title: HIV-1 subtype B polymerase 9 clones mixture
design: HIV2exp is a mixture of two variants. We designed nine in silico plasmid constructs comprising a 950-bp region of the HIV-1 subtype B polymerase (pol) gene that were then synthesized and cloned into pUCIDT-Amp (Integrated DNA Technologies, Skokie, IL). Each clone was  confirmed by Sanger sequencing. This 950-bp region at the beginning of polcontains known protease and reverse transcriptase genes that are monitored for drug-resistant mutations and is monitored with sequence analysis for patient care. Each of these plasmids contains a specific set of point mutations chosen using mutation profiles from a realclinical study to create nine unique synthetic HIV-1 pol haplotypes. Different proportions of these plasmids were mixed and then sequenced using an Illumina MiSeq protocol to obtain 2x300-bp reads. We used 50,000 total copies of plasmid DNA from these nine constructs as input for a nested PCR reaction to amplify the polymerase region using the following primary and nested primers respectively: HIV-B PRO-OUT.3F, 5 CCT CAG ATC ACT CTT TGG CAA CG 3 and HIV-RT 215/219.3R, 5 CTT CTG TAT GTC ATT GAC AGT CC 3 Nested PCR: HIV-B PR/RT.2F, 5 CTT TGG CAA CGA CCC CTY GTC CA 3 and HIV-RT 181-190.1.4R, 5 ATC AGG ATG GAG TTC ATA ACC CA 3. The primary and nested PCRs were done using 94C for four minutes, followed by 40 cycles of 94C for one minute, 50C for 30 seconds, and 72C for two minutes and a final extension at 72C for five minutes1. We created two plasmid mixtures to generate artificial mixtures simulating clinical specimens containing many variants at different virus levels. The mixtures comprised nine and two plasmids with varying copy numbers of each plasmid. PCR reactions were generated and purified using the QIAquick PCR purification kit. (Qiagen, Valencia CA) The purified amplicons (10 ng) were subsequently used for NGS library construction using the Nextera XT DNA Library Prep kit (Illumina Inc., San Diego, CA). Libraries were pooled, and enriched for 900-1,000-bp fragments using magnetic bead based size selection (AMPure XP, Beckman Coulter, Brea, CA) and sequenced on a MiSeq v3 (600-cycle) flow cell on the Miseq system ( Illumina Inc., San Diego CA).