Deep sequencing of ZIKV stocks and sequence alignment

ZIKV RNA was extracted from virus working stocks using the QIAmp viral RNA minikit (Qiagen, Hilden, Germany). Isolated RNA was then digested with DNase I and purified using the Qiagen RNeasy kit. RNA quality was analyzed on the 2100 bioanalyzer (Agilent, Santa Clara, CA, USA) using the Agilent RNA 6000 Pico assay and quantified on a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA) prior to rRNA depletion and RNA library prep using the KAPA HyperPrep kit (Roche Diagnostics Corporation, Indianapolis, IN, USA). cDNA library qualities were evaluated on the Agilent 2100 bioanalyzer and quantified with the Qubit fluorometer before sequencing on a NextSeq 500 sequencing platform (Illumina, San Diego, CA, USA). 2 × 76-nucleotide stranded paired-end reads were generated. Raw RNA sequencing (RNA seq) data were depleted of adapters, low quality bases, and human reads. Sequencing files were then loaded onto the de novo assembler Trinity (version 2.9.0) and assembled and aligned as previously described (27, 111). Each sample had approximately 25 million raw reads. Viral sequence alignments were loaded into the JALView software (version 2.11.0) to identify amino acid differences between ZIKV strains (112).