Does universal 16S rRNA gene amplicon sequencing of environmental communities provide an accurate description of nitrifying guilds?

Abstract

Universal (i.e., targeting most bacteria/prokaryotes) 16S rRNA gene based amplicon sequencing is widely used for assessing microbial communities due to its low cost, time efficiency, and ability to provide a full overview of the community. However, it is currently unclear if it can yield reliable information on specific microbial guilds, as obtained by using primer sets targeting functional genes or specific16S rRNA gene sequences. Here, we compared the relative abundance, diversity, richness, and composition of selected guilds (nitrifiers), obtained from universal 16S rRNA gene based amplicon sequencing and from guild targeted approaches. The universal amplicon sequencing provided 1) accurate estimates of nitrifier composition, 2) clustering of the samples based on these compositions consistent with sample origin, 3) estimates of the relative abundance of the guilds correlated with those obtained from the targeted approaches and within ~1.2 orders of magnitude of them, but with measurable bias that should be considered when comparing estimates from both approaches. In contrast, the diversity and richness estimations using the universal 16S rRNA based amplicon sequencing were likely limited by the sequencing depth; therefore, we suggest preferring targeted approaches for assessing nitrifiers diversity and richness or using sequencing depth larger than those currently typically practiced. Overvall, we conclude that universal amplicon sequencing provides, in a single analysis, useful information on the abundance and composition of diverse guilds in complex environmental communities.

Use of Newly Designed Primers for Quantification of Complete Ammonia-Oxidizing (Comammox) Bacterial Clades and Strict Nitrite Oxidizers in the Genus Nitrospira

“…differ substantially, an accurate estimation of their individual abundance is crucial for understanding the roles of Nitrospira in environments. Previously, the nxrB gene (encoding NXR ␤-subunit), which is phylogenetically congruent with the 16S rRNA gene, was consistently chosen as a biomarker for sNOB (16) and applied to the estimation of diversity and abundance of Nitrospira in various environments (17)(18)(19)(20). However, since the nxrB gene of comammox bacteria and sNOB are affiliated with Nitrospira sublineage II, neither the nxrB gene nor the 16S rRNA gene can be effectively used for differentiation of sNOB from comammox Nitrospira (21).…”

Extended spectrum β-lactamase and carbapenemase genes are substantially and sequentially reduced during conveyance and treatment of urban sewage

“…(2) by considering the cell concentration N (cells ml −1 ), the average 16S rRNA gene copies per cell (16Scell, copies cell −1 ) and relative abundance of ARG or MGE (RA, copies (16S rRNA gene copy) −1 ). For each amplicon library, universal 16S rRNA gene amplicon sequencing data was used to perform CaRcone analysis to obtain the average 16S rRNA gene copies per cell (R script https://github.com/ardagulay/CaRcone-Community-average-rRNA-gene-copy-nrestimator) 57 . …”

“…by considering the cell concentration N (cells ml -1 ), the average 16S rRNA gene copies per cell (16Scell, copies cell -1 ) and relative abundance of ARG or MGE (RA, copies (16S rRNA gene copy) -1 ). For each amplicon library, universal 16S rRNA gene amplicon sequencing data was used to perform CaRcone analysis to obtain the average 16S rRNA gene copies per cell (R scripthttps://github.com/ardagulay/CaRcone-Community-average-rRNA-gene-copynrestimator)57 .Concentration of ARG or MGE (copies ml-1 ) = RA × 16Scell × N(2)The observed flux of each gene or taxon (copies hour -1 or cells hour -1 ) at the STP entrance was obtained by multiplying the concentration of ARG or MGE (copies ml -1 or cell ml-1 ) with the average flow rate (ml hour -1 ) of the sewage entering STP. This was compared with the expected flux calculated from the abundances measured in the hospital and residential sewers, under the assumption of simple conveyance of the genes or the taxa from the sewer sampling points to the entrance of STP, and taking into account the respective contribution of both sources to the STP influent.…”

Recovery of human gut microbiota genomes with third-generation sequencing

“…There are two main methods for bacteria metagenome sequencing: 16S rRNA-based sequencing and whole-metagenome shotgun sequencing (WGS). The 16S rRNA-based sequencing is widely used to assess microbial communities due to its low cost, time efficiency, and ability to provide a full overview of the community 11 , 12 . However, as only a single region of 16s rRNA in the genome is detected, it provides limited information about the microbial community 13 , 14 .…”