The p8.9NSX *gag-pol* expression vector, which contains a unique *Apa*I restriction site in *gag* (upstream of the *PR* start codon) and an *EcoR*I restriction site in *vif* (downstream of the *IN* stop codon), was modified by introducing a unique *Cla*I restriction site at the beginning of *IN* (flanking amino acids 4/5), creating the p8.9NSXClaI+ vector. In parallel, mutagenesis was used to introduce the same *Cla*I and *EcoR*I sites in nine of the single genomes amplified from the patient in the single genome assay. The nine single genome variants were selected from different on- and off-RAL treatment time points, which included all the different RAL resistance mutation combinations observed in the patient, with these being: N155H + V151I, Q148R + G140A, Y143R + G163R, Y143R + G163K, Y143C + E92Q, Y143C + T97A, Y143C + G163R, Y143G + G163R as well as a wild-type *IN* from the pre-RAL time point (used as a control and designated ptA\_WT*IN*).

In addition, the *Apa*I restriction site in *gag* and the *Cla*I restriction site at the beginning of *IN* were introduced into a single genome containing L10F, V32I, L33F, M46I, I47V, I54L, A71T, I84V, L89V and L90M in *PR*, and M41L, D67N, L74V, M184V, T215Y, K219E, L100I, K103N and N348I in *RT*. These resistance mutations were present in all single genomes chosen for the analysis. The unique restriction sites were then used for the subcloning of patient-derived *PR* + *RT* (*Apa*I and *Cla*I) or *IN* (*Cla*I and *EcoR*I) into p8.9NSXClaI+, to generate ptA*\_PR* + *RT* and patient-derived *IN* only vectors, respectively (Figure 2). The generation of full-length *pol* vectors was achieved by subcloning patient-derived *IN* fragments into ptA*\_PR* + *RT*, using the *Cla*I and *EcoR*I restriction sites. All mutagenesis was carried out using the QuikChange Lightning Multi Site-Directed Mutagenesis kit or QuikChange II XL Site-Directed Mutagenesis kit (Agilent Technologies), according to the manufacturer's protocol. The presence of mutations was verified by sequencing.

The two other vectors used in this assay are the retroviral expression vector, pCSFLW, which encodes the firefly luciferase reporter gene, and pMDG, which encodes the vesicular stomatitis virus G protein. Viruses were generated by cotransfection of 293T cells as previously described, and then used to infect fresh 293T cells in the presence of serially diluted RAL or EVG [41,42]. The replication fitness of the virus was determined by p24 ELISA (Genscreen™ HIV-1 Ag Assay, Bio-Rad) and expressed as a percent of the ptA\_WT*IN* control, as described previously [13].
