2.3. Expression profiling based on short-reads
Sequencing quality was assessed using FastQC software
(http://www.bioinformatics.babraham.ac.uk/). After RNA-Seq, Illumina
adaptors and poly-A segments were excised using the Trimmomatic tool
v.0.39 . Reads shorter than 120 nucleotides (nt) and with a quality
score PHRED < 20 were removed from the dataset. Next,
high-quality reads were mapped to the draft genome (preprint Mazdziarz
et al., 2023) using the STAR v.2.7.11a tool. Obtained
BAM
files were used to create annotations using the stringtie v.2.2.1
software . Splicing variants of individual genes were obtained using the
genomic annotations (GTF file) and the count values for genes and
transcripts were calculated by featureCounts v.2.0.6 . For transcript
expression level, Salmon v.0.13.1 tool was implemented as a mapper . The
numeric values of expressed transcripts were estimated by the tximport
v.1.30.0 package . The statistical test (based on a negative binomial
model) implemented in the DESeq2 v.1.42.0 R library was used to compare
expression profiles of water and land transcripts . The following
cut-off values for significant differentially expressed genes (DEGs) and
transcripts were set: logarithmic fold change (log2FoldChange)
> 1 and adjusted p-value (padj) < 0.05.