Evaluation of 16S rRNA gene sequencing for species and strain-level microbiome analysis

By Jethro S. Johnson, Daniel J. Spakowicz, B. Hong, Lauren M. Petersen, Patrick C. Demkowicz, Lei Chen, Shana Leopold, B. Hanson, Hanako Agresta, Mark Gerstein and 2 more. Nature Communications, 2019

1,833 citations recorded by the source corpus.

The 16S rRNA gene has been a mainstay of sequence-based bacterial analysis for decades. However, high-throughput sequencing of the full gene has only recently become a realistic prospect. Here, we use in silico and sequence-based experiments to critically re-evaluate the potential of the 16S gene to provide taxonomic resolution at species and strain level. We demonstrate that targeting of 16S variable regions with short-read sequencing platforms cannot achieve the taxonomic resolution afforded by sequencing the entire (~1500 bp) gene. We further demonstrate that full-length sequencing platforms are sufficiently accurate to resolve subtle nucleotide substitutions (but not insertions/deletions) that exist between intragenomic copies of the 16S gene. In consequence, we argue that modern analysis approaches must necessarily account for intragenomic variation between 16S gene copies. In particular, we demonstrate that appropriate treatment of full-length 16S intragenomic copy variants has the potential to provide taxonomic resolution of bacterial communities at species and strain level. Here, the authors explore the potential of the 16S gene for discriminating bacterial taxa and show that full-length sequencing combined with appropriate clustering of intragenomic sequence variation can provide accurate representation of bacterial species in microbiome datasets.

Subfields: Ecology, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/a14e48e0ccdabbcafeac5be9e266f6b8aabed3a4

How researchers see this paper

The researchers whose trusted networks rank it highest, strongest advocate first. Every researcher gets a different order.

In Ecology

In Molecular Biology

Citations in the corpus

Primer, Pipelines, Parameters: Issues in 16S rRNA Gene Sequencing (2021, methodology): “Those methods seemingly offer taxonomic identification down to species or even strain level (27).”
The Gut Microbiota and Inflammation: An Overview (2020, background): “Doing so compromises the reliability of species-level classification as a small portion of the gene can have a highly variable ability to detect species [15].”
EzBioCloud: a genome-driven database and platform for microbiome identification and discovery (2024, neutral): “However, limitations arise when these methods are applied at the species level, especially in the case of nearly identical 16S rRNA gene sequences that do not guarantee species congruence [2].”