Nanopore Sequencing Data Processing

The fast5 files generated during sequencing were submitted to the pipeline defined by Black and colleagues [25 (link)] with minor modifications. In brief, the sequencing data were basecalled on the high-accuracy model performed by Guppy v.3.4.4 (Oxford Nanopore Technologies, UK). The basecalled fastq files with a minimum Q score of 7 were selected for the subsequent demultiplex process using Guppy v.3.4.4 (Oxford Nanopore Technologies, UK). A re-demultiplex process, trimming adapters, and chimeras were performed by Porechop v.0.2.4 (https://github.com/rrwick/Porechop, accessed on 29 September 2022). Furthermore, the assembly was performed by Burrows–Wheeler Aligner (BWA) v.0.7.17-r1188 [26 (link)] using NCBI Genbank accession number KJ776791.1 as genome reference. The primer sequences were trimmed with align_trim.py. The assembly was then polished, and the variant calling was performed by nanopolish v.0.11.3 (https://github.com/jts/nanopolish, accessed on 29 September 2022). The consensus sequences were then masked with “N” at regions with coverage depth <20, and the variant candidates were incorporated into the consensus genome using VCFtools v.0.1.16 [27 (link)]. The assembly statistics were calculated with SAMtools v.1.10 (using HTSlib 1.10.2) [28 (link)] and Seqtk v.1.3-r106 (https://github.com/lh3/seqtk, accessed on 29 September 2022).