Integrated whole-genome and transcriptome profiling of antithrombotic genes in Asian buffalo leech (Hirudinaria manillensis)

Leech-derived antithrombotic molecules remain of strong biomedical interest because blood-feeding leeches simultaneously suppress coagulation and platelet activation while enhancing fibrinolysis, inflammation, and local tissue barriers. Hirudinaria manillensis has recently emerged as a key genomic reference in this field; however, sample-level integration of whole-genome and RNA-seq data remains limited. Here, we applied an integrated workflow combining whole-genome read mapping, RNA-seq alignment and counting, COI -based species confirmation, phylogenetic analysis, reference-guided antithrombotic gene cataloging, transcript-supported discovery, and selected-gene sequence comparison. All four samples were confirmed to be H. manillensis via COI BLAST (top hit MN882684 with 99.842% identity across 1266 bp), and each sample was phylogenetically placed within the H. manillensis reference clade. The whole-genome mapping rates ranged from 72.17% to 92.75% with a mean depth of 108.0×–134.4×, and the RNA-seq alignment rates ranged from 67.62% to 70.49%. Reference-guided analysis recovered 72 known antithrombotic genes matching the published framework, whereas transcript discovery added 38 transcript-supported candidates, of which 30 represent novel isoforms of known loci (gffcompare class code “j”) and 8 represent overlap-type candidates (class codes “o” and “x”). Hirudinaria manillensis elastase inhibitor ( HMEI ) represented the largest supported family, followed by hirustasin -related genes. The expression was markedly heterogeneous across samples, with lefaxin_Hman2 being the most abundant gene and clear sample-specific patterns among HMEI loci. The variant burden was highest for hyaluronidase_Hman2 and progranulin_Hman , indicating that the most highly expressed genes were not always the most variable. Discovery analysis primarily supported class-code “j”, “o”, and “x” transcripts, whereas no high-confidence class-code “u” antithrombotic candidates remained after filtering. Selected-gene alignments confirmed localized substitutions and indel-rich regions in informative loci. This work provides a sample-level genomic and transcriptomic view of antithrombotic diversity in H. manillensis and a practical framework for prioritizing candidates for future functional work.