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The vast majority, exceeding 99%, of natural microorganisms elude isolation and cultivation through conventional methods, rendering environmental samples indispensable for the exploration of microbial diversity. CD Genomics addresses this challenge by offering Full-Length 16S/18S/ITS Sequencing utilizing advanced PacBio SMRT and Oxford Nanopore technologies. This innovative approach delivers comprehensive long-read coverage of entire rRNA and ITS regions, facilitating precise species identification and accurate community profiling. By encompassing the full length of these regions, this methodology surpasses traditional short-read techniques, thereby enhancing microbial diversity analysis and mitigating biases to yield more reliable and robust results.

Our Advantages:

• Comprehensive Microbial Profiling: Captures the entire gene, offering a complete view of microbial diversity.
• High Taxonomic Resolution: Enables precise identification at species and strain levels.
• Enhanced Data Quality: Provides high-accuracy results with self-corrected sequencing errors.
• Reduced Amplification Bias: Minimizes PCR-related biases for more reliable data.
• Versatile Sample Compatibility: Adaptable to various sample types with tailored extraction methods.
• High Sensitivity: Offers more precise species information compared to single-region sequencing.

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Introduction to Full-Length 16S/18S/ITS Sequencing

Full-length 16S/18S/ITS sequencing represents a significant advancement in microbial diversity analysis, enabling comprehensive characterization of microbial communities at both species and strain levels. Unlike traditional amplicon sequencing, which focuses on short fragments of the 16S, 18S, or ITS regions, full-length sequencing covers the entire length of these regions. This method provides a more detailed and precise representation of microbial taxonomy, phylogeny, and functional potential.

The 16S rRNA gene has long been the cornerstone of bacterial identification, owing to its presence across all bacteria and the combination of conserved and hypervariable regions that facilitate species and genus differentiation. Similarly, 18S rRNA gene sequencing is used to investigate eukaryotic microorganisms, such as fungi and protozoa, leveraging conserved regions for broader taxonomic classification. The Internal Transcribed Spacer (ITS) regions, located between the 18S and 28S rRNA genes in eukaryotes, are particularly valuable in fungal taxonomy due to their significant variability between species.

The emergence of third-generation sequencing technologies, such as PacBio SMRT and Oxford Nanopore platforms, has transformed full-length 16S/18S/ITS sequencing. These technologies overcome the limitations of short-read sequencing by generating long reads that span the entire rRNA gene or ITS region, providing more comprehensive data for microbial diversity analysis, species classification, and functional profiling.

Applications of Full-Length 16S/18S/ITS Sequencing

The applications of full-length 16S/18S/ITS sequencing include, but are not limited to, the following areas:

• Microbial Diversity and Community Structure Analysis
• Phylogenetic Studies and Evolutionary Biology
• Environmental and Ecological Research
• Integrated Multi-Omics Studies

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Partial results of our full-length 16S/18S/ITS sequencing service are shown below:

Full-Length 16S/18S/ITS Sequencing FAQ

• 1. What are the benefits of using PacBio SMRT or Oxford Nanopore platforms for full-length sequencing?

  • Third-generation sequencing platforms, such as PacBio SMRT and Oxford Nanopore, offer the significant capability of producing long reads that span entire rRNA genes or ITS regions. This capacity effectively reduces sequencing biases and enhances accuracy. Consequently, these platforms facilitate more detailed analyses of microbial diversity, phylogenetic relationships, and functional profiles, providing superior data quality compared to traditional short-read methods.

• 2. Can full-length 16S/18S/ITS sequencing be used for all types of samples?

  • Indeed, full-length sequencing is highly adaptable to a diverse array of sample types, including soil, water, feces, and tissues. CD Genomics customizes its extraction and sequencing protocols to meet the specific requirements of each sample type, ensuring the acquisition of high-quality data across various research contexts.

• 3. How does full-length sequencing improve the accuracy of microbial identification?

  • Full-length sequencing achieves a comprehensive genetic profile by encompassing the entire 16S, 18S, or ITS regions. This inclusive approach allows for more precise species and strain identification, thereby reducing the likelihood of misclassification and providing a more accurate depiction of microbial community structures.

• 4. How can CD Genomics' full-length sequencing service benefit my research?

  • CD Genomics' full-length sequencing service offers extensive coverage, high-resolution taxonomic classification, and minimized biases. When combined with our multi-omics integration, this service significantly enhances the accuracy and depth of microbial diversity analyses, thereby supporting more impactful scientific research and outcomes.

Case Study

Influence of Peanut, Sorghum, and Soil Salinity on Microbial Community Composition in Interspecific Interaction Zone
Journal: Frontiers in Microbiology
Impact factor: 5.64
Published: 24 May 2021

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Background

Soil salinization affects crop productivity globally, reducing yields and increasing soil-borne pathogens. Intercropping, such as peanut and sorghum, can improve resource use and stress tolerance. This study uses 16S rDNA and ITS sequencing to analyze bacterial and fungal communities in peanut-sorghum intercropping systems under saline conditions, aiming to enhance soil stability and optimize cropping systems.

Materials & Methods

Sample preparation:

• Soil
• DNA extraction

Method:

• Full-Length 16S rDNA genes Sequencing
• Full-Length ITS Sequencing

Data Analysis:

• Annotation
• Microbial diversity analysis
• PCoA analysis
• Linear discriminant analysis (LDA)
• Effect size measurements (LEfSe) analysis

Results

OTU Analysis: Sequencing depth was adequate, revealing 1,531 bacterial and 284 fungal OTUs with high coverage. Venn diagrams showed common OTUs across samples, with reduced overlap under salt stress.

Community Composition: Salt treatment altered bacterial and fungal communities, increasing Bacteroidota and Ascomycota while decreasing Acidobacteriota and Mucoromycota.

Fig 1. Relative abundance of the peanut rhizosphere (IP), sorghum rhizosphere (IS), and interspecific interaction zone (II) microbial communities at the phylum and genus levels under different soil conditions.

Beta Diversity: Distinct bacterial and fungal community structures were observed between salt-treated and normal soils, with different clustering patterns under varying conditions.

Fig 2. Principal coordinate analysis and unweighted pair group method with arithmetic mean analysis.

Microbial Taxa Abundance: Core bacterial taxa shifted under salt stress, with increased Bacteroidetes and Verrucomicrobiota. Fungal communities showed significant changes, especially in Ascomycota and Mucoromycota, with salt treatment.

Fig 3. Linear discriminant analysis effect size.

Conclusions

Soil salinity significantly shapes microbial communities, with peanut driving changes more than sorghum. While fungal communities in different soil conditions shared main phyla, their taxonomic compositions differed. Under salt stress, interspecies interactions promote specific microbial recruitment. Future research should clarify these mechanisms and their applicability to natural ecosystems.

References

1. Callahan B J, Wong J, Heiner C, et al. High-throughput amplicon sequencing of the full-length 16S rRNA gene with single-nucleotide resolution. BioRxiv, 2019: 392332.
2. Shi X, Zhao X, Ren J, et al. Influence of peanut, sorghum, and soil salinity on microbial community composition in interspecific interaction zone. Frontiers in Microbiology. 2021, 12:678250.