Design of 16S rRNA gene primers for 454 pyrosequencing of the human foregut microbiome

Abstract

Aim: To design and validate broad-range 16S rRNA primers for use in high throughput sequencing to classify bacteria isolated from the human foregut microbiome.

Methods: A foregut microbiome dataset was constructed using 16S rRNA gene sequences obtained from oral, esophageal, and gastric microbiomes produced by Sanger sequencing in previous studies represented by 219 bacterial species. Candidate primers evaluated were from the European rRNA database. To assess the effect of sequence length on accuracy of classification, 16S rRNA genes of various lengths were created by trimming the full length sequences. Sequences spanning various hypervariable regions were selected to simulate the amplicons that would be obtained using possible primer pairs. The sequences were compared with full length 16S rRNA genes for accuracy in taxonomic classification using online software at the Ribosomal Database Project (RDP). The universality of the primer set was evaluated using the RDP 16S rRNA database which is comprised of 433 306 16S rRNA genes, represented by 36 phyla.

Results: Truncation to 100 nucleotides (nt) downstream from the position corresponding to base 28 in the Escherichia coli 16S rRNA gene caused misclassification of 87 (39.7%) of the 219 sequences, compared with misclassification of only 29 (13.2%) sequences with truncation to 350 nt. Among 350-nt sequence reads within various regions of the 16S rRNA gene, the reverse read of an amplicon generated using the 343F/798R primers had the least (8.2%) effect on classification. In comparison, truncation to 900 nt mimicking single pass Sanger reads misclassified 5.0% of the 219 sequences. The 343F/798R amplicon accurately assigned 91.8% of the 219 sequences at the species level. Weighted by abundance of the species in the esophageal dataset, the 343F/798R amplicon yielded similar classification accuracy without a significant loss in species coverage (92%). Modification of the 343F/798R primers to 347F/803R increased their universality among foregut species. Assuming that a typical polymerase chain reaction can tolerate 2 mismatches between a primer and a template, the modified 347F and 803R primers should be able to anneal 98% and 99.6% of all 16S rRNA genes in the RDP database.

Conclusion: 347F/803R is the most suitable pair of primers for classification of foregut 16S rRNA genes but also possess universality suitable for analyses of other complex microbiomes.

Figure 1

Classification accuracy is dependent on amplicon size. Full length sequences were trimmed to 900, 800, 700, 600, 500, 400, 350, 300, 200,100, and 50 bases with each amplicon starting at Escherichia coli base 28. Each sequence trim was uploaded onto Ribosomal Database ProjectII classifier and the results at each taxonomical level were compared to results obtained using the full length sequence. Percent classification accuracies at the genus level for each amplicon size trim are shown.

Figure 2

Design of amplicons for in silico evaluation. Amplicons designed using the 6 universal primer sets as described in Table 3 were evaluated for theoretical forward and reverse reads. The schematic shows the relative position of each amplicon read and read direction, as well as which bases would be included in the sequence. The positional template is the Escherichia coli (E. coli) 16S rDNA gene with the approximate locations of the hypervariable regions labeled.

Figure 3

Optimization of primers used to generate amplicon B. European Ribosome Database primers 343F (A) and 798R (B) were optimized to generate maximal % match with corresponding region in the 16S sequences of foregut species on a base by base manner. Each primer base was analyzed for the number of matches with the corresponding base of all 219 foregut species studied [base # assigned by position within Escherichia coli (E. coli) 16S sequence]. Most bases for both 343F and 798R showed 100% match (219/219), however some bases had slight mismatch % and a few bases had significant mismatch % (base 798, 784, and 359, 360). To have a better homology between the primers and the foregut 16S sequences, the bases with significant mismatch were adjusted to result in lower % mismatch. This was accomplished by changing 798A to R (increasing match from 197 to 213/219) and by changing 359G and 360G to R (increasing match from 169 and 163 to 212 and 219/219, respectively). Further modifications of primers were made to make them more suitable for polymerase chain reaction (PCR) reactions. R primer 5’ end was shifted from 798 to 803, and 3’ end from 784 to 785. F primer 5’ end was shifted from 343 to 347 and 3’ end from 357 to 365. These changes provided suitable melting and annealing temperatures for the designed primer pairs in our PCR reactions. Resulting primers were designated 347F and 803R.