Fig. 14

Workflow for genome assembly, annotation, and comparative analysis of Microsporidia sp. MB. This flowchart outlines the step-by-step process used in the genome annotation pipeline. The workflow begins with sample collection and sequencing of Anopheles arabiensis mosquito ovaries using DNBSEQ technology, followed by genome assembly using SPAdes. The quality of the assembled genome was assessed using QUAST and visualized with BlobToolKit to check for contamination. Genome completeness was evaluated using BUSCO by identifying conserved single-copy orthologs specific to microsporidia. Repeat sequences in the genome were detected using RepeatMasker and RepeatModeler2 to annotate known and novel repeats. Gene prediction was performed using GeneMark-ES, optimized for eukaryotes with intronless genomes. Functional annotation of predicted genes was carried out with InterProScan, assigning proteins to families and domains from databases such as Pfam and CDD. Phylogenomic analysis of the genome was conducted using OrthoFinder for ortholog identification and IQ-TREE for maximum likelihood phylogenetic tree construction. Finally, predicted proteins were mapped to biological pathways using KEGG, and physicochemical properties, including transmembrane regions, were characterized using ProtParam and DeepTMHMM. This comprehensive workflow provides a detailed overview of the computational tools used in the experimental design and their roles in the analysis of the Microsporidia sp. MB genome