Recent improvements in ONT sequencing chemistry and basecalling have substantially increased the accuracy of full-length 16S rRNA gene

The gap

Evidence profile

Sourced from the future work of the source papers, classified as general, drawn from work published between 2025 and 2026, spanning 3 journals. Those papers have been cited 12 times in total.

Supporting evidence — 3 representative gaps

• Microbial Composition of Carious Dentin and the Impact of Minimally Invasive Excavation Techniques: A Narrative Review (2026) · International Journal of Molecular Sciences · doi

  Contemporary research on carious dentin is gradually shifting from descriptive micro- biology to a functional and ecological interpretation of the oral microbiome. Future studies should, therefore, aim for higher-resolution microbial analyses. Moreover, they should better distinguish metabolically active populations and clarify the relationship between microbiological data and clinical outcomes [7,15,46]. One promising direction is the refinement of 16S rRNA sequencing by analyzing different hypervariable regions, such as V1–V2 and V3–V4. Another important step is the adoption of full-length 16S sequencing technologies. These approaches allow more accurate taxonomic classification and reduce errors in the identification of closely related species. This is particularly relevant in carious dentin, where many anaerobic taxa show functional similarities but may have distinct pathogenic roles [26,39]. In parallel, shotgun metagenomics is emerging as the next step in oral microbiome re- search. Unlike targeted PCR and 16S-based approaches, it enables taxonomic and functional characterization of microbial communities. It also provides information on metabolic path- ways involved in acid production, biofilm formation, proteolytic activity, and adaptation to low-pH environments. These processes play a central role in caries progression [3,45]. Another important area of development is the distinction between microbial compo- sition and microbial activity. Metatranscriptomic analyses remain relatively uncommon in dental research. However, they can potentially identify metabolically active processes within the biofilm, which may allow for a more accurate assessment of the pathogenic potential of residual microflora after treatment [37,43]. Future research is also expected to increasingly adopt integrative multi-omics ap- proaches that combine genomics, transcriptomics, and metabolomics. They can provide information about the composition of microbial communities and their functional dynam- ics. Such insights may improve our understanding of different stages of carious lesion development and the microbial response to treatment approaches [44]. From a clinical perspective, future studies will likely focus on biomarkers of caries activity. These biomarkers could improve the prediction of lesion progression and support personalized minimally invasive treatment. They may also facilitate risk stratification and help guide the selection of the most appropriate excavation strategy [1,22]. https://doi.org/10.3390/ijms27135648 Int. J. Mol. Sci. 2026, 27, 5648 15 of 19 Although the findings from pilot and experimental studies are promising, the in- tegration of these technologies into routine clinical practice remains limited. High costs, analytical complexity, and the lack of standardized protocols continue to repre- sent major barriers. Nevertheless, the overall trend is clear: caries microbiology is mov- ing beyond culture-based descriptions toward functional, ecological, and personalized diagnostic models [18,75].

  generalfuture work
  Keywords: microbial functional carious future clinical approaches activity caries treatment dentin ecological oral microbiome analyses metabolically
• Comparative analysis of full-length 16s ribosomal RNA gene sequencing in human oropharyngeal swabs using primer sets with different degrees of degeneracy (2025) · Frontiers in Cellular and Infection Microbiology · cited 4× · doi

  Recent improvements in ONT sequencing chemistry and basecalling have substantially increased the accuracy of full-length 16S rRNA gene sequencing, thereby enhancing its potential to resolve complex microbial communities with higher taxonomic resolution than conventional short-read approaches. Given its cost-efficiency, scalability, and ability to sequence full-length amplicons in real time, the ONT platform is poised to gain increasing importance in oral and oropharyngeal microbiome research. Our study presents a comparative analysis of two primer sets with different levels of degeneracy for nanopore-based 16S rRNA gene sequencing of human oropharyngeal swabs. We demonstrate that the widely used standard 27F primer (27F-I) introduces measurable amplification bias, whereas a more degenerate variant (27F-II) yields richer and more representative taxonomic profiles. These findings underscore the critical role of primer selection in shaping microbiome readouts and support the broader use of degenerate primers for accurate and unbiased profiling in complex oral environments. Looking ahead, future studies should aim for greater methodological harmonization, particularly in the design and selection of primer sets. The current lack of interoperability among primer strategies remains a major obstacle to reproducibility and cross-study comparability. Establishing community-wide standards for primer choice, as well as unified guidelines for the selection of taxonomic reference databases across anatomical niches and sequencing platforms, will be essential for advancing microbiome research toward clinical and translational applications.

  generalfuture work
  Keywords: primer sequencing taxonomic microbiome selection full length rrna gene complex oral oropharyngeal sets degenerate recent
• Exploring the Molluscan Microbiome: Diversity, Function, and Ecological Implications (2025) · Biology · cited 8× · doi

  A central limitation is the lack of standardized methodologies for sample collection, DNA extraction, sequencing, and bioinformatics pipelines. While 16S rRNA gene se- quencing enables broad community profiling, inconsistencies in study designs hinder the comparability and reproducibility across taxa and systems [27,195]. Furthermore, amplicon- based studies exclude non-bacterial taxa and lack functional resolution. Adoption of multi-omics approaches—such as metagenomics, metatranscriptomics, metabolomics, and metaproteomics—is crucial for holistic characterization of the microbiome function and dynamics [196,197], as fungal and archaeal taxa may participate in immune regulation and nutrient cycling yet remain poorly characterized. Expanding analyses beyond bacterial taxa will offer a more ecologically realistic view of the microbial networks in mollusks. Even among bacteria, several key genera—such as Alcanivorax, Vibrio, and Pseudomonas—are recurrently detected but undercharacterized functionally in mollusks [198]. Predictive tools like PICRUSt2, HUMAnN3, and KEGG orthology pipelines should be systematically employed to infer potential microbial functions, followed by culture-based validation [171]. The host’s genetics, evolutionary history, and habitat specificity are additional factors influencing the microbiome composition, yet few studies integrate host transcriptomes or immune gene expression profiles with microbial data [170]. A combined eco-evolutionary framework is needed to understand how host filters shape microbial colonization, per- sistence, and co-evolution. Finally, while microbial monitoring and probiotic-based inter- ventions show promise for use in aquaculture, their application is limited by their cost, a lack of field validation, and their scalability. Most studies remain laboratory-bound, with short-term endpoints. Longitudinal, in situ trials are needed to assess the effects of microbiome manipulation on host health, microbial stability, ecosystem resilience, and antimicrobial resistance [199]. The development of portable diagnostic tools and training programs for aquaculture practitioners is equally essential for real-world implementa- tion [27]. Advancing molluscan microbiome research through interdisciplinary, integrative approaches will not only enhance our understanding of host–microbe co-evolution but also unlock novel applications for sustainable aquaculture, conservation translocations, and environmental biomonitoring.

  generalfuture work
  Keywords: microbial host taxa microbiome lack based aquaculture pipelines gene bacterial approaches immune remain mollusks tools

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