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

Housekeeping genes have proven to be effective taxonomic markers for characterizing bacterial microbiota in short-read amplicon metabarcoding studies. A region of the <i>rpoB</i> 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 <i>rpoB</i> 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 optimize the <i>rpoB</i> procedure. The first PCR amplifies a region of the <i>rpoB</i> gene with the outer primers, and the second PCR then uses primers incorporating Illumina adapters, the inner primers, to amplify the taxonomic marker for metabarcoding. We first used <i>in silico</i> approaches to evaluate the universality of the outer and inner <i>rpoB</i> primers. We then tested the nested <i>rpoB</i> PCR method on commercial mock samples. The nested PCR approach increased amplification efficiency for dilute samples without biasing the bacterial composition of the mock sample revealed by metabarcoding relative to single-step PCR. We also tested the nested <i>rpoB</i> PCR method on field-collected samples of insects. The nested PCR outperformed single-step PCR, increasing amplification efficiency for bacterial DNA present at low concentrations (insect oral secretions) or embedded in eukaryotic DNA matrices (insect larvae). This method provides a promising new strategy for characterizing host-associated microbiota.IMPORTANCEIn this study, we improved an existing method for characterizing host-associated bacterial microbiota using a genetic marker targeting a specific subregion of the <i>rpoB</i> gene. The two-step PCR approach enables more efficient amplification of bacterial DNA at low concentrations or within complex host DNA matrices, such as those found in insects. We optimized the cycle number in each PCR step to minimize bias in bacterial community composition, validated through tests on mock communities. This method provides a robust and reliable solution for analyzing host-associated microbiota, enhancing amplification efficiency and improving result accuracy.