Linking 16S rRNA Gene Classification to amoA Gene Taxonomy Reveals Environmental Distribution of Ammonia-Oxidizing Archaeal Clades in Peatland Soils

Abstract

The recently established phylogeny of amoA provides a finer resolution than previous studies, allowing clustering of AOA beyond the order level and thus revealing novel clades. While the 16S rRNA gene is mostly appreciated in microbiome studies, this novel phylogeny is in limited use.

Fine-scale evaluation of two standard 16S rRNA gene amplicon primer pairs for analysis of total prokaryotes and archaeal nitrifiers in differently managed soils

“…This resulted in 89,103 ± 3,615 and 21,752 ± 529 reads per sample from primers of 515F-806R and 515F-926R, respectively, which were then taxonomically classified against SILVA database release 138.1 (Quast et al, 2013) using the scikit-learn classifier (Pedregosa et al, 2011) embedded in QIIME2. For more detailed taxonomic classification, sequences of ASVs belonging to class Nitrososphaeria (excluding non-AOA group 1.1c) were picked out and re-classified into putative family-level clades using BLASTN against a custom AOA database (Wang et al, 2021). A total of 4,302 ± 217 and 1,130 ± 50 reads AOA per sample were obtained from primers of 515F-806R and 515F-926R, respectively.…”

“…The theoretical coverage of the two primer pairs for analyzing total prokaryotes was tested in silico by using the TestProbe function of the SILVA database 138.1 server. 3 Additionally, the primers were also evaluated for the coverage of specific AOA clades by Probe Match function of ARB (Ludwig et al, 2004) against the AOA 16S rRNA gene database after removing reads that do not contain the primer locus region (Wang et al, 2021).…”

“…Oxygen availability, pH and temperature have been recognized to drive 1 https://earthmicrobiome.org/protocols-and-standards/16s/ the radiative evolutionary diversification of terrestrial AOA lineages and their differential niche adaptation (Gubry-Rangin et al, 2015;Ren et al, 2019;Yang et al, 2021), leading to a consortium of phylogenetically distinct phylotypes co-existing and competing in the same soil (Gubry-Rangin et al, 2011;Zhao et al, 2015;Huang et al, 2021). Due to the low rate of horizontal gene transfer within Nitrososphaeria (Sheridan et al, 2020), the congruence is well supported between genome-resolved phylogeny and some single-copy marker gene phylogenies, including the 16S rRNA gene (Oton et al, 2016;Sheridan et al, 2020;Wang et al, 2021). So far, 12 putative family-level AOA clades can be confidently identified from established 16S rRNA gene database (Wang et al, 2021), and continuous expansion of metagenome assembled genomes (MAGs) of AOA allow characterization of novel lineages using 16S rRNA genes as biomarker.…”

“…Due to the low rate of horizontal gene transfer within Nitrososphaeria (Sheridan et al, 2020), the congruence is well supported between genome-resolved phylogeny and some single-copy marker gene phylogenies, including the 16S rRNA gene (Oton et al, 2016;Sheridan et al, 2020;Wang et al, 2021). So far, 12 putative family-level AOA clades can be confidently identified from established 16S rRNA gene database (Wang et al, 2021), and continuous expansion of metagenome assembled genomes (MAGs) of AOA allow characterization of novel lineages using 16S rRNA genes as biomarker. However, there is little information about the potential bias toward specific AOA clade by using different 16S rRNA gene primers.…”

Stochastic factors drive dynamics of ammonia-oxidizing archaeal and bacterial communities in aquaculture pond sediment

“…The ammonia monooxygenase subunit A ( amoA ) gene has been widely used to study ammonia oxidizers, and is considered to be second only to the 16S rRNA gene as the most frequently sequenced maker gene in microbial ecology studies ( Alves et al, 2018 ). In addition, it has been acknowledged that a finer resolution of phylogeny can be provided by using the amoA gene ( Wang et al, 2021 ). In this study, to reveal the spatiotemporal dynamics of AOA and AOB communities in response to environmental changes in aquaculture pond sediment, we analyzed amoA gene dynamics in the sediment of aquaculture ponds across three different representative aquaculture areas in China.…”