What is High-Throughput Phenotyping?

Author: Hannah Styers

Learning Objectives

1. Describe high-throughput phenotyping.
2. List the pros and cons of using high-throughput phenotyping.
3. List actual and theoretical applications of high-throughput phenotyping.
4. Evaluate why it may be important to commercialize and simplify the use of high-throughput phenotyping.

 

Graphical Abstract

 

Legend. High-throughput phenotyping is away to discover genetic traits that are desired or expressed. These phenotypes can be genetically targeted through genes and environmental conditions to create optimal growth factors, for applications that span from cells to humans to plants. As more researchers are turning to high-throughput technologies to perform experiments for efficiency, more practical and useful methods are being developed and utilized to ensure the maximum quality in the phenotyping data. This lesson is an overview of common high-throughput phenotyping applications and methods. The top image¹ shows how there is a connection from the cellular level to the organ system-level.  The bottom left image² describes the genotype-phenotype relationship. Lastly, the bottom right image³ portrays that gathering high-throughput data that can apply to many different pathways in investigations and projects. (These images are reused for educational purposes only.) Image created with Canva Pro.

Summary

It is theorized that all cells come from a common ancestor, which is one of the reasons our genes are ubiquitous.⁴ The focus of researchers are shifting to determine what makes individuals unique in the sense of their genetic make-up as well as their appearance.⁴ In order to do this, experimenters have turned to high-throughput discovery to perform a multitude of experiments at one time to better understand these differences. High-throughput screening has been used to initiate more efficient experimentation that has been used to determine high-yield results in many different studies for large populations and samples. There are two different types of high-throughput screening: target-based and phenotypic based.  This lesson focuses on phenotypic screening, which uses high-throughput technology to selectively focus on traits of interest to be expressed.⁵ High-throughput phenotyping can be applied from the cellular level up to the production- and large-scale growth level.⁶ Initially, studies must focus on single cells and organisms to determine the phenotype of interest and then apply these methods to larger growth centers.⁷ By starting at the most basic level, researchers are able to identify the direct gene of interest, which allows for more efficient growth with a higher and more useful yield to generate a specific phenotype.⁶ Manual phenotyping can lead to errors and lack reproducibility in methodology.⁶ Application of high-throughput phenotyping technology can lead to less variability across data and unbiased results when compared to manual methods.⁴ One of the main goals of high-throughput phenotyping focuses on the genetic gain, by which researchers focus on expanding the knowledge of different genomes and genetic variation.⁶ Using high-throughput phenotyping can lead to exponential gain in knowledge about genetics of populations.⁶

Two main focuses of developing strategies for high-throughput phenotyping differ in the approach to how to identify the genotype-phenotype relationship.^8,9 Generally, strategies employed on human and behavioral studies approach the phenotyping ‘backwards’, where the data is looked at afterwards to determine the key traits that link back to a gene. This is usually referred to as RNAi approaches.⁹ Strategies focused on crop and biofuel production take a ‘forward’ approach where gene manipulations are studied to determine if a key or desired trait can be created. This protocol focuses on mutagenesis and genetically modifying genes.⁶

Applications of high-throughput phenotyping are endless but include a few of the main categories of focus right now, such as: plant phenotyping, behavioral studies, cellular focused phenotyping, and phenotyping using medical databases. Through these applications, traits have been focused on in small enclosures where growth can occur.⁷ These applications can hopefully be expanded to entire fields and multicellular subjects in the near future to better understand growth factors.⁷ The use of high-throughput phenotyping for plants focuses on engineering specific, ideal traits. These traits are what are used to selectively grow plants in the future. Using these methods can also assist in accelerating plant breeding to contain the desired traits for consumption and distribution.⁹ Additionally, advances in phenotypic information of crops, especially those under different environmental stressors, can be applied to help prevent major reduction in productivity during extreme environmental conditions, such as climate change.⁶ Behavioral studies have used high-throughput phenotyping to discover genes of variants and relate that to specific actions, such as preferring dark environments.¹⁰ Developmental studies have used cellular level phenotypic studies to try to better understand the change in phenotype as genes are expressed in embryos.⁸ Understanding the development of specific traits from gene expression in embryos could assist in determining where mutations occur and how to prevent them in the fetus before birth.⁸ Electronic health records and online databases have been used to discover phenotypic traits in patients with specific diseases and conditions.¹¹ The use of these databases have allowed researchers to find correlations in similar phenotypes and symptoms exemplified by patients.¹¹ Setting the standards for phenotypes of interest could assist physicians in predicting certain conditions in advance.^8,11 Applications of high-throughput phenotyping lead to resourcefulness and efficiency in terms of yield and selection, as well as expanding the reach of the genetic gain.⁶

Because of the ever-evolving ecosystem and need for valuable genetic gain, this technology has been applied to uses such as crop production⁷ and clinical trials^4,8 to try to find a universally practical approach to phenotyping. The methods of phenotyping include sensing changes, either remotely or directly, which correlate to the type of approach used, may it be forward or backward. Remote-sensing uses devices such as multispectral and thermal sensors, much of what is used in plant phenotyping.^6,7 Red-green-blue cameras are widely used in both applications of sensing.⁶ Florescent microscopy is one of the methods for generating quantitative data about gene expression in different samples.⁸ In clinical trials and cellular studies, varying assays are used with the assistance of automated systems to facilitate the high-throughput discovery processes.¹² Phenotyping allows for the development of key statistical models to predict the genome of and ideal traits of a species.⁶ Many different companies are generating software and programs to assist in the automaton and proficiency of high-throughput phenotyping. For example, small-animal sorter machines have been developed to analyze and produce florescent emission profiles of up to 100 live animals (or cells) per second.⁸ Additionally, databases of phenotypic information can be used by software and programs to run comparative analysis in order to determine phenotypic differences in traits.^4,10,11 Most of these methods used in order to gain phenotypic information were designed to address specifics about research hypothesis and do not yet encompass all of the fields that this phenotyping can be applied.⁸

In order to develop a more widespread, practical application of these methods, the technology must be improved to be applied with high-throughput phenotyping. The current methodology allows for sourcing of one particular trait of interest but does not allow for selectivity through multiple expressed traits, which shows little correlation to the total phenotypic variance that is expressed.^8,9,12 This is one of the major applications that researchers aim to reach with this technology, however no such methods have been documented.^7,12 The use of high-throughput phenotyping can be beneficial with software and a database that is able to run the statistical tests and data analysis on the sets of data.¹² Additionally, for many of the non-commercial researchers, the cost of running high-throughput phenotyping, along with the set-up fees, may not be worth the benefits of the novel technology. There is a large up-front cost associated with any high-throughput application.⁶ Lastly, many researchers feel that this technology will not be practical until methods and practices are standardized and normalized across all fields.⁴ This is a substantial challenge, as many different facilities use non-standard lingo and data analysis methods; however, this must be done in order to generate a global database at the disposal of education and additional research. ⁸ This will aid immensely in the gap of knowledge as well as ensuring that practices are uniform, reproducible, and publicly available.

By applying these technologies, scientists and farmers alike are being more resourceful and efficient by gathering information on traits that experimenters are already selecting for. The use of high-throughput phenotyping can change many aspects of research and medicine. Though some shortcomings in the current approaches do exist, the focus of integrating and utilizing this technology will lead to improvements and further applications of high-throughput discovery.

Additional Resources

Link to audio files: https://drive.google.com/drive/folders/1GXvhaEIUITM93BR9qKNG-NAMQEXSP2el?usp=sharing

References

1. Gloria, L. Cell To System – Lessons – Tes Teach. Tes Teach with Blendspace https://www.tes.com/lessons/trkpPl8uYkqRgQ/cell-to-system.
2. Phenotype vs Genotype – Difference Between. They Differ https://theydiffer.com/difference-between-phenotype-and-genotype/.
3. Allen, L. High-throughput Phenotyping: Sensing Technologies and Data Mining. Maximum Yield https://www.maximumyield.com/high-throughput-phenotyping-sensing-technologies-and-data-mining/2/17228.
4. Finak, G. et al. High-throughput flow cytometry data normalization for clinical trials: Flow Cytometry Data Normalization for Clinical Trials. Cytometry A 85, 277–286 (2014).
5. Wang, X. et al. High-throughput phenotyping with deep learning gives insight into the genetic architecture of flowering time in wheat. http://biorxiv.org/lookup/doi/10.1101/527911 (2019) doi:10.1101/527911.
6. Araus, J. L., Kefauver, S. C., Zaman-Allah, M., Olsen, M. S. & Cairns, J. E. Translating High-Throughput Phenotyping into Genetic Gain. Trends Plant Sci. 23, 451–466 (2018).
7. Andrade-Sanchez, P. et al. Development and evaluation of a field-based high-throughput phenotyping platform. Funct. Plant Biol. 41, 68 (2014).
8. Sozzani, R. & Benfey, P. N. High-throughput phenotyping of multicellular organisms: finding the link between genotype and phenotype. Genome Biol. 12, 219 (2011).
9. Singh, D. et al. High-Throughput Phenotyping Enabled Genetic Dissection of Crop Lodging in Wheat. Front. Plant Sci. 10, 394 (2019).
10. Hossain, S. M., Wong, B. K. Y. & Simpson, E. M. The dark phase improves genetic discrimination for some high throughput mouse behavioral phenotyping. Genes Brain Behav. 3, 167–177 (2004).
11. Yu, S. et al. Toward high-throughput phenotyping: unbiased automated feature extraction and selection from knowledge sources. J. Am. Med. Inform. Assoc. 22, 993–1000 (2015).
12. Sturino, J. et al. Statistical Methods for Comparative Phenomics Using High-Throughput Phenotype Microarrays. Int. J. Biostat. 6, (2010).

 

Questions

1. What is high-throughput phenotyping?
   1. High-throughput phenotyping is a method of screening that uses high-throughput technology to generate data about specific traits and characteristics of large populations.
2. What applications have allowed high-throughput phenotyping to be beneficial?
   1. High-throughput phenotyping has aided in efficient plant breeding to generate crops that are ideal for production and consumption. It has also aided in helping discover genes that can be targeted to try to manipulate or change specific traits. These applications can apply to medicine and human cells by allowing screening of a multitude of humans for specific characteristics such as a disease or condition. There are many applications, with new studies being published daily where high-throughput phenotyping can be utilized.
3. List the pros and cons to using high-throughput phenotyping.
   1. Pros- Resourceful in terms of being efficient and reducing waste in time and materials, allows for rapid discoveries with rapid data analysis to be able to understand more about specific traits, generates more accurate results through automation and reproducibility
   2. Cons- Can be expensive to initiate these studies, can only analyze one phenotype at a time, need for technology that has large data-analyzing capabilities, no current universal dataset or library containing this information
4. Reflect and consider a company that grows tobacco that is considering applying these high-throughput phenotyping methods to use because the company has heard that this can increase the crop yield. What should he consider before implementing these methods to try to produce a higher yield of crops?
   1. The company should consider the price of setting up these materials, as sometimes they are not efferent in terms of the overall benefit. The company should also look at the environment in which they are growing the crops to see if it is constant or changing. Additionally, previous studies should be considered to determine if there will be an increase in yield from enhancements or other changes that may be made to their current methodology. The equipment and technology that is used in high-throughput phenotyping of crops must also be considered, as there will need to be equipment that can analyze the tobacco that the company grows. (There may be other conditions that should be considered that are not listed here.)