Q5 high fidelity dna polymerase 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.

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.

For the viroid library preparation, circular PSTVd molecules were purified from the pooled total RNA of each week by separating 10 μg of total RNA by 5% polyacrylamide-8 M urea denaturating gel electrophoresis (5%-dPAGE). The circular PSTVd (cPSTVd) molecules were eluted from the gel and precipitated with 2.5 vol. of ethanol. To prepare the two complementary DNA (cDNA) libraries, 1 μg of purified cPSTVd RNA was separately subjected to reverse transcription (RT) using a primer binding at either position 280–269 (L1) or 10–354 (L2) of PSTVd-RG1, respectively, for each week. The resulting cDNA libraries were amplified by polymerase chain reaction (PCR) with Q5 High-fidelity DNA polymerase enzyme (New England Biolabs) in the presence of primers F1/R1 for L1 and F2/R2 for L2 followed by indexing of each library. The 8 libraries (2 libraries per week × 4 weeks) were sequenced using the Illumina MiSeq sequencer at the Laboratoire de Génomique Fonctionnelle de l’Université de Sherbrooke¹. All the primers used in this study are listed in Supplementary Table 1. The deep sequence data generated in this study is deposited in the Gene Expression Omnibus under accession number GSE147577.