Superscript 4 reverse transcriptase enzyme

Virus populations replicating in macaque plasma or mosquito tissues were sequenced in duplicate using a method adapted from Quick et. al. [45 (link)]. Viral RNA was isolated from mosquito tissues or plasma using the Maxwell 16 Total Viral Nucleic Acid Purification kit, according to manufacturer’s protocol. Viral RNA then was subjected to RT-PCR using the SuperScript IV Reverse Transcriptase enzyme (Invitrogen, Carlsbad, CA). Theoretical input viral template numbers are shown in Tables 1–3 and 5. For sequencing the entire ZIKV genome, the cDNA was split into two multi-plex PCR reactions using the PCR primers described in Quick et. al with the Q5 High-Fidelity DNA Polymerase enzyme (New England Biolabs, Inc., Ipswich, MA). For sequencing solely the barcode region, individual PCR reactions were performed that either used a primer pair generating a 131 bp amplicon (131F: 5’-TGGTTGGCAATACGAGCGATGGTT-3’; 131R: 5’-CCCCCGCAAGTAGCAAGGCCTG-3’) or a 178bp amplicon (178F: 5’-CCTTGGAAGGCGACCTGATGGTTCT-3’; 178R (same as 131R): 5’-CCCCCGCAAGTAGCAAGGCCTG-3’). Purified PCR products were tagged with the Illumina TruSeq Nano HT kit or the and sequenced with a 2 x 300 kit on an Illumina MiSeq.

Viral RNA from positive deer mouse samples was prepared for Next-generation sequencing. Briefly, cDNA was generated using SuperScript IV Reverse Transcriptase enzyme (Invitrogen) with random hexamers. PCR amplification was performed using ARTIC network V3 tiled amplicon primers in two separate reactions by Q5 High-fidelity polymerase (NEB). First-round PCR products were purified using Ampure XP beads (Beckman Coulter). Libraries were prepared using the Nextera XT Library Preparation Kit (Illumina) according to manufacturer protocol. Unique Nextera XT i7 and i5 indexes for each sample were incorporated for dual indexed libraries. Indexed libraries were again purified using Ampure XP bead (Beckman Coulter). Final libraries were pooled and analyzed for size distribution using the Agilent High Sensitivity D1000 Screen Tape on the Agilent Tapestation 2200, final quantification was performed using the NEBNext Library Quant Kit for Illumina (NEB) according to manufacture protocol. Libraries were then sequenced on the Illumina MiSeq V2 using 2 x 250 paired end reads.

Virus populations replicating in macaque plasma or mosquito saliva were sequenced in duplicate using a method adapted from Quick et al.^{55 (link)}. Viral RNA was isolated from mosquito saliva or plasma using the Maxwell 16 Total Viral Nucleic Acid Purification kit, according to manufacturer’s protocol. Viral RNA then was subjected to RT–PCR using the SuperScript IV Reverse Transcriptase enzyme (Invitrogen). Input viral RNA ranged from 294 to 64745 viral RNA templates per cDNA reaction (Supplementary Table 3). The cDNA was then split into two multiplex PCR reactions using the PCR primers described in Quick et. al with the Q5 High-Fidelity DNA Polymerase enzyme (New England Biolabs, Inc., Ipswich, MA). PCR products were tagged with the Illumina TruSeq Nano HT kit and sequenced with a 2 × 300 kit on an Illumina MiSeq.