16S/18S/ITS Amplicon Sequencing for Microbial Diversity (50k+ Reads)

Reference

1. Callahan, B.J., Grinevich, D., Thakur, S. et al. Ultra-accurate microbial amplicon sequencing with synthetic long reads. Microbiome 9, 130 (2021). https://doi.org/10.1186/s40168-021-01072-3
2. De Santiago, Alejandro, et al. "Dataset complexity impacts both MOTU delimitation and biodiversity estimates in eukaryotic 18S rRNA metabarcoding studies." Environmental DNA 4.2 (2022): 363-384. https://doi.org/10.1002/edn3.255
3. Newbold, Lindsay K., et al. "DNA extraction methodology has a limited impact on multitaxa riverine benthic metabarcoding community profiles." Environmental DNA 7.3 (2025): e70102. https://doi.org/10.1002/edn3.70102
4. Yadev, Brijesh Singh, Pallavi Chauhan, and Sandeep Kushwaha. "Bioinformatics resources for microbial research in biological systems." Microbial Genomics in Sustainable Agroecosystems: Volume 2 (2019): 45-60. https://doi.org/10.1007/978-981-32-9860-6_3
5. Ogundolie, Frank Abimbola, et al. "Microbiome characterization and identification: key emphasis on molecular approaches." An Introduction to the Microbiome in Health and Diseases. Academic Press, 2024. 49-69. https://doi.org/10.1016/B978-0-323-91190-0.00004-7

Demo Results

These demo figures illustrate typical bioinformatics deliverables—rarefaction/depth QC, alpha/beta diversity (PCoA/NMDS), taxonomy profiles, and differential abundance (LEfSe)—formatted for publication-ready reporting.

16S/18S/ITS Amplicon Sequencing FAQs

1. How do I choose the right amplicon region (e.g., V3–V4, ITS2) for my sequencing project?

The ideal region depends on your sample type and research goal. Below are common use-case recommendations:

• Gut microbiome (human or animal): Use V3–V4 for broad microbial coverage and balanced taxonomic resolution.
• Oral or skin microbiota: Try V1–V3 or V1–V2 to better distinguish dominant surface-associated bacteria.
• Soil, water, or other environmental samples: Choose V3–V4 or V4–V5, both effective for high-diversity microbial communities.
• Fungal profiling: Use ITS2 for general fungal community studies, or ITS1 when analyzing air or plant-associated fungi.
• Eukaryotic microbes (e.g., protists): The 18S rRNA SSU region is preferred for soil and aquatic samples.

✅ These are starting points — the best choice ultimately depends on your taxonomic resolution needs and study design. We recommend a brief consultation during project planning to select the most appropriate region and primers.

2. Do I need to include biological replicates? If so, how many?

Yes — replicates are highly recommended, especially for studies involving statistical comparisons or differential abundance analysis.

• Minimum suggestion: At least 3 biological replicates per group for standard LEfSe or ANOSIM tests.
• For complex or heterogeneous samples: Use 5–6 replicates to improve data reliability and capture community variability.

3. What if my sample quantity or quality doesn't meet requirements?

All incoming samples undergo quality checks. If the DNA is too low in concentration or purity, we'll alert you and suggest alternatives.

• Low-yield rescue: In many cases, we can offer optimized protocols for limited samples — contact our support team for options.

4. Can you process difficult or unconventional sample types?

Absolutely. We routinely handle:

• Soil, sediment, and sludge
• Water (fresh or marine)
• Faeces, swabs (skin, nasal), fermentation broths
• Other high-contamination or low-biomass samples

⚠️ For extreme environments or low-abundance communities, let us know in advance — we'll tailor the workflow accordingly.

5. Is the bioinformatics analysis customisable?

Yes. Our standard pipeline includes:

• Sequence quality control
• Taxonomic annotation
• α- and β-diversity metrics
• Differential abundance (e.g., LEfSe)

For advanced requests — like metabolic pathway prediction, network analysis, or absolute quantification — we offer bespoke analysis packages. Pricing and timelines are quoted case by case.

6. Can I send multiple samples at once? Will they contaminate each other?

Yes, we support high-throughput processing of multiple samples. Each sample is barcoded with a unique identifier.

7. Do I need to supply primers, or are they provided?

We typically provide validated primer sets optimised for commonly used regions like V3–V4 or ITS2. If you need a custom region or primer design, simply share the target and we'll assist with optimization and synthesis.

8: What is the difference between relative and absolute abundance in amplicon sequencing?

Relative abundance shows the proportion of taxa within a sample, while absolute abundance estimates calibrated load (e.g., copies per unit sample) to help distinguish true biomass changes from compositional shifts.

16S/18S/ITS Amplicon Sequencing Case Studies

Customer Publication Highlight

Enhanced Co-Cultivation of Stone Pine and Truffle through Mycorrhizal Inoculation in Southern Hemisphere Climates

Journal: Agroforestry Systems

Impact factor: ~2.8 (2022)

Published: 7 October 2023

DOI: https://doi.org/10.1007/s10457-023-00915-2

Background

Mediterranean stone pine (Pinus pinea), valued for its pine nuts, and Tuber borchii (bianchetto truffle) represent a promising agroforestry combination. However, the persistence of T. borchii mycorrhization and its impact on pine growth under non-native conditions remained unexplored. This study evaluated the symbiotic relationship between P. pinea and T. borchii across a 2000 km latitudinal gradient in Chile, focusing on tree growth, vigor, and mycorrhizal colonization over three years.

Project Objective

The client aimed to:

1. Validate the growth-enhancing effects of T. borchii inoculation on P. pinea.
2. Assess long-term mycorrhizal persistence under diverse climatic conditions.
3. Identify environmental factors influencing truffle-pine symbiosis.

CD Genomics' Services

As a trusted partner in molecular authentication, CD Genomics provided:

• ITS Sequencing: Sanger sequencing of the ITS region confirmed T. borchii identity in inoculated roots, with >96% sequence similarity to reference strains.
• High-Resolution Analysis: DADA2 pipeline processed sequencing data to ensure taxonomic accuracy.

Key Findings

1. Mycorrhization Enhances Pine Growth:
   • Inoculated trees outperformed controls: +6.9% height, +10% root collar diameter, +8.3% crown diameter (P < 0.0001).
   • Vigor increased by 14.1%, with extreme environments (e.g., Exploradores) showing the highest gains (+33.6% height, +75.6% root diameter).
2. High Mycorrhizal Persistence:
   • Over 60% of root apexes remained colonized by T. borchii after 3 years (score ≥4.9/5).
   • Molecular authentication via CD Genomics' sequencing confirmed truffle identity in field samples.
3. Climate-Driven Symbiosis:
   • PCA revealed T. borchii colonization thrived at >5°C minimum temperatures, with no negative impact from high rainfall (up to 2,450 mm/year).
   • Competing fungi (e.g., Suillus luteus) showed limited interference, indicating robust truffle dominance.
4. Agroforestry Scalability:
   • Survival rates exceeded 86% across all sites, demonstrating adaptation to diverse soils (pH 5.8–8.1) and climates.

Figures Referenced

Biplot of the principal component analysis by sites, T. borchii mycorrhization and climatic variables.

Implications

This study pioneers T. borchii–P. pinea co-cultivation in the Southern Hemisphere, offering a dual-income model (pine nuts + truffles) for marginal lands. CD Genomics' sequencing validated the stability of this symbiosis, supporting scalable agroforestry innovations in non-native habitats.

Reference

1. Loewe-Muñoz, V., Delard, C., del Río, R. et al. Effects of Tuber borchii inoculation on Pinus pinea 3 years after establishment along a latitudinal gradient in the Southern Hemisphere. Agroforest Syst 98, 369–381 (2024). https://doi.org/10.1007/s10457-023-00915-2