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.