HPIV3 Genome Consensus Assembly Protocols

Reference-guided genome consensus assemblies were generated for each sample by aligning the sequencing reads to HPIV3 strain 14072 (GenBank accession no. EU424062.1) using BWA-MEM v0.7.5a-r405, removing multimapped reads, and resolving conflicts using a majority rule. (Priority was given in the order A > C > G > T in the event of a tie.) Sequence coverage was determined using the SAMtools depth tool v1.1 (29 (link)). mNGS libraries for day 28 lung organoids and day 14 CV1 passage experiments were prepared as described previously (30 (link)). RNA was extracted from viral supernatant using the Zymo Viral RNA kit and treated with Turbo DNase I at 37°C for 20 min (Thermo Fisher). RNA was reverse transcribed using random hexamers SuperScript IV (Thermo Fisher), and double-stranded cDNA was produced using the same random hexamers with Sequenase 2.0 (Thermo Fisher) and cleaned using a Zymo DNA Clean and Concentrator. Sequencing libraries were generated using one-third volumes of Nextera XT tagmentation followed by 20 cycles of dual-indexed PCR and a 0.8X Ampure bead cleanup (Beckman Coulter). Libraries were sequenced on an Illumina MiSeq. Sequencing reads are deposited in NCBI under BioProject PRJNA524147.