In silico evaluation and selection of the best 16S rRNA gene primers for use in next-generation sequencing to detect oral bacteria and archaea.

BACKGROUND: Sequencing has been widely used to study the composition of the oral microbiome present in various health conditions. The extent of the coverage of the 16S rRNA gene primers employed for this purpose has not, however, been evaluated in silico using oral-specific databases. This paper analyses these primers using two databases containing 16S rRNA sequences from bacteria and archaea found in the human mouth and describes some of the best primers for each domain. RESULTS: A total of 369 distinct individual primers were identified from sequencing studies of the oral microbiome and other ecosystems. These were evaluated against a database reported in the literature of 16S rRNA sequences obtained from oral bacteria, which was modified by our group, and a self-created oral archaea database. Both databases contained the genomic variants detected for each included species. Primers were evaluated at the variant and species levels, and those with a species coverage (SC) ≥75.00% were selected for the pair analyses. All possible combinations of the forward and reverse primers were identified, with the resulting 4638 primer pairs also evaluated using the two databases. The best bacteria-specific pairs targeted the 3-4, 4-7, and 3-7 16S rRNA gene regions, with SC levels of 98.83-97.14%; meanwhile, the optimum archaea-specific primer pairs amplified regions 5-6, 3-6, and 3-6, with SC estimates of 95.88%. Finally, the best pairs for detecting both domains targeted regions 4-5, 3-5, and 5-9, and produced SC values of 95.71-94.54% and 99.48-96.91% for bacteria and archaea, respectively. CONCLUSIONS: Given the three amplicon length categories (100-300, 301-600, and >600 base pairs), the primer pairs with the best coverage values for detecting oral bacteria were as follows: KP_F048-OP_R043 (region 3-4; primer pair position for Escherichia coli J01859.1: 342-529), KP_F051-OP_R030 (4-7; 514-1079), and KP_F048-OP_R030 (3-7; 342-1079). For detecting oral archaea, these were as follows: OP_F066-KP_R013 (5-6; 784-undefined), KP_F020-KP_R013 (3-6; 518-undefined), and OP_F114-KP_R013 (3-6; 340-undefined). Lastly, for detecting both domains jointly they were KP_F020-KP_R032 (4-5; 518-801), OP_F114-KP_R031 (3-5; 340-801), and OP_F066-OP_R121 (5-9; 784-1405). The primer pairs with the best coverage identified herein are not among those described most widely in the oral microbiome literature. Video Abstract.

Impact of 16S rRNA Gene Redundancy and Primer Pair Selection on the Quantification and Classification of Oral Microbiota in Next-Generation Sequencing.

This study aimed to evaluate the number of 16S rRNA genes in the complete genomes of the bacterial and archaeal species inhabiting the human mouth and to assess how the use of different primer pairs would affect the detection and classification of redundant amplicons and matching amplicons (MAs) from different taxa. A total of 518 oral-bacterial and 191 oral-archaeal complete genomes were downloaded from the NCBI database, and their complete 16S rRNA genes were extracted. The numbers of genes and variants per genome were calculated. Next, 39 primer pairs were used to search for matches in the genomes and obtain amplicons. For each primer, we calculated the number of gene amplicons, variants, genomes, and species detected and the percentage of coverage at the species level with no MAs (SC-NMA). The results showed that 94.09% of oral bacteria and 52.59% of oral archaea had more than one intragenomic 16S rRNA gene. From 1.29% to 46.70% of bacterial species and from 4.65% to 38.89% of archaea detected by the primers had MAs. The best primers were the following (SC-NMA; region; position for Escherichia coli [GenBank version no. J01859.1]): KP_F048-OP_R030 for bacteria (93.55%; V3 to V7; 342 to 1079), KP_F018-KP_R063 for archaea (89.63%; V3 to V9; undefined to 1506), and OP_F114-OP_R121 for both domains (92.52%; V3 to V9; 340 to 1405). In addition to 16S rRNA gene redundancy, the presence of MAs must be controlled to ensure an accurate interpretation of microbial diversity data. The SC-NMA is a more useful parameter than the conventional coverage percentage for selecting the best primer pairs. The pairs used the most in the oral microbiome literature were not among the best performers. IMPORTANCE Hundreds of publications have studied the oral microbiome through 16S rRNA gene sequencing. However, none have assessed the number of 16S rRNA genes in the genomes of oral microbes, or how the use of primer pairs targeting different regions affects the detection of MAs from different taxa. Here, we found that almost all oral bacteria and more than half of oral archaea have more than one intragenomic 16S rRNA gene. The performance of the primer pairs in not detecting MAs increases as the length of the amplicon augments. As none of those most employed in the oral literature were among the best performers, we selected a series of primers to detect bacteria and/or archaea based on their percentage of species detected without MAs. The intragenomic 16S rRNA gene redundancy and the presence of MAs between distinct taxa need to be considered to ensure an accurate interpretation of microbial diversity data.

In situ substrate-formed biofilms using IDODS mimic supragingival tooth-formed biofilms.

This study aimed to compare the bacterial viability and diversity of a substrate-formed biofilm (SF-biofilm) in situ to a supragingival tooth-formed biofilm (TF-biofilm) in the same group of individuals. The impact of the device/disc position and toothbrushing during the formation of SF-biofilm was also assessed. Two tests were run. In test 1, 15 volunteers wore two hemi-splints carrying six discs of human enamel, glass, and hydroxyapatite for 2 days, and were instructed to not perform any oral hygiene measure. Biofilm samples were collected from the substrates and the contralateral tooth and were analysed using CLSM. In five volunteers, half of the biofilm present on the discs and their contralateral teeth were scraped and analysed using 16S  pyrosequencing. In test 2, the microscopic analysis was repeated only on the SF-biofilm samples, and the volunteers were allowed to brush their teeth. Multivariate analyses revealed that the donors had a significant effect on the composition of the biofilm, confirming its subject-dependent character. The bacterial composition of the SF-biofilm was similar to the TF-biofilm, with significant differential abundance detected in very few taxa of low abundance. The toothbrushing during the formation of SF-biofilm was the only factor that conditioned the thickness or bacterial viability.