2.7 Differential gene expression analysis
Differences in gene expression among groups were identified by differential expression analysis performed using DESeq2 on raw read counts (non-normalized, as suggested by DESeq2). To allow comparison between 3’ Tag-Seq reads and whole mRNA-Seq (NEB) reads data, for the latter we only used one sequencing direction as suggested for the DESeq2 program. The false discovery rate (FDR) was adjusted to 0.05, corresponding to a recovery at most of 5% of false positives. We use the default options for all the other parameters. We look at differences in gene expression for the following comparisons between: 1.sampling methods (dip netting vs electrofishing; data analyzed with 3’ Tag-Seq, NEB, and Tag-Seq vs. NEB), 2. delaying the tissue harvesting to 5 min after death or harvesting immediately (only for samples obtained by electrofishing, immediate sampling vs delayed of 5 min, data analyzed with 3’ Tag-Seq, NEB, and 3’ Tag-Seq vs. NEB),3. comparisons 1 and 2 per tissue type (blood, gills, muscle; 3’ Tag-Seq data), 4. 3’ Tag-Seq vs NEB for all 14 samples for which we have both data independently of sampling method and tissue harvesting time, 5. tissue type within each group (sampling method or tissue harvesting time; 3’ Tag-Seq data) (see Supporting Information Table S1 per information about sample size for each comparison; minimum N = 4). The log fold changes obtained from DESeq2 were used as a measure of how many more (or less) genes are expressed in one group versus the other. We considered genes having different expression if the adjusted p-value (using the adjusted p-value results in less false positives) was < 0.05.
Finally, previous work has indicated an increase in read count for longer transcripts using the whole mRNA library (NEB) than 3’ Tag-Seq (Ma et al. 2019). To further address the relationship between gene length and genes differentially expressed between 3’ Tag-Seq and whole mRNA library (NEB), we conducted an assessment using the known transcript length from orthologous genes in zebrafish in Ensembl 101 (Yates et al. 2020) based on gene name for genes that were recovered to be differentially expressed between the two library methods. We also used the same approach to specifically assess if transcript length could influence absence of gene expression (expression = 0) in one but not the other technique, 3’ Tag-Seq or NEB.