The plasmid sequences of infectious clones and the identities of virus stocks as well as selected samples from mice and mosquitoes were verified by Sanger sequencing, using primers flanking the entire genome or position 1404 (see Table S1 in the supplemental material). Extracted RNA samples were amplified using a Qiagen (Redwood City, CA) one-step reverse transcription-PCR (RT-PCR) kit, forward primer AGCTGTTGGCCTGATATGCG, and reverse primer AGCTGCAAAGGGTATGGCTA. The cycling conditions were as follows: 50°C for 30 min, 95°C for 15 min, and 40 cycles of 94°C for 1 min, 57°C for 1 min, and 72°C for 1 min, followed by 72°C for 10 min and a hold at 4°C. Samples were sequenced at the University of California, Davis, Sequencing Core Facility. Chromatograms were visualized and sequences were called using the Sequencher program (Gene Codes, Ann Arbor, MI). The complete ZIKV genome from the inoculum and samples from pregnant rhesus macaque 5338, whose fetus died at 7 dpi, was sequenced at both the University of California, San Francisco, using an established protocol (
66), and Lawrence Livermore National Laboratories, using a different established protocol (
67). Samples from all other rhesus macaques and other samples in this study were sequenced using a different next-generation sequencing protocol that was previously described (
68,
69) and adapted here to flank the sequence from position 1404. After viral RNA isolation using QIAzol solution and RNA quantification by qRT-PCR, at least 1,000 genome copies/sample were used to generate libraries for sequencing. Five microliters of ZIKV RNA was used in a cDNA synthesis reaction with a SuperScript IV (SSIV) kit (Invitrogen, Thermo Fisher Scientific, Emeryville, CA), in addition to 6 μl of nuclease-free water, 1 μl of a 10 mM deoxynucleoside triphosphate (dNTP) mix, and 1 μl of random hexamers. The mixture was heated to 70°C for 7 min and immediately placed on ice. A new mixture containing 4 μl of 5× SSIV buffer with 1 μl of 100 mM dithiothreitol, 1 μl of RNase inhibitor, and 1 μl of SSIV reverse transcriptase was added, and cDNA synthesis occurred under thermocycler conditions of 23°C for 10 min, 50°C for 45 min, 55°C for 15 min, and 80°C for 10 min and then a hold at 4°C until further use. The sequence at position 1404 was amplified using 1 μl each of 10 μM forward primer CCCTAGCGAAGTACTCACAGCT and reverse primer TACACTCCATCTGTGGTCTCCC, 2.5 μl of cDNA, 15 μl of nuclease-free water, 0.5 μl of Q5 high-fidelity DNA polymerase (New England Biolabs, Ipswich, MA), 1 μl of 10 mM dNTPs, and 5 μl of 5× Q5 reaction buffer, followed by 98°C for 30 s, 95°C for 15 s, and 65°C for 5 min, then repetition of steps 2 and 3 for 34 additional cycles, and then a hold at 4°C until further use. The PCR products were purified using Agencourt AMPure XP magnetic beads (Beckman Coulter, San Jose, CA) at a 1.8:1 ratio of beads to sample. Sequencing libraries were next generated using a Kapa Hyper preparation kit (Roche, Pleasanton, CA). Specifically, the ends were repaired by mixing 1.75 μl of end-repair and A-tailing buffer, 0.75 μl of end-repair A-tailing enzyme mix, and 12.5 μl of amplified DNA, followed by incubation at 20°C for 30 min and then 65°C for 30 min. For adaptor ligations, 2.5 μl of 250 nM NEXTflex dual-index DNA barcodes (Bioo Scientific, Austin, TX) was used with 15 μl of the end-repair reaction product, 2.5 μl of DNA ligase, and 7.5 μl of ligation buffer, which were incubated at 20°C for 15 min. This procedure was followed by a postligation cleanup using Agencourt AMPure XP magnetic beads at a ratio of 0.8:1 beads to sample. The sequencing library was then amplified using 17 μl of 2× Kapa HiFi HotStart ReadyMix (Roche, Pleasanton, CA), 2 μl of Illumina (Redwood City, CA) primer mix, and 15 μl of adaptor-ligated library, followed by 98°C for 45 s, 98°C for 15 s, 60°C for 30 s, and 72°C for 30 s and then a repetition of steps 2 to 4 for 8 cycles, followed by 72°C for 1 min and a hold at 4°C until further use. The amplified samples were then cleaned using Agencourt AMPure XP (Beckman Coulter, San Diego, CA) magnetic beads in a ratio of 0.8:1 beads to sample. DNA library sizes were then analyzed using a Bioanalyzer DNA 1000 kit (Agilent Santa Clara, CA), and the DNA concentration was quantified using a Qubit high-sensitivity DNA kit (Thermo Fisher Scientific, Emeryville, CA). Libraries were diluted to 2 nM in 10 mM Tris-EDTA (TE) buffer, and samples were sequenced with a MiSeq apparatus (Illumina, Hayward, CA), using a paired-end approach. We used a previously described work flow (
63) to determine M1404I allele frequencies. Briefly, sequence reads were trimmed to remove the primer sequences and low-quality base calls before they were aligned to the Zika virus WT SPH2015 reference genome using the BWA-mem program (
70). Mutants with over a 3% minor allele frequency were called using the SAMtools mpileup tool (
71) and were filtered according to frequency and strand biases. After sequencing, the ratios of G (encoding M1404) versus A (encoding I1404) were calculated and are represented as a percentage of the total sequencing depth at the locus.