Genotyping by Sequencing: Principles, Protocols, and Applications

The genomic era has ushered in a plethora of technologies to facilitate high-throughput genotyping. Among these, genotyping by sequencing (GBS) has emerged as a prominent and cost-effective approach that has gained immense popularity in the realm of plant breeding and genetics. In essence, GBS entails sequencing a subset of the genome using a restriction enzyme to generate a library of reduced representation fragments, which are then sequenced using next-generation sequencing (NGS) technologies. However, the principles and protocols of GBS are not without their intricacies, and proper attention must be paid to ensure optimal results.

Despite its relative simplicity, GBS harbors both advantages and disadvantages that must be considered when employing this technology. On one hand, GBS is highly efficient and capable of producing high-quality genotyping data at a fraction of the cost of traditional genotyping methods. Furthermore, GBS is highly scalable, enabling the interrogation of large numbers of samples simultaneously, which is particularly useful in the context of plant breeding and genetic studies. On the other hand, GBS has limitations, such as the potential for missing data due to non-random distribution of restriction enzyme sites throughout the genome. Additionally, GBS requires careful consideration of the bioinformatic pipeline for variant calling, which can be complex and computationally demanding.

GBS represents a powerful tool in the genotyping arsenal, with numerous applications in plant breeding and genetics. By providing a comprehensive overview of the principles, protocols, advantages, and disadvantages of GBS, we hope to equip readers with the knowledge necessary to effectively utilize this technology in their research endeavors.

Principles and Protocols of GBS

The principle of GBS involves sequencing genomic regions that are flanked by restriction sites. This method involves digesting genomic DNA with a restriction enzyme, ligating adapters to the ends of the resulting fragments, and amplifying the library using PCR. The resulting library is then sequenced using high-throughput sequencing technologies. The sequencing reads are then aligned to a reference genome or assembled de novo to identify genetic variations such as single nucleotide polymorphisms (SNPs) and insertion-deletions (INDELs).

DNA extraction: High-quality DNA must be extracted from the organism of interest. This DNA is then sheared into fragments of a specific size range. DNA extraction is a critical step in the GBS protocol, as high-quality DNA is required for successful library preparation and sequencing. There are several methods for DNA extraction, including CTAB (cetyltrimethylammonium bromide), silica-based columns, and magnetic beads. The choice of method depends on the organism of interest and the quality and quantity of DNA required.

Library preparation: The fragments are then ligated to adapters that allow for amplification and sequencing. This step involves PCR amplification of the fragments to create a library of DNA fragments that can be sequenced. The choice of adapter and PCR primers can affect the quality and quantity of the resulting library. There are several commercially available GBS library preparation kits, such as the Illumina TruSeq DNA PCR-Free Library Preparation Kit, which can simplify the library preparation process.

Sequencing: The library is then sequenced using high-throughput sequencing technologies. The resulting reads are then mapped to a reference genome, if available, or de novo assembled into contigs. GBS libraries can be sequenced using high-throughput sequencing technologies, such as Illumina or Ion Torrent. The choice of sequencing platform depends on the requirements of the study, such as read length, depth of coverage, and cost. Illumina sequencing is widely used for GBS due to its high accuracy and low cost.

SNP calling: The aligned reads are then analyzed to identify SNPs and genotypes. This can be done using a variety of software tools, such as TASSEL, GATK, and Stacks. The aligned reads are then analyzed to identify SNPs and genotypes. The choice of software depends on the requirements of the study, such as accuracy, speed, and ease of use.

Data analysis: The resulting genotypic data can then be analyzed using various statistical methods, such as principal component analysis (PCA) and genome-wide association studies (GWAS). PCA can be used to visualize population structure and identify genetic clusters, while GWAS can be used to identify associations between genotypes and phenotypes. Other data analysis methods include marker-trait association analysis and genomic selection.

Schematic steps of the genotyping-by-sequencing (gbs) protocol for plant breeding.Schematic steps of the genotyping-by-sequencing (gbs) protocol for plant breeding.

Applications of GBS

In recent years, Genotyping by Sequencing has emerged as a powerful technique for genotyping and discovering genetic variation. GBS is a cost-effective and scalable approach that allows researchers to analyze large numbers of samples and obtain high-density genotyping data.

Plant Breeding

In the realm of plant breeding, the pursuit of crop improvement is a never-ending quest that necessitates the development and utilization of cutting-edge technologies. One such technology that has emerged as an essential tool in this quest is genotyping by sequencing. In recent years, GBS has gained widespread adoption in the plant breeding community due to its ability to identify genetic markers that are associated with agronomic traits and disease resistance, which can subsequently be utilized in marker-assisted selection (MAS) to accelerate the breeding process.

It is worth noting, however, that the successful implementation of GBS in plant breeding necessitates careful consideration of numerous factors, including the choice of restriction enzyme, sequencing depth, and bioinformatic analysis pipeline. Moreover, the accuracy and reliability of GBS-derived markers must be rigorously validated to ensure their suitability for MAS. Despite these challenges, GBS represents a powerful tool for accelerating crop improvement efforts in the plant breeding community.

Animal Breeding

The field of animal breeding is replete with challenges and complexities that necessitate the development and application of innovative technologies. Among these, genotyping by sequencing (GBS) has emerged as a powerful and versatile tool for identifying genetic markers associated with production traits such as milk yield, meat quality, and disease resistance. In addition, GBS can be used to estimate genetic diversity and population structure, which can inform breeding strategies and optimize the efficacy of breeding programs.

In a study, GBS was used to identify SNP markers that were significantly associated with resistance to sea lice. Moreover, the study also estimated the genetic diversity and structure of the salmon population, which can inform the design of breeding programs aimed at enhancing disease resistance. By utilizing GBS in this manner, animal breeders can enhance the efficiency of breeding programs and accelerate the progress of animal improvement efforts.

Population Genetics

Genotyping by sequencing (GBS) has ushered in a new era of precision and sophistication in the field of population genetics. GBS has enabled researchers to analyze thousands of genetic markers across multiple individuals, thereby allowing for the accurate estimation of genetic diversity, population structure, and gene flow. These insights can shed light on the evolutionary history and demographic processes of populations, as well as provide valuable information for conservation and management efforts.

One striking example of the potential of GBS in the realm of population genetics can be seen in the study of the endangered New Zealand Falcon (Falco novaeseelandiae). By leveraging the power of GBS, researchers were able to estimate the genetic diversity and structure of the falcon population, illuminating the underlying genetic architecture of the species. Furthermore, the study identified regions of the genome under selection, which can provide crucial insights into the adaptation of the falcon to its environment and help guide conservation efforts. With its capacity for high-throughput, cost-effective, and informative genotyping, GBS is poised to continue revolutionizing the field of population genetics in the years to come.

Evolutionary Biology

In the multifaceted realm of evolutionary biology, the advent of GBS has enabled researchers to tackle a litany of vexing queries, such as the elusive origins of species and the intricate genetic underpinnings of adaptation.

GBS has opened up new vistas of investigation by empowering scientists to scrutinize genetic variation across a panoply of species or populations with remarkable precision. Through such analyses, researchers can detect those regions of the genome that have diverged due to the inexorable forces of natural selection. These cutting-edge insights into the complex genetic underpinnings of evolutionary processes have transformed our understanding of the intricate mechanisms governing the evolution of species on this planet.

Genotyping by Sequencing in Plants

The burgeoning field of plant genetics and breeding has undergone a profound transformation, thanks to the advent of GBS. This cutting-edge methodology has ushered in a new era of genetic analysis, providing an efficient and cost-effective way to generate an abundance of genetic information.

The sweeping potential of GBS has unleashed a wave of novel applications, including plant diversity and conservation, crop improvement, and genome-wide association studies. The colossal treasure trove of thousands of genetic markers offered by GBS has paved the way for the rapid development of new crop varieties with enhanced traits, bolstering the sustainable use of precious plant resources.

The synergistic blend of GBS with other innovative technologies equipping researchers with a powerful set of tools to unravel the complex genetic architecture of plants. These advances have kindled a burgeoning sense of optimism about the transformative impact that GBS will have on the future of plant genetics and breeding.

Advancements in Plant Breeding Using GBS

The advent of GBS technology has upended traditional paradigms in the realm of plant breeding, by providing plant breeders with unprecedented access to thousands of markers with high-throughput sequencing, all the while maintaining a cost-effective and efficient workflow. The remarkable accuracy and profusion of markers offered by GBS technology have made it possible to identify and isolate genetic variants, including the elusive single-nucleotide polymorphisms (SNPs), insertions, deletions, and structural variants. This cutting-edge technology has opened up new vistas for researchers to explore and unravel the genomic diversity, population structure, and evolutionary history of plants, thereby unveiling a treasure trove of genes and markers that are closely associated with critical agronomic traits.

Plant Diversity And Conservation

The multifaceted and complex nature of the study of genetic diversity is vital not only for scientific advancement but also for conservation purposes, especially for the preservation of endangered and threatened plant species. Through the use of GBS technology, it is possible to identify unique genetic markers that are specific to certain plant populations, geographic regions or habitats, thereby enabling the design and implementation of conservation strategies that are tailored to the specific needs of rare and endangered plant species.

Crop Improvement

The innovative technology of GBS has revolutionized the field of crop improvement, by facilitating the identification of genes and molecular markers that are closely associated with critical traits such as disease resistance, yield and quality. The wealth of information garnered from GBS analysis can be harnessed to develop new varieties of crops with enhanced traits that are tailor-made for specific environmental conditions, as well as to devise novel genomic selection models for plant breeding. The applications of GBS technology in crop improvement are far-reaching and multifarious, and its ability to identify markers for resistance to diseases such as Fusarium head blight in wheat, late blight in potato, and soybean cyst nematode has been nothing short of phenomenal.

Genome-wide Association Studies (GWAS)

GWAS have emerged as a powerful tool for uncovering the genetic basis of complex traits in plants, by identifying loci that control variation in specific traits and gaining a comprehensive understanding of the genetic factors driving phenotypic variation.

Despite its great potential, GWAS can be challenging in plants with complex genomes, where traditional genotyping methods may prove ineffective. In this context, GWAS has been revolutionized by the advent of GBS, which enables the identification of genetic variation in plants with polyploidy or high levels of heterozygosity by harnessing the power of high-throughput sequencing.

Advantages and Disadvantages of Genotyping by Sequencing

Advantages Disadvantages
Provides large amounts of genetic data Requires high initial investment in equipment
Can be used with a variety of organisms Data analysis can be complex and time-consuming
More cost-effective than traditional genotyping methods Requires high computational resources
Has the potential to improve breeding efficiency and accuracy Quality of data can be affected by DNA quantity and quality
Enables identification of novel genomic regions and markers Error rate can be higher than traditional genotyping methods
Can detect rare genetic variations Presence of sequencing errors can lead to false positives and false negatives
Provides a high level of reproducibility and accuracy Requires careful consideration of sample size and coverage depth
Offers high-throughput capabilities Can lead to biased results if certain genomic regions are overrepresented
Can be customized to target specific genomic regions The use of PCR can introduce amplification bias and errors


The world of genotyping-by-sequencing has undergone a seismic shift, revolutionizing a myriad of disciplines such as plant and animal breeding, population genetics, and evolutionary biology. The proliferation of GBS has been instrumental in enabling researchers to sift through a vast swath of samples and acquire high-density genotyping data. Through this process, genetic markers linked to traits of interest can be identified, providing crucial insights into the complex genetic architecture underpinning diverse biological phenomena. Moreover, GBS has facilitated the estimation of genetic diversity and population structure, granting researchers the capacity to tease apart the evolutionary dynamics that shape life on earth.

At CD Genomics, we understand the immense potential of GBS to usher in a new era of discovery in these fields. We are steadfast in our commitment to delivering the highest quality GBS services to researchers worldwide, empowering them to make breakthroughs that transform our understanding of the natural world.


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  2. Wang, N., Yuan, Y., Wang, H. et al. Applications of genotyping-by-sequencing (GBS) in maize genetics and breeding. Sci Rep 10, 16308 (2020)
  3. Deschamps S, Llaca V, May GD. Genotyping-by-Sequencing in Plants. Biology (Basel). 2012 Sep 25;1(3):460-83.
  4. Jiangfeng He et al,. Genotyping-by-sequencing (GBS), an ultimate marker-assisted selection (MAS) tool to accelerate plant breeding FSec. Plant Genetics and Genomics ront. Plant Sci., 30 September 2014.
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