Version 1
: Received: 25 May 2023 / Approved: 26 May 2023 / Online: 26 May 2023 (11:38:01 CEST)
How to cite:
Ng, W. Difficulty in Achieving High Identification Accuracy and High Amplification Efficiency in Bacterial 16s rRNA Amplicon Metagenomics. Preprints2023, 2023051931. https://doi.org/10.20944/preprints202305.1931.v1
Ng, W. Difficulty in Achieving High Identification Accuracy and High Amplification Efficiency in Bacterial 16s rRNA Amplicon Metagenomics. Preprints 2023, 2023051931. https://doi.org/10.20944/preprints202305.1931.v1
Ng, W. Difficulty in Achieving High Identification Accuracy and High Amplification Efficiency in Bacterial 16s rRNA Amplicon Metagenomics. Preprints2023, 2023051931. https://doi.org/10.20944/preprints202305.1931.v1
APA Style
Ng, W. (2023). Difficulty in Achieving High Identification Accuracy and High Amplification Efficiency in Bacterial 16s rRNA Amplicon Metagenomics. Preprints. https://doi.org/10.20944/preprints202305.1931.v1
Chicago/Turabian Style
Ng, W. 2023 "Difficulty in Achieving High Identification Accuracy and High Amplification Efficiency in Bacterial 16s rRNA Amplicon Metagenomics" Preprints. https://doi.org/10.20944/preprints202305.1931.v1
Abstract
Bacterial phylogenetics has largely been determined via 16S rRNA gene sequencing and phylogenetic tree reconstruction. Observed utility of this approach has driven the popularity of the 16S rRNA gene amplicon metagenomics method for profiling and identifying diverse microbes from specific habitats. This work sought to develop universal primers for amplifying the 16S rRNA gene from a consortium of disparate microbial species. Using multiple sequence alignment of the 16S rRNA gene of a variety of microbes, the resulting highly conserved region of the consensus sequence was used for design of universal polymerase chain reaction (PCR) primers for 16S rRNA gene. Application of the universal primers in simulated PCR reveals poor amplification efficiency where only 12 species out of 31 generated an amplicon. BLAST analysis of the resulting amplicons reveals a classification error of 50%. More significantly, analysis of the amplicon length indicates variable read length ranging from 81 to 122 base pair compared to the predicted read length of 100 base pairs. This suggests that the 16S rRNA gene harbours significant hitherto underappreciated sequence diversity, and may have unknown alternative splicing and recombination mechanisms. Overall, results from this study suggests that primer design for 16S rRNA amplicon metagenomics may be application and habitat specific, where it is difficult to design universal primers for all bacterial species. Conceptually, this meant that there may be sequence co-evolution in 16S rRNA gene for microbial species in the habitat where environmental and nutritional conditions impact on 16S rRNA gene structure and sequence. In essence, 16S rRNA gene may habour epigenetics signals at the gene level.
Biology and Life Sciences, Biochemistry and Molecular Biology
Copyright:
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
