Probe Capture Enrichment Sequencing of amoA Genes Improves the Detection of Diverse Ammonia-Oxidising 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-oxidising archaeal (AOA) and bacterial (AOB), which contribute significantly to the nitrogen cycle in various ecosystems. Amplicon sequencing of amoA is a widely used method; however, it 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 abundance of target genes in heterogeneous environmental DNA restricts a comprehensive analysis to a realisable sequencing depth. In this study, we developed a probe set and bioinformatics workflow for amoA enrichment sequencing using a hybridisation capture technique. Using metagenomic mock community samples, our approach effectively enriched amoA genes with low compositional changes, outperforming amplification and meta-omics sequencing analyses. Following the analysis of metatranscriptomic marine samples, we predicted 80 operational taxonomic units (OTUs) assigned to either AOA or AOB, of which 30 OTUs were unidentified using simple metatranscriptomic or amoA gene amplicon sequencing. Mapped read ratios to all the detected OTUs were significantly higher for the capture samples (50.4 ± 27.2%) 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.