Probe capture enrichment sequencing of amoA genes discloses diverse ammonia-oxidizing archaeal and bacterial populations

The ammonia monooxygenase subunit A ( amoA ) gene has been used to investigate the phylogenetic diversity, spatial distribution, and activity of ammonia-oxidizing archaeal (AOA) and bacterial (AOB) populations that contribute greatly to the nitrogen cycle in various ecosystems. Amplicon sequencing of amoA genes is a widely used method; however, it generally produces inaccurate results owing to the lack of a ‘universal’ primer set. Moreover, currently available primer sets suffer from amplification biases, which can lead to severe misinterpretation. Although shotgun metagenomic and metatranscriptomic analyses are alternative approaches without amplification bias, the low concentration of target genes in heterogeneous environmental DNA restricts comprehensive analysis to a realizable sequencing depth. Here, we developed a method for amoA gene enrichment sequencing using a probe capture hybridization technique. Using mock community samples, our approach effectively enriched amoA genes with low compositional changes, outperforming amplification and meta-omics sequencing analyses. Following the analysis of marine metatranscriptome samples, we predicted 79 AOA and AOB operational taxonomic units (OTUs), of which 33 (42%) and 60 (76%) were unidentified by simple metatranscriptomic and amplicon sequencing, respectively. Mapped read ratios were significantly higher for the capture samples (50.4 ± 27.6%) than for non-capture samples (0.05 ± 0.02%), demonstrating the high enrichment efficiency of the method. The analysis also revealed the spatial diversity of AOA ecotypes with high sensitivity and phylogenetic resolution, which are difficult to examine using conventional approaches. Importance The nitrogen cycle is largely driven by prokaryotes via nitrogen metabolism, including ammonia oxidation. To understand the phylogenetic diversity, spatial distribution, and potential activity of these ammonia-oxidizing prokaryotes, the ammonia monooxygenase subunit A ( amoA ) gene has been used. However, current conventional approaches, such as amoA amplicon sequencing and meta-omics (metagenomics and metatranscriptomics), generally produce inaccurate results owing to PCR biases and low concentrations of target genes in the samples. Here, as an alternative, we developed a novel enrichment sequencing method that condenses the amoA gene in metagenomic/metatranscriptomic samples via probe capture hybridization for high-throughput sequencing. We demonstrated that this method is applicable to both artificial mock and oceanic samples and generates reliable results with high phylogenetic resolution and sensitivity. ### Competing Interest Statement Minoru Ijichi is an employee of QIAGEN (Chuo-ku, Tokyo, JAPAN) and a former employee of the Bioengineering Lab (Sagamihara, Kanagawa, Japan). The remaining authors declare no conflicts of interest.