In-Silico Evaluation and Selection of the Best 16S rRNA Gene Primers for Use in Next-Generation Sequencing to Detect Oral Bacteria and Archaea.

Abstract

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 97.14-98.83%; meanwhile, the optimum archaea-specific primer pairs amplified regions 5-6, 3-5 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 94.54-95.71% and 96.91-99.48% for bacteria and archaea, respectively. Conclusions: Given the three amplicon length categories (100-300, 301-600 and >600 bps), the primer pairs with the best coverage values for detecting oral bacteria were: 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: 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.

Impact of 16S rRNA gene redundancy and primer pair selection on the quantification and classification of oral microbiota in next-generation sequencing

“…An initial list of 177 different oral archaea and their corresponding GenBank identifiers, obtained as part of a previous investigation conducted by our research group (20), enabled us to access their complete sequences in the NCBI database.…”

“…We selected the primer pairs with the best in silico coverage values, as identified in previous research by our group, as well as those used most in the oral-microbiome literature (20). This left us with 33 and 6 primer pairs, respectively, for this stage of the study, which were classified according to the average length of the amplicons into: short primer pairs (100-300 bps), medium primer pairs (301-600 bps), and large primer pairs (>600 bps) (20) (Additional files 3 and 4).…”

“…The amplicon length category and gene regions were determined in a previous investigation according to the mean size of the amplicons generated by a given primer and to the mode first position of the forward primer and the mode last of the reverse primer, respectively. The most commonly used primer pairs in the literature were selected in previous research (20) The amplicon length category and gene regions were determined in a previous investigation according to the mean size of the amplicons generated by a given primer and to the mode first position of the forward primer and the mode last of the reverse primer, respectively. The most commonly used primer pairs in the literature were selected in previous research (20) Tables 4 and 5 show the percentages of detected taxa with and without matching amplicons and overabundance estimators obtained by the primer pairs tested on the oralbacteria and oral-archaea genomes.…”

“…The most commonly used primer pairs in the literature were selected in previous research (20) The amplicon length category and gene regions were determined in a previous investigation according to the mean size of the amplicons generated by a given primer and to the mode first position of the forward primer and the mode last of the reverse primer, respectively. The most commonly used primer pairs in the literature were selected in previous research (20) Tables 4 and 5 show the percentages of detected taxa with and without matching amplicons and overabundance estimators obtained by the primer pairs tested on the oralbacteria and oral-archaea genomes. Our selected primer pairs detected 16S rRNA gene amplicons in a range from 99.46% to 88.71% for the bacterial species and 99.26% to 90.37% for the archaeal species; these percentages were lower for the primer pairs used most in the oral microbiome literature (95.16%-74.19% for the bacteria, and 63.70% and 30.37% for the archaea).…”

“…However, the low number of species evaluated (n= 32) and the focus on a specific niche and health condition within the mouth limit the applicability of the findings to the oral microbiota more generally. In contrast, the present study evaluated all of the complete bacterial genomes described in an oral-specific database (18) and a series of genomes taken from archaeal species previously identified in the human mouth (20); all these bacterial and archaeal genomes were downloaded from the NCBI website (19). Moreover, for the first time in the oral microbiome literature, we extracted the 16S rRNA genes using a special and easily accessible tool based on Edgar's algorithm, which has an estimated sensitivity >99% for identifying known genes (27).…”