Phylogenetics traces the evolutionary relationships between organisms in the same or across habitats [
1]. At the molecular level, efforts to delineate phylogenetic relationships between different microbial species typically rests on the 16S rRNA gene [
2,
3]. Indeed, 16S rRNA gene is the anchor for the current three domains of life phylogenetic tree, and is the bedrock of phylogenetics effort [
4].
This work starts off with the hypothesis of whether it is possible to use the highly conserved regions of the 16S rRNA gene to design universal primers for the polymerase chain reaction (PCR) amplification of amplicons for subsequent sequencing or for direct identification via quantitative polymerase chain reaction (qPCR). Such an effort may result in universal primers useful for profiling the microbial diversity in many habitats via amplicon metagenomics or direct identification via Sanger sequencing of the amplicon.
Efforts were first expended to download the 16S rRNA gene sequence of a variety of disparate microbial species on the de Silva database portal linked to the European Nucleotide Archive. Using an in-house MATLAB phylogenetic analysis software, multiple sequence alignment was conducted for the downloaded 16S rRNA gene sequences to yield a consensus sequence. Visual inspection of the multiple sequence alignment reveals stretches of the gene where there is high level of sequence conservation across different species. This region was used to design the universal primers.
Next, universal primers designed were used in another in-house MATLAB simulated PCR software to conduct computational biology experiments examining whether the primers could amplify a large number of amplicons from different microbial species, and how well the amplicons could be used in accurate identification of the microbial species.
Table 1 shows the identification accuracy and amplification efficiency of the universal primers designed in this study for different microbial species in a simulated microbiota. Results show poor amplification efficiency where only 12 out of 31 microbial species yielded a 16S rRNA gene amplicon. More importantly, 50% of the amplified amplicons could be used for accurate identification of the original microbial species. This suggests that the concept of universal primers for disparate microorganisms may need a rethink, and there is hitherto unknown gene architecture and genetic recombination mechanisms in 16S rRNA gene that impact on the results we observed.
Specifically,
Table 1 also reveals that the amplicon read length for 16S rRNA gene ranges from 81 to 122 base pair for a target 100 base pair region. This suggests that there is substantial sequence diversity and genetic recombination such as alternative splicing occurring in the 16S rRNA gene of different microbial species which has eluded our understanding of the gene thus far. Implications for this observation include: (i) the architecture of the 16S rRNA gene may be affected by environmental and nutritional conditions where similarities in these conditions lead to similar gene architecture and read length, (ii) 16S rRNA gene of microbial species in the same habitat may have similar gene architecture and read length, and (iii) epigenetic effects may be encoded in the 16S rRNA gene at the gene level.
Overall, universal primers for amplifying the 16S rRNA gene in amplicon metagenomics is highly sought after. Results from this work reveals that the concept of universal primers in microbial amplicon metagenomics may need a rethink especially for microbes in divergent environments with different nutritional conditions. Observations of poor amplification efficiencies, poor identification accuracies, and variable amplicon read lengths point to a complex 16S rRNA gene architecture with a larger than anticipated sequence space. A complex 16S rRNA gene architecture would exert tight constraints on yielding a similar read length amplicon for identification, while an expanded sequence space meant that regions with high sequence conservation may be too small for yielding amplicon of sufficient length to afford accurate identification of microbial species. Collectively, 16S rRNA gene is a complex gene with epigenetic encodings, and its use in amplicon metagenomics may require care such as using it to identify species in a particular habitat where microbes with similar metabolic demands and environmental adaptations reside. Such metabolic and environmental adaptations would select for particular 16S rRNA gene architecture and sequence space as the molecule plays an important role in affecting the stability of the large and small ribosome macromolecular complex, which in turn, affects protein synthesis rate and cell growth rate.
Funding
No funding was used in this work.
Conflicts of Interest
The author declares no conflicts of interest.
References
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Table 1.
Identification accuracy and amplification efficiency of universal 16S rRNA primer designed in this study for a simulated microbiota.
Table 1.
Identification accuracy and amplification efficiency of universal 16S rRNA primer designed in this study for a simulated microbiota.
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