Development of an integrated workflow (NosZRef) for rapid and accurate nosZ gene profiling and its application to...

The widespread adoption of next-generation sequencing technologies and the rapid growth of publicly available sequence data have generated an unprecedented resource for investigating nitrous oxide (N₂O) reducing microorganisms. However, the lack of standardized practices has hindered cross-study comparisons and limited our ability to thoroughly assess the community structure, diversity and biogeography of N₂O-reducing microorganisms. To address these knowledge gaps, a bioinformatic workflow was developed to standardize the processing of nosZ gene datasets originating from high-throughput sequencing. We first manually curated and constructed a comprehensively annotated reference protein sequence database containing 3,361, 5,665, and 96 sequences from the NosZ-I, NosZ-II, and NosZ-III clades, respectively. Integrated with this database, we developed NosZRef, a fast, accurate and scalable analytical pipeline for nosZ gene profiling. With one single command, NosZRef provides users full-pipeline analysis from raw reads to statistical and visualization outputs. We employed NosZRef to profile nosZ genes across diverse depths in the Tara Oceans dataset, revealing that the three nosZ clades exhibit distinct depth-dependent distribution patterns and ecological strategies. In summary, NosZRef offers high specificity and comprehensive coverage for accurate profiling of N₂O-reducing microorganisms in oceanic ecosystems from high-throughput sequencing data, providing a useful tool for investigating microbially mediated N₂O reduction in oceanic environments. NosZRef is available at https://github.com/ZhangBaoshan668/NosZRef.