Emergence of biased hypermutation in a heterologous additional transcription unit in recombinant lentogenic Newcastle disease virus

For NGS, recombinant NDV strains were grown to early cytopathic effects in Vero E6 cells. Supernatants were harvested, treated with benzonase (Sigma) and MgCl2 (Applied Biosystems) and stored in Trizol-LS (Invitrogen). RNA was isolated using Direct-zol RNA Microprep (Zymo Research). cDNA was prepared using random primers, and sequencing was performed on an Illumina MiSeq. We performed reference-based assembly using BBTools Suite (v.39.01: https://jgi.doe.gov/data-and-tools/software-tools/bbtools/). After trimming of artefacts, adapters and homopolymers reads were mapped against the reference sequence followed by variant calling and generation of a consensus sequence. This consensus was subsequently used to map the trimmed reads, and Freebayes (V0.9.21: https://github.com/freebayes/freebayes) was used to generate a variant calling file, using a threshold setting of 5%. Sequence results were analyzed using Integrative Genomics Viewer (IGV - https://igv.org/). Raw NGS sequence reads, bam/bai files and consensus sequences are available from the NCBI SRA database: BioProject accession number PRJNA936109. The files in this repository are the VCF reports that belong to these sequences.