4.7. Read Mapping

FastQC v.0.11.5 was used to assess the quality of forward and reverse raw read files, granting a PASS status for most fastQC statistics. Then, STAR (v.2.5.3a) was used to map paired-end reads to the most recent assembly of the Atlantic salmon genome (ICSASG_v2; GenBank Accession No. GCF_000233375.1) [41]. Mapping parameters included a maximum of 2 mismatched nucleotides and 20 multiple alignments per read; if exceeded, the read was considered unmapped. Uniquely mapped paired reads were counted and assigned to genes (NCBI S. salar Annotation Release 100) using FeatureCounts (SourceForge Subread package v.1.6.2). Only reads with both ends mapped to the same gene were considered in downstream analyses.

For each sample, forward and reverse reads were also mapped against the POMV genome isolate POMV14-01514 (GenBank Accession Numbers {"type":"entrez-nucleotide-range","attrs":{"text":"MN241407-MN241414","start_term":"MN241407","end_term":"MN241414","start_term_id":"1838318681","end_term_id":"1838318697"}}MN241407-MN241414) using Bowtie2 v.2.2.9. SAMtools v.0.1.19 was used to convert the resulting SAM files to BAM files. Segment coverage of the POMV genome was quantified by converting sorted and indexed BAM files into BED files and then using the genomeCoverageBed command in BEDtools v.2.26.0. The POMV per base coverage was normalized by the proportion of viral reads among the total reads in each sample. Then, mean normalized per base coverage was calculated for each viral segment expressed in each tissue and disease stage.