End-to-End Genotyping by Sequencing (GBS) Service - Low-Cost SNP Discovery

Partial results are shown below:

1. What is the definition of a GBS Tag?

A GBS Tag refers to a sequence of reads adjacent to a restriction enzyme cut site. The genomic coverage captured by GBS is determined by multiplying the number of Tags by the length of a single read. For instance, utilizing HiSeq 4000 PE150 sequencing, the genomic coverage can be calculated as follows:

GBS captured genomic range=100,000 Tag x 150bp/Tag=100000X150=15 M

If the average sequencing depth per sample is 10x per Tag, the sequencing data volume per sample would be:

15Mx 10=150 M

2. How to select the number of Tags?

The required number of Tags varies depending on the specific research objectives. For example, GWAS might necessitate tens of thousands of high-density molecular markers, whereas studies on phylogenetic relationships or linkage analysis may only require a few hundred to a few thousand molecular markers to achieve satisfactory results. Hence, it is crucial to first evaluate the necessary number of Tags based on the study's requirements and then select an appropriate number of Tags accordingly.

For species with a genome size less than 1 Gb undergoing genetic linkage map studies, a common recommendation is to utilize approximately 100,000 Tags. Adjustments to the number of Tags can be made based on specific research needs.

Table 1. Commonly used GBS Tag numbers (e.g., for genetic linkage maps).

3. Can GBS be used for non-reference species?

GBS can indeed be utilized for non-reference species to obtain SNP markers. However, the lack of annotated genomic information in non-reference species presents a significant challenge, often rendering the identification of candidate genes unfeasible. For tasks such as quantitative trait loci (QTL) mapping, association studies, or the mining of genes related to domestication traits, it is advisable to utilize reference species to achieve more accurate and informative results.

4. Can GBS be applied to polyploid species?

GBS technology is applicable to polyploid species. A salient example is the successful application of GBS to hexaploid oat species for genetic mapping in 2014. Polyploid species are characterized by their complex ploidy levels, which can include both autopolyploids and allopolyploids, as well as tetraploids and hexaploids. Each scenario requires specific analytical considerations. Current research efforts have already begun to employ GBS for genetic mapping in polyploid crops such as wheat and cotton.

5. Is GBS suitable for inter-species research?

GBS leverages restriction enzymes for genome capture, thereby facilitating the development of SNP markers needed for genetic linkage and population genetic analyses. Significant genetic divergence between samples can result in non-uniform capture of restriction fragments across samples and a paucity of shared SNPs. Consequently, GBS is predominantly suited for studies at the intra-species level. Nonetheless, in rare cases where there is a close phylogenetic relationship between different species within the same genus and minimal genetic divergence, GBS can be employed effectively for phylogenetic studies.

6. Can GBS data be integrated with other omics datasets (e.g., transcriptomics)?

Absolutely. We offer multi-omics integration to help uncover deeper genetic mechanisms.

Available analyses include:

• eQTL mapping (requires RNA-seq data)
• Epigenetic association studies (integrating DNA methylation or ChIP-seq data)

7. My DNA samples don't meet the recommended input or concentration. Can I still use GBS?

While we recommend ≥300 ng of DNA per sample at a concentration of ≥10 ng/μL for optimal results, we’re flexible.

• What to do: Contact our technical team for a quick feasibility assessment.
• Need help? We offer in-house DNA extraction services if your sample quantity or quality is suboptimal.

8. Can I request only sequencing data without bioinformatics analysis?

Yes, we offer modular GBS services.

• Choose the level of support you need—from library prep and sequencing only, to full bioinformatics analysis.
• This flexibility allows you to integrate our services seamlessly into your own research pipeline.

9. Do you support large-scale GBS projects involving thousands of samples?

Yes, we specialise in high-throughput GBS services.

• Our automated library prep and advanced sequencing platforms (e.g., NovaSeq) can handle thousands of samples in parallel.
• This is ideal for large-scale breeding programs, GWAS, or population genetics studies.

Customer Publication Highlight

Use of biostimulants for water stress mitigation in two durum wheat (Triticum durum Desf.) genotypes with different drought tolerance

Journal: Plant Stress  

Published: December 2024

DOI: https://doi.org/10.1016/j.stress.2024.100566

Background

Durum wheat (Triticum durum Desf.) is a staple crop in the Mediterranean region, but its productivity is severely threatened by drought stress. Biostimulants have emerged as a promising agronomic strategy to enhance drought tolerance. This study evaluated the efficacy of two biostimulants (B1 and B2) in mitigating water stress effects on two durum wheat genotypes—drought-tolerant Svems16 and drought-sensitive Iride—through physiological, morphological, and genomic analyses.

Project Objective

The research aimed to:

1. Assess the impact of biostimulants on growth performance under drought stress.
2. Identify genetic variants associated with drought tolerance using Genotyping-by-Sequencing (GBS).
3. Elucidate the mechanisms of biostimulant-induced stress mitigation.

CD Genomics' Services

As a leader in genomic solutions, CD Genomics provided:

• GBS Analysis: High-throughput sequencing using the NovaSeq platform (5M PE150 reads per sample) to identify SNPs and InDels.
• Variant Calling: Alignment to the Svevo.v1 reference genome, quality control, and annotation using GATK and SnpEff.
• Functional Profiling: Gene Ontology (GO) enrichment analysis to pinpoint drought-responsive genes and pathways.

Key Findings

1. Biostimulants Mitigate Drought Stress in Sensitive Genotypes

• Iride (sensitive): Drought reduced shoot biomass by 25% and root biomass by 29%, but biostimulant application (B1/B2) restored growth by up to 37%.
• Svems16 (tolerant): Minimal biomass loss under stress; biostimulants showed limited efficacy, confirming innate tolerance.

2. Genomic Basis of Drought Tolerance

• GBS Analysis: Revealed 7,000 shared variants between Iride and Svems16, with distinct missense mutations in dehydrin and histidine kinase genes (e.g., TRITD6Bv1G204160 in Svems16).
• GO Enrichment: Svems16 exhibited variants in water deprivation-response genes (e.g., GO:0042631), explaining its superior tolerance.

3. Physiological Adaptations

• Stomatal Density: Drought reduced stomata by 15–16%; biostimulants increased density by 26–30% in Iride.
• Oxidative Stress: MDA (lipid peroxidation marker) spiked in Iride under drought (+165% in roots), but biostimulants partially alleviated oxidative damage.

4. Root Architecture Modulation

• Biostimulants induced thicker, shorter roots in control plants. Under drought, Iride developed longer roots with more tips, while Svems16 maintained stable morphology.

Figures Referenced

Figure 7: Genomic variant distribution and GO enrichment highlights drought-responsive genes in Svems16

Figure 5: Stomatal density changes under biostimulant and drought treatments.

Implications

This study demonstrates that biostimulants can effectively mitigate drought stress in sensitive durum wheat cultivars by modulating root morphology and stomatal density. CD Genomics' GBS analysis provided critical insights into the genetic basis of drought tolerance, enabling targeted breeding strategies. The findings support the use of biostimulants as a sustainable tool to enhance crop resilience in water-limited environments.

CD Genomics' Contribution: By delivering high-resolution genomic data and variant annotation, CD Genomics enabled the identification of key genetic markers for drought tolerance, paving the way for precision agriculture in cereal crops.

Reference

1. Spada, Matteo, et al. "Use of biostimulants for water stress mitigation in two durum wheat (Triticum durum Desf.) genotypes with different drought tolerance." Plant Stress 14 (2024): 100566. https://doi.org/10.1016/j.stress.2024.100566