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.