Fig. 3.

Schematic of experimental design. a Method 1: viral RNA was isolated from original stock and quantified via qRT-PCR. SIVmac239 and SIVmac239-24 × were diluted to 10⁶ copies/reaction and mixed at the following SIVmac239:SIVmac239-24 × ratios: 100:0, 95:5, 90:10, 75:25, 40:60, and 0:100. Serial dilutions were preformed to 10⁵, 10⁴, and 10³ copies per reaction. Viral cDNA was generated from viral RNA mixes, with one cDNA reaction per vRNA mix. b Method 2: viral RNA was isolated and approximately 10⁷ viral RNA copies were added to each cDNA synthesis reaction. Viral cDNA copies were quantified using qRT-PCR and each was diluted to 10⁶ copies per reaction. SIVmac239:SIVmac239-24 × mixes were generated at the following ratios: 100:0, 95:5, 90:10, 75:25, 50:50, and 0:100. cDNA mixes were then serially diluted to 10⁵, 10⁴, and 10³ copies per reaction. c cDNA was used for multiplex PCR. PCR products were then combined at equimolar ratios and library prepped according to TruSeq Library Preparation documentation (Illumina). Libraries were quantified, pooled, and sequenced using a 2 × 250 v2 MiSeq cartridge