RNA sequencing and transcriptome assembly
A total of 18 gut tissue samples from one family, but from all 3
treatments (6 fish per treatment), were used for transcriptome analyses
by RNAseq. Fish from one family were used to minimize differences due to
genetic variability among individuals. RNA quality was assessed using
the Eukaryotic RNA 6000 Nano assay on a 2100 Bioanalyzer (Agilent,
Mississauga, ON). All samples had an RIN > 7 and a 28S:18S
rRNA ratio >1.0. RNAseq libraries were prepared and
sequenced at the McGill University and Genome Quebec Innovation Centre
using the Illumina NovaSeq 6000 S4 PE100 protocol and 100-bp paired-end
sequencing. To remove potentially contaminating rRNA sequences, raw
sequences were filtered against eight default rRNA databases using
SortMeRNA v2.1 (Kopylova et al., 2012). The sequences were then
quality-filtered using Trimmomatic v0.38 (Bolger et al., 2014). The
non-rRNA sequences were aligned to the Chinook salmon
(GCF_002872995.1_Otsh_v1.0;
https://www.ncbi.nlm.nih.gov/assembly/GCF_002872995.1/) reference
genome using the splicing aligner HISAT2 (Kim et al., 2015).
FeatureCounts (Liao et al., 2014), was used to calculate the number of
transcript sequence fragments assigned to each gene.