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• Explore Complex Microbiomes with 5R 16S Microbial Diversity Sequencing

Achieve species-level microbial profiling even from degraded or low-biomass samples

The 5R 16S Microbial Diversity Sequencing service offers comprehensive and high-resolution microbial community profiling by targeting five hypervariable regions (V2, V3, V5, V6, V8) of the 16S rRNA gene. Through multiplex PCR amplification combined with the advanced Short MUltiple Regions Framework (SMURF) algorithm, this method provides enhanced taxonomic resolution and coverage, especially for challenging sample types such as clinical tissues, FFPE blocks, and blood.

For standard environmental or fecal microbiome studies, our Bacterial 16S rRNA Sequencing service based on the V3-V4 region remains a widely used baseline solution.

Technical Principle

Unlike conventional single- or dual-region 16S sequencing, 5R 16S simultaneously amplifies five distinct variable regions of the 16S rRNA gene, covering approximately 68% of the full-length sequence. The resulting data is processed using the SMURF algorithm, enabling reconstruction of microbial profiles with species-level resolution and broad taxonomic coverage.

Figure 1. Graphic representation of bacterial 16S rRNA gene, showing conserved and variable regions

Figure 2. Comparison of taxonomic resolution and coverage between single-region, dual-region, and 5R 16S sequencing approaches

For projects requiring maximum read length or rare taxon recovery, consider our Full-Length 16S/18S/ITS Sequencing service using long-read technologies.

Key Features and Applications

For metagenomic profiling of ultra-low biomass environments such as sterile tissues, cerebrospinal fluid, or even cell-free DNA, we also recommend 2bRAD-M Analysis for its robustness and species-level output.

Technical Comparison: 5R 16S vs. V3-V4 Sequencing

Workflow

Advanced Bioinformatics Analysis Summary

Sample Requirements

Please ship all samples on dry ice to ensure integrity.

Case Study: Mouse Lung Tissue Microbiome

To evaluate the performance of 5R 16S sequencing, we analyzed the same mouse lung tissue sample using both 16S V3-V4 dual-region sequencing and 5R 16S. The top six microbial taxa identified by the V3-V4 method included several potential environmental contaminants. In contrast, 5R 16S sequencing provided a more accurate and comprehensive profile of the actual microbial population present in the lung tissue.

Figure 3. Comparison of microbial composition results:

Left: 16S V3-V4 sequencing | Right: 5R 16S sequencing

To complement sequencing data with spatial localization, we offer custom 16S and 18S FISH probe design, synthesis, and in situ detection. This is especially useful in histological studies where localization within tissues is critical.

Explore our FISH Probe Design & Detection Service to visualize microbial distributions directly in tissue sections.

Why Choose Us?

Creative Proteomics combines robust laboratory protocols with advanced informatics to provide accurate, sensitive, and reproducible 16S microbiome sequencing results for even the most difficult sample types. With our 5R 16S Microbial Diversity Sequencing service, you'll uncover microbial insights that conventional methods might miss.

Frequently Asked Questions (FAQs)

• 1. What advantages does 5R 16S sequencing offer over conventional V3-V4 sequencing?

  • 5R 16S sequencing targets five variable regions (V2, V3, V5, V6, V8) instead of two, offering broader coverage (~68% of full-length 16S) and species-level resolution. It also performs better on low-biomass and degraded samples.

• 2. Is this method suitable for FFPE samples or old tissue archives?

  • Yes. 5R 16S sequencing is optimized for partially degraded DNA, making it ideal for FFPE tissues or aged clinical samples where DNA integrity may be compromised.

• 3. How much DNA is required for library construction?

  • We recommend ≥15 μL of DNA with a concentration above 20 ng/μL. However, lower amounts can sometimes be accommodated-please consult us for evaluation.

• 4. Can I use this method for blood, tumor tissue, or cerebrospinal fluid?

  • Yes. The high sensitivity of 5R 16S makes it suitable for extremely low microbial biomass samples, such as blood, solid tumors, or sterile body fluids.

• 5. Do you provide downstream analysis and visual reports?

  • Absolutely. We provide a complete bioinformatics pipeline, including taxonomic annotation, diversity analysis, differential abundance, and publication-ready charts.

• 6. How is this different from full-length 16S sequencing?

  • Full-length 16S typically requires third-generation sequencing and longer library prep. 5R 16S uses short-read platforms but achieves similar taxonomic resolution with lower cost and better sample compatibility.

• 7. Can I combine this with other services like ITS or 2bRAD?

  • Yes! We support multi-kingdom microbiome profiling. 5R 16S can be combined with ITS sequencing for fungi, or 2bRAD-M for ultra-low biomass metagenomics.

• 8. Do you help with publication or result interpretation?

  • Yes. Our bioinformatics team can assist with scientific interpretation, figure layout, and even methods section writing support for publications.

References

1. Nejman D, et al. Science. 2020;368(6494):973-980. https://doi.org/10.1126/science.aay9189
2. Fuks G, et al. Microbiome. 2018;6(1):17. https://doi.org/10.1186/s40168-017-0396-x