HPIV3 Genome Sequencing from Nasal Swabs

A 500-μl volume of nasal swab-inoculated viral transport medium was filtered through a 0.45 μm Ultrafree-MC spin filter (Millipore), and 200 μl of filtrate was used as input for extraction in a ZR viral RNA kit (Zymo Research). Extracted RNA was treated with Turbo DNase (LifeTech) and first- and second-strand synthesis was performed using random hexamers and SuperScript III (Life Tech) and Sequenase (Agilent) enzymes (28 (link)). cDNA was purified using a DNA clean and concentrator-5 kit (Zymo) and used as input for Nextera XT library generation with 20 cycles of PCR amplification. Sequencing libraries were purified using 1.0× AMPure XP beads (Beckman Coulter), quantified on a Qubit 3.0 (LifeTech) and a Bioanalyzer 2100 (Agilent) (29 (link)). Samples were mixed in equimolar to achieve approximately 2 million reads per sample and sequenced using a 1×180 bp run on a MiSeq desktop sequencer (Illumina).
Sequencing reads were adapter- and quality-trimmed using cutadapt and mapped to the NCBI reference genome for HPIV3 (NC_001796) using Geneious v9.1 software (30 (link)). Consensus sequences of regions with >1× coverage were called by majority-voting base with hand curation. Genome alignments were made using MAFFT and phylogenetic trees were created using MrBayes (31 (link), 32 (link)).