Integrative 16S rRNA Gene Sequencing Characterization of Four Bacterial Isolates: Phylogenetic Placement, Comparative ATGC Content Analysis and BLAST–EzBioCloud–VSEARCH Bioinformatics Pipeline

For decades, the 16S rRNA gene has been a cornerstone of bacterial taxonomy and community analysis. Modern long-read sequencing is accurate enough to detect subtle single-nucleotide differences among multiple 16S gene copies within the same genome (though small insertion/deletion variants remain challenging to resolve). These findings highlight that microbiome analyses must account for intragenomic 16S variation. By leveraging full-length 16S intragenomic sequence variants, it is possible to resolve bacterial community composition at the species and even strain level. In this study, the nearly full-length 16S rRNA gene sequences (~1.2-1.5 kb) of four bacterial isolates (ASW5, SED11, A13, and S14) were analyzed to determine their taxonomic identities and evolutionary relationships. BLAST analysis via the EzBioCloud database identified isolates SED11, A13, and S14 as members of the genus Bacillus, with 16S sequence similarities of 99.54%, 100%, 100%, and 99.8% to the type strains of Bacillus thuringiensis, Bacillus paranthracis, Bacillus halotolerans, and Bacillus velezensis, respectively. These high identity values indicate that each isolate’s 16S sequence is nearly identical to a known species sequence, facilitating confident species-level assignment (typically, ≥98.7% 16S similarity is considered indicative of the same species). Quality filtering and chimera checking using VSEARCH v2.21.1 (with the GOLD/UCHIME database) revealed no chimeric sequences (0% chimera detected) in any of the four 16S rRNA genes, confirming that each sequence was suitable for downstream analysis. Nucleotide composition analysis showed that the sequences ranged from 1,235 to 1,500 bp in length and had very similar base frequency profiles. Each 16S rRNA Gene comprised approximately 20–21% Adenine (A), 24-26% Thymine (T), 22-24% Guanine (G), and 30-32% Cytosine (C). Accordingly, all four isolates exhibited a relatively high GC content in the ~53-55% range. Notably, Isolate ASW5 had a 16S GC content (~53%) comparable to those of the Bacillus isolates (~53-55%), indicating only minor variation in 16S GC content despite the organisms’ distinct genera. In silico secondary structure prediction of each 16S rRNA (using the RNAfold tool) yielded the typical bacterial 16S rRNA stem-loop structure for all isolates. Phylogenetic analysis was performed using MEGA X to construct 16S rRNA gene trees. The resulting neighbor-joining phylogeny clustered the three Bacillus isolates (ASW5, SED11, A13, S14) together within the Bacillus clade. This clustering aligns with the taxonomic identifications and the high 16S sequence similarities to the respective type strains. In summary, all four isolates were unambiguously identified by full-length 16S rRNA sequencing. They showed high GC contents and conserved 16S secondary structures, and each isolate grouped phylogenetically with the expected genus-underscoring the consistency between their molecular composition and phylogenetic affiliations.