title: Black Barley
design: Following cluster creation, paired-end reads were produced by sequencing the library preparations using the Illumina HiSeq X Ten platform. Low-quality, adapter-containing, and poly-N reads were eliminated from the raw data to provide clean reads. Additionally, the clean data's Q20, Q30, and GC contents were computed. With Tophat2 (parameters: -N 5 -p 30 -i 20), all downstream analyses were based on clean, high-quality data that was aligned to the reference genome: Hv_IBSC_PGSB_v2 (Elakhdar et al., 2023). The reference genome is 4.83 Gbp in length, with N50 values of 4 Mbp for contigs and 7 pseudomolecules for scaffolds, respectively (Guan et al., 2016).The longest transcripts of each gene were selected as unigenes. To investigate the activities of mRNA, all constructed unigenes were compared to the NR (NCBI-non-redundant protein sequences) database using the BLAST method, which also found homologs of genes with established functions (Altschul et al., 1997). Blast2GO was used to scan the NR database for the best gene ontology (GO) terms that were obtained (Gtz et al., 2008). To identify and forecast functional classifications and molecular pathways, the assembled unigenes were also compared to the Swiss-Prot (a manually annotated and reviewed protein sequence database), NT (NCBI nucleotide sequences), Pfam (Protein family), KOG (euKaryotic Ortholog Groups)/COG (Clusters of Orthologous Groups of proteins), and KEGG (Kyoto Encyclopedia of Genes and Genomes) Orthology databases.