rpoB, a promising marker for analyzing the diversity of bacterial communities by amplicon sequencing

Abstract

Background Microbiome composition is frequently studied by the amplification and high-throughput sequencing of specific molecular markers (metabarcoding). Various hypervariable regions of the 16S rRNA gene are classically used to estimate bacterial diversity, but other universal bacterial markers with a finer taxonomic resolution could be employed. We compared specificity and sensitivity between a portion of the rpoB gene and the V3V4 hypervariable region of the 16S rRNA gene. Results We first designed universal primers for rpoB suitable for use with Illumina sequencing-based technology and constructed a reference rpoB database of 45,000 sequences. The rpoB and V3V4 markers were amplified and sequenced from (i) a mock community of 19 bacterial strains from both Gram-negative and Gram-positive lineages; (ii) bacterial assemblages associated with entomopathogenic nematodes. In metabarcoding analyses of mock communities with two analytical pipelines (FROGS and DADA2), the estimated diversity captured with the rpoB marker resembled the expected composition of these mock communities more closely than that captured with V3V4. The rpoB marker had a higher level of taxonomic affiliation, a higher sensitivity (detection of all the species present in the mock communities), and a higher specificity (low rates of spurious OTU detection) than V3V4. We applied both primers to infective juveniles of the nematode Steinernema glaseri. Both markers showed the bacterial community associated with this nematode to be of low diversity (< 50 OTUs), but only rpoB reliably detected the symbiotic bacterium Xenorhabdus poinarii. Conclusions Our results confirm that different microbiota composition data may be obtained with different markers. We found that rpoB was a highly appropriate marker for assessing the taxonomic structure of mock communities and the nematode microbiota. Further studies on other ecosystems should be considered to evaluate the universal usefulness of the rpoB marker. Our data highlight two crucial elements that should be taken into account to ensure more reliable and accurate descriptions of microbial diversity in high-throughput amplicon sequencing analyses: i) the need to include mock communities as controls; ii) the advantages of using a multigenic approach including at least one housekeeping gene (rpoB is a good candidate) and one variable region of the 16S rRNA gene.

Entomopathogenic nematode-associated microbiota: from monoxenic paradigm to pathobiome

“…Rarefaction curves generated on quality-filtered reads indicated that sequencing depth was sufficient (Additional file 2). As previously observed [41], the rpoB marker gave a better taxonomic assignment, with 56% and 32% of the OTUs associated with a genus or species name, respectively (Additional file 5). Based on these results, we pursued the two-marker approach.…”

“…*OTU present in the control samples Steinernema infective stages. We first molecularly characterised the microbiota of four Steinernema species with two markers, the V3V4 region of the 16S rRNA gene and a 435 bp region of the rpoB gene, because multigene approaches have been shown to improve the reliability of bacterial community description in ecosystems of low complexity [41]. We also characterised the Steinernema microbiota by culture-based methods.…”

“…In metabarcoding analysis based on the V3V4 region of the 16S marker, only 57 and 12% of the OTUs could be associated with a genus or species name, respectively (Additional file 5). For refinement of the taxonomic assignment of OTUs, we used the rpoB marker, a 435-bp region of the housekeeping rpoB gene [41]. The sequencing of rpoB marker amplicons generated 96,825 reads (mean = 16,137 reads per sample; number of samples = 6).…”

“…The first amplification step was performed on 10 to 100 ng of DNA, with iProof TM DNA Polymerase (Bio-Rad), in a Bio-Rad thermocycler. The hyper variable V3V4 region of the 16S rRNA gene was targeted with the universal primers F343 (5′-CT TTCCCTACACGACGCTCTTCCGATCTTACGGRAG GCAGCAG-3′) and R784 (5′-GGAGTTCAGACGTG TGCTCTTCCGATCTTACCAGGGTATCTAATCCT-3′), and the rpoB fragment was targeted with the primers Univ_rpoB_F_deg (5′-CTTTCCCTACACGA CGCTCTTCCGATCTGGYTWYGAAGTNCGHGAC GTDCA-3′) and Univ_rpoB_R_deg (5′-GGAGTTCA GACGTGTGCTCTTCCGATCTTGACGYTGCATGT TBGMRCCCATMA-3′) [41]. The Illumina adapters are indicated in non-bold characters.…”

Carbon steel corrosion by bacteria from failed seal rings at an offshore facility

“…Genomic DNA was extracted using DNeasy PowerBiofilm Kit (Qiagen), as described by the manufacturer. The number of total bacteria was estimated by quantifying the number of copies of the rpoB gene, which is a single-copy gene 102 . PCR reaction was conducted as described elsewhere 83,103 .…”