CurateMake: an auditable workflow for multi-source ITS reference database harmonisation and phylogenetic validation

O_LIReference databases shape the taxonomic resolution, uncertainty, and reproducibility of metabarcoding analyses. For ITS barcodes, public references are distributed across repositories with different taxonomic conventions, geographic coverage, and annotation practices, creating conflicts, missing ranks, and misannotations when databases are merged or compared. C_LIO_LIWe introduce CurateMake, a reproducible Snakemake workflow for ITS reference database construction, harmonisation, and validation. It integrates four public sources (UNITE, BOLD, PLANiTS, and CALeDNA) and user-supplied databases, combines Catalogue of Life name harmonisation with ITSx-based region standardisation, MSA/HMM-based alignment grouping, and SATIVA phylogenetic validation. Raw, CoL-harmonised, and SATIVA-validated annotation layers are retained throughout to compare curation effects while preserving flagged records for review. C_LIO_LIWe evaluated CurateMake on 3.58 million ingested sequences and controlled error-injection simulations. ITSx expanded the final harmonised database to 5.19 million barcode-resolved entries by recovering ITS1 and ITS2 sub-regions from full-length ITS records. Across the full dataset, normalised intra-cluster entropy decreased from Raw to CoL-harmonised to SATIVA-validated annotations, consistent with improved taxonomic coherence. In simulations, CurateMake achieved the highest correction rate across 1%-50% corruption and, at 15% corruption, corrected 42% {+/-} 1% of introduced errors, compared with 28% {+/-} 1% for CoL alone and 0% for SATIVA without the workflows alignment infrastructure. C_LIO_LIThese results show that nomenclatural harmonisation and phylogeny-informed validation address complementary error classes, with phylogenetic validation contributing measurably only within taxon-coherent alignments in this benchmark. CurateMake therefore provides a reproducible, provenance-tracked framework for auditable ITS reference database curation in metabarcoding workflows. C_LI