Probe capture enrichment sequencing ofamoAgenes discloses diverse ammonia-oxidizing archaeal and bacterial populations

Abstract

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 ofamoAgenes 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 foramoAgene enrichment sequencing using a probe capture hybridization technique. Using mock community samples, our approach effectively enrichedamoAgenes 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.