Nested PCR to optimize <i>rpoB</i> metabarcoding for low-concentration and host-associated bacterial DNA.

ABSTRACT: Housekeeping genes have proven to be effective taxonomic markers for characterizing bacterial microbiota in short-read amplicon metabarcoding studies. A region of the rpoB gene has been shown to minimize Operational Taxonomic Unit (OTU) overestimation bias with a high degree of accuracy, providing better species-level taxonomic resolution (J.-C. Ogier, S. Pagès, M. Galan, M. Barret, and S. Gaudriault, S., BMC Microbiol. 19:171, https://doi.org/10.1186/s12866-019-1546-z). However, the primers for rpoB are highly degenerate, leading to potential problems in the amplification of bacterial DNA present at low concentration in the sample or embedded within eukaryotic matrices, as for the host-associated microbiota. We addressed these limitations by using a two-step PCR approach to

SUBMITTER: Leclerc L 

PROVIDER: S-EPMC12403564 | biostudies-literature | 2025 Sep

REPOSITORIES: biostudies-literature