An optimized workflow for accurate taxonomic annotation of high-throughput comammox Nitrospira sequences

High-throughput sequencing of the amoA gene is increasingly used to investigate comammox Nitrospira , but reliable annotation is hindered by the absence of a rigorously curated, phylogeny-consistent reference set. This can lead to unresolved or inconsistent sequence assignments and obscure phylogenetic distinctions. Although these issues can be resolved through phylogenetic analysis, such analyses do not readily scale to the large sequence counts produced by high-throughput sequencing. Here, we constructed a systematically curated and phylogeny-validated reference database of comammox Nitrospira amoA genes by compiling and filtering sequences from published phylogenetic studies. We integrated this database into a streamlined QIIME 2 workflow and evaluated its classification performance using internal validation of 506 phylogenetically validated reference sequences and external validation across independent environmental datasets. The workflow achieved high concordance with phylogenetically validated lineage assignments across independent environmental benchmark datasets and maintained robust classification performance during internal validation. By combining phylogenetically validated reference assignments with automated classification, this workflow enables rapid, reproducible, and large-scale profiling of comammox Nitrospira communities and provides a standardized framework for taxonomic analysis in nitrification research.