High-throughput screening
Encyclopedia
High-throughput screening (HTS) is a method for scientific experiment
ation especially used in drug discovery
and relevant to the fields of biology
and chemistry
. Using robotics
, data processing and control software, liquid handling devices, and sensitive detectors, High-Throughput Screening allows a researcher to quickly conduct millions of chemical, genetic or pharmacological tests. Through this process one can rapidly identify active compounds, antibodies or genes which modulate a particular biomolecular pathway. The results of these experiments provide starting points for drug design and for understanding the interaction or role of a particular biochemical process in biology.
: a small container, usually disposable and made of plastic, that features a grid of small, open divots called wells. Modern (circa 2008)
microplates for HTS generally have either 384, 1536, or 3456 wells. These are all multiples of 96, reflecting the original 96 well microplate with 8 x 12 9mm spaced wells. Most of the wells contain experimentally useful matter, often an aqueous solution
of dimethyl sulfoxide
(DMSO) and some other chemical compound
, the latter of which is different for each well across the plate. (The other wells may be empty, intended for use as optional experimental controls
.)
A screening facility typically holds a library of stock plates, whose contents are carefully catalogued, and each of which may have been created by the lab or obtained from a commercial source. These stock plates themselves are not directly used in experiments; instead, separate assay plates are created as needed. An assay plate is simply a copy of a stock plate, created by pipette
ing a small amount of liquid (often measured in nanoliters) from the wells of a stock plate to the corresponding wells of a completely empty plate.
, or an animal embryo
. After some incubation time has passed to allow the biological matter to absorb, bind to, or otherwise react (or fail to react) with the compounds in the wells, measurements are taken across all the plate's wells, either manually or by a machine. Manual measurements are often necessary when the researcher is using microscopy
to (for example) seek changes or defects in embryonic development caused by the wells' compounds, looking for effects that a computer could not easily determine by itself. Otherwise, a specialized automated analysis machine can run a number of experiments on the wells (such as shining polarized light on them and measuring reflectivity, which can be an indication of protein binding). In this case, the machine outputs the result of each experiment as a grid of numeric values, with each number mapping to the value obtained from a single well. A high-capacity analysis machine can measure dozens of plates in the space of a few minutes like this, generating thousands of experimental datapoints very quickly.
Depending on the results of this first assay, the researcher can perform follow up assays within the same screen by "cherrypicking" liquid from the source wells that gave interesting results (known as "hits") into new assay plates, and then re-running the experiment to collect further data on this narrowed set, confirming and refining observations.
is an important element in HTS's usefulness. Typically, an integrated robot
system consisting of one or more robots transports assay-microplates from station to station for sample and reagent addition, mixing, incubation, and finally readout or detection. An HTS system can usually prepare, incubate, and analyze many plates simultaneously, further speeding the data-collection process. HTS robots currently exist which can test up to 100,000 compounds per day. The term uHTS or ultra high throughput screening refers (circa 2008) to screening in excess of 100,000 compounds per day.
s or siRNA
s) to identify active compounds, HTS has led to an explosion in the rate of data generated in recent years
.
Consequently, one of the most fundamental challenges in HTS experiments is to glean biochemical significance from mounds of data, which relies on the development and adoption of appropriate experimental designs and analytic methods for both quality control and hit selection
.
HTS research is one of the fields which have a feature described by Eisenstein as follows: soon, if a scientist does not understand some statistics or rudimentary data-handling technologies, he or she may not be considered to be a true molecular biologist and thus will simply become a dinosaur
.
. A good plate design helps to identify systematic errors (especially those linked with well position) and determine what normalization should be used to remove/reduce the impact of systematic errors on both QC and hit selection
.
Effective analytic QC methods serve as a gatekeeper for excellent quality assays. In a typical HTS experiment, a clear distinction between a positive control and a negative reference such as a negative control is an index for good quality. Many quality assessment measures have been proposed to measure the degree of differentiation between a positive control and a negative reference. Signal-to-background ratio, signal-to-noise ratio, signal window, assay variability ratio, and Z-factor
have been adopted to evaluate data quality
.
Strictly standardized mean difference (SSMD) has recently been proposed for assessing data quality in HTS assays
.
.
For hit selection in primary screens without replicates, the easily interpretable ones are average fold change, mean difference, percent inhibition, and percent activity. However, they do not capture data variability effectively. The z-score method or SSMD, which can capture data variability based on an assumption that every compound has the same variability as a negative reference in the screens
.
However, outliers are common in HTS experiments, and methods such as z-score are sensitive to outliers and can be problematic. Consequently, robust methods such as the z*-score method, SSMD*, B-score method, and quantile-based method have been proposed and adopted for hit selection
.
In a screen with replicates, we can directly estimate variability for each compound; consequently, we should use SSMD or t-statistic that does not rely on the strong assumption that the z-score and z*-score rely on. One issue with the use of t-statistic and associated p-values is that they are affected by both sample size and effect size
. They come from testing for no mean difference, thus are not designed to measure the size of compound effects. For hit selection, the major interest is the size of effect in a tested compound. SSMD directly assesses the size of effects
. SSMD has also been shown to be better than other commonly used effect sizes
.
The population value of SSMD is comparable across experiments and thus we can use the same cutoff for the population value of SSMD to measure the size of compound effects
.
For example, imagine a plate where compound A is in wells 1-2-3, compound B is in wells 2-3-4, and compound C is in wells 3-4-5. In an assay of this plate against a given target, a hit in wells 2, 3, and 4 would indicate that compound B is the most likely agent, while also providing three measurements of compound B's efficacy against the specified target. Commercial applications of this approach involve combinations in which no two compounds ever share more than one well, to reduce the (second-order) possibility of interference between pairs of compounds being screened.
In 2010 researchers developed a silicon sheet of lenses that can be placed over microfluidic arrays to allow the fluorescence measurement of 64 different output channels simultaneously with a single camera. This process can analyze 200,000 drops per second.
There is a trend in academia to be their own drug discovery enterprise. ( High-throughput screening goes to school) Facilities which normally only industry had can now increasingly be found as well at universities. UCLA for example, features an HTS laboratory ( Molecular Screening Shared Resources (MSSR, UCLA) which can screen more than 100,000 compounds a day on a routine basis. The University of Illinois also has a facility for HTS, as does the University of Minnesota. The Rockefeller University, has an open access (infrastructure)
HTS Resource Center HTSRC (The Rockefeller University, HTSRC) which offers a library of over 165,000 compounds. Northwestern University's High Throughput Analysis Laboratory supports target identification, validation, assay development, and compound screening.
In the United States, the National Institute of Health or NIH has created a nationwide consortium of small molecule screening centers that has been recently funded to produce innovative chemical tools for use in biological research. The Molecular Libraries Screening Center Network or MLSCN performs HTS on assays provided by the research community, against a large library of small molecules maintained in a central molecule repository.(http://www.ncgc.nih.gov/index.html)
For more information see Laboratory automation
Experiment
An experiment is a methodical procedure carried out with the goal of verifying, falsifying, or establishing the validity of a hypothesis. Experiments vary greatly in their goal and scale, but always rely on repeatable procedure and logical analysis of the results...
ation especially used in drug discovery
Drug discovery
In the fields of medicine, biotechnology and pharmacology, drug discovery is the process by which drugs are discovered or designed.In the past most drugs have been discovered either by identifying the active ingredient from traditional remedies or by serendipitous discovery...
and relevant to the fields of biology
Biology
Biology is a natural science concerned with the study of life and living organisms, including their structure, function, growth, origin, evolution, distribution, and taxonomy. Biology is a vast subject containing many subdivisions, topics, and disciplines...
and chemistry
Chemistry
Chemistry is the science of matter, especially its chemical reactions, but also its composition, structure and properties. Chemistry is concerned with atoms and their interactions with other atoms, and particularly with the properties of chemical bonds....
. Using robotics
Robotics
Robotics is the branch of technology that deals with the design, construction, operation, structural disposition, manufacture and application of robots...
, data processing and control software, liquid handling devices, and sensitive detectors, High-Throughput Screening allows a researcher to quickly conduct millions of chemical, genetic or pharmacological tests. Through this process one can rapidly identify active compounds, antibodies or genes which modulate a particular biomolecular pathway. The results of these experiments provide starting points for drug design and for understanding the interaction or role of a particular biochemical process in biology.
Assay plate preparation
The key labware or testing vessel of HTS is the microtiter plateMicrotiter plate
A Microtiter plate or microplate or microwell plate, is a flat plate with multiple "wells" used as small test tubes. The microplate has become a standard tool in analytical research and clinical diagnostic testing laboratories...
: a small container, usually disposable and made of plastic, that features a grid of small, open divots called wells. Modern (circa 2008)
microplates for HTS generally have either 384, 1536, or 3456 wells. These are all multiples of 96, reflecting the original 96 well microplate with 8 x 12 9mm spaced wells. Most of the wells contain experimentally useful matter, often an aqueous solution
Aqueous solution
An aqueous solution is a solution in which the solvent is water. It is usually shown in chemical equations by appending aq to the relevant formula, such as NaCl. The word aqueous means pertaining to, related to, similar to, or dissolved in water...
of dimethyl sulfoxide
Dimethyl sulfoxide
Dimethyl sulfoxide is an organosulfur compound with the formula 2SO. This colorless liquid is an important polar aprotic solvent that dissolves both polar and nonpolar compounds and is miscible in a wide range of organic solvents as well as water...
(DMSO) and some other chemical compound
Chemical compound
A chemical compound is a pure chemical substance consisting of two or more different chemical elements that can be separated into simpler substances by chemical reactions. Chemical compounds have a unique and defined chemical structure; they consist of a fixed ratio of atoms that are held together...
, the latter of which is different for each well across the plate. (The other wells may be empty, intended for use as optional experimental controls
Scientific control
Scientific control allows for comparisons of concepts. It is a part of the scientific method. Scientific control is often used in discussion of natural experiments. For instance, during drug testing, scientists will try to control two groups to keep them as identical and normal as possible, then...
.)
A screening facility typically holds a library of stock plates, whose contents are carefully catalogued, and each of which may have been created by the lab or obtained from a commercial source. These stock plates themselves are not directly used in experiments; instead, separate assay plates are created as needed. An assay plate is simply a copy of a stock plate, created by pipette
Pipette
A pipette is a laboratory tool used to transport a measured volume of liquid.-Use and variations:Pipettes are commonly used in molecular biology, analytical chemistry as well as medical tests...
ing a small amount of liquid (often measured in nanoliters) from the wells of a stock plate to the corresponding wells of a completely empty plate.
Reaction observation
To prepare for an assay, the researcher fills each well of the plate with some logical entity that he or she wishes to conduct the experiment upon, such as a proteinProtein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
, or an animal embryo
Embryo
An embryo is a multicellular diploid eukaryote in its earliest stage of development, from the time of first cell division until birth, hatching, or germination...
. After some incubation time has passed to allow the biological matter to absorb, bind to, or otherwise react (or fail to react) with the compounds in the wells, measurements are taken across all the plate's wells, either manually or by a machine. Manual measurements are often necessary when the researcher is using microscopy
Microscopy
Microscopy is the technical field of using microscopes to view samples and objects that cannot be seen with the unaided eye...
to (for example) seek changes or defects in embryonic development caused by the wells' compounds, looking for effects that a computer could not easily determine by itself. Otherwise, a specialized automated analysis machine can run a number of experiments on the wells (such as shining polarized light on them and measuring reflectivity, which can be an indication of protein binding). In this case, the machine outputs the result of each experiment as a grid of numeric values, with each number mapping to the value obtained from a single well. A high-capacity analysis machine can measure dozens of plates in the space of a few minutes like this, generating thousands of experimental datapoints very quickly.
Depending on the results of this first assay, the researcher can perform follow up assays within the same screen by "cherrypicking" liquid from the source wells that gave interesting results (known as "hits") into new assay plates, and then re-running the experiment to collect further data on this narrowed set, confirming and refining observations.
Automation systems
AutomationAutomation
Automation is the use of control systems and information technologies to reduce the need for human work in the production of goods and services. In the scope of industrialization, automation is a step beyond mechanization...
is an important element in HTS's usefulness. Typically, an integrated robot
Robot
A robot is a mechanical or virtual intelligent agent that can perform tasks automatically or with guidance, typically by remote control. In practice a robot is usually an electro-mechanical machine that is guided by computer and electronic programming. Robots can be autonomous, semi-autonomous or...
system consisting of one or more robots transports assay-microplates from station to station for sample and reagent addition, mixing, incubation, and finally readout or detection. An HTS system can usually prepare, incubate, and analyze many plates simultaneously, further speeding the data-collection process. HTS robots currently exist which can test up to 100,000 compounds per day. The term uHTS or ultra high throughput screening refers (circa 2008) to screening in excess of 100,000 compounds per day.
Experimental design and data analysis
With the ability of rapid screening of diverse compounds (such as small moleculeSmall molecule
In the fields of pharmacology and biochemistry, a small molecule is a low molecular weight organic compound which is by definition not a polymer...
s or siRNA
Sírna
Sírna Sáeglach , son of Dian mac Demal, son of Demal mac Rothechtaid, son of Rothechtaid mac Main, was, according to medieval Irish legend and historical tradition, a High King of Ireland...
s) to identify active compounds, HTS has led to an explosion in the rate of data generated in recent years
.
Consequently, one of the most fundamental challenges in HTS experiments is to glean biochemical significance from mounds of data, which relies on the development and adoption of appropriate experimental designs and analytic methods for both quality control and hit selection
.
HTS research is one of the fields which have a feature described by Eisenstein as follows: soon, if a scientist does not understand some statistics or rudimentary data-handling technologies, he or she may not be considered to be a true molecular biologist and thus will simply become a dinosaur
.
Quality control
High-quality HTS assays are critical in HTS experiments. The development of high-quality HTS assays requires the integration of both experimental and computational approaches for quality control (QC). Three important means of QC are (i) good plate design, (ii) the selection of effective positive and negative chemical/biological controls, and (iii) the development of effective QC metrics to measure the degree of differentiation so that assays with inferior data quality can be identified. A good plate design helps to identify systematic errors (especially those linked with well position) and determine what normalization should be used to remove/reduce the impact of systematic errors on both QC and hit selection
.
Effective analytic QC methods serve as a gatekeeper for excellent quality assays. In a typical HTS experiment, a clear distinction between a positive control and a negative reference such as a negative control is an index for good quality. Many quality assessment measures have been proposed to measure the degree of differentiation between a positive control and a negative reference. Signal-to-background ratio, signal-to-noise ratio, signal window, assay variability ratio, and Z-factor
Z-factor
The Z-factor is a measure of statistical effect size. It has been proposed for use in high-throughput screening to judge whether the response in a particular assay is large enough to warrant further attention.-Background:...
have been adopted to evaluate data quality
.
Strictly standardized mean difference (SSMD) has recently been proposed for assessing data quality in HTS assays
.
Hit selection
A compound with a desired size of effects in an HTS screen is called a hit. The process of selecting hits is called hit selection. The analytic methods for hit selection in screens without replicates (usually in primary screens) differ from those with replicates (usually in confirmatory screens). For example, the z-score method is suitable for screens without replicates whereas the t-statistic is suitable for screens with replicate. The calculation of SSMD for screens without replicates also differs from that for screens with replicates.
For hit selection in primary screens without replicates, the easily interpretable ones are average fold change, mean difference, percent inhibition, and percent activity. However, they do not capture data variability effectively. The z-score method or SSMD, which can capture data variability based on an assumption that every compound has the same variability as a negative reference in the screens
.
However, outliers are common in HTS experiments, and methods such as z-score are sensitive to outliers and can be problematic. Consequently, robust methods such as the z*-score method, SSMD*, B-score method, and quantile-based method have been proposed and adopted for hit selection
.
In a screen with replicates, we can directly estimate variability for each compound; consequently, we should use SSMD or t-statistic that does not rely on the strong assumption that the z-score and z*-score rely on. One issue with the use of t-statistic and associated p-values is that they are affected by both sample size and effect size
. They come from testing for no mean difference, thus are not designed to measure the size of compound effects. For hit selection, the major interest is the size of effect in a tested compound. SSMD directly assesses the size of effects
. SSMD has also been shown to be better than other commonly used effect sizes
.
The population value of SSMD is comparable across experiments and thus we can use the same cutoff for the population value of SSMD to measure the size of compound effects
.
Techniques for increased throughput and efficiency
Unique distributions of compounds across one or many plates can be employed to increase either the number of assays per plate, or to reduce the variance of assay results, or both. The simplifying assumption made in this approach is that any N compounds in the same well will not typically interact with each other, or the assay target, in a manner that fundamentally changes the ability of the assay to detect true hits.For example, imagine a plate where compound A is in wells 1-2-3, compound B is in wells 2-3-4, and compound C is in wells 3-4-5. In an assay of this plate against a given target, a hit in wells 2, 3, and 4 would indicate that compound B is the most likely agent, while also providing three measurements of compound B's efficacy against the specified target. Commercial applications of this approach involve combinations in which no two compounds ever share more than one well, to reduce the (second-order) possibility of interference between pairs of compounds being screened.
Recent advances
In March 2010 research was published demonstrating an HTS process allowing 1,000 times faster screening (100 million reactions in 10 hours) at 1 millionth the cost (using 10−7 times the reagent volume) than conventional techniques using drop-based microfluidics. Drops of fluid separated by oil replace microplate wells and allow analysis and hit sorting while reagents are flowing through channels.In 2010 researchers developed a silicon sheet of lenses that can be placed over microfluidic arrays to allow the fluorescence measurement of 64 different output channels simultaneously with a single camera. This process can analyze 200,000 drops per second.
Increasing lab utilization of HTS
HTS is a relatively recent innovation, made lately feasible through modern advances in robotics and high-speed computer technology. It still takes a highly specialized and expensive screening lab to run an HTS operation, so in many cases a small-to-moderately sized research institution will use the services of an existing HTS facility rather than set up one for itself.There is a trend in academia to be their own drug discovery enterprise. ( High-throughput screening goes to school) Facilities which normally only industry had can now increasingly be found as well at universities. UCLA for example, features an HTS laboratory ( Molecular Screening Shared Resources (MSSR, UCLA) which can screen more than 100,000 compounds a day on a routine basis. The University of Illinois also has a facility for HTS, as does the University of Minnesota. The Rockefeller University, has an open access (infrastructure)
Open Access (Infrastructure)
In the context of infrastructure, open access involves physical infrastructure such as railways and physical telecommunications network plant being made available to clients other than the owners, for a fee....
HTS Resource Center HTSRC (The Rockefeller University, HTSRC) which offers a library of over 165,000 compounds. Northwestern University's High Throughput Analysis Laboratory supports target identification, validation, assay development, and compound screening.
In the United States, the National Institute of Health or NIH has created a nationwide consortium of small molecule screening centers that has been recently funded to produce innovative chemical tools for use in biological research. The Molecular Libraries Screening Center Network or MLSCN performs HTS on assays provided by the research community, against a large library of small molecules maintained in a central molecule repository.(http://www.ncgc.nih.gov/index.html)
For more information see Laboratory automation
Laboratory Automation
Laboratory automation is a multi-disciplinary strategy to research, develop, optimize and capitalize on technologies in the laboratory that enable new and improved processes...
See also
- Drug discovery hit to leadDrug discovery hit to leadEarly drug discovery involves several phases from target identification to preclinical development. The identification of small molecule modulators of protein function and the process of transforming these into high-content lead series are key activities in modern drug discovery. The Hit-to-Lead...
- Virtual high throughput screeningVirtual screeningVirtual screening is a computational technique used in drug discovery research. By using computers, it deals with the quick search of large libraries of chemical structures in order to identify those structures which are most likely to bind to a drug target, typically a protein receptor or...
- High-content screening
- Drug discoveryDrug discoveryIn the fields of medicine, biotechnology and pharmacology, drug discovery is the process by which drugs are discovered or designed.In the past most drugs have been discovered either by identifying the active ingredient from traditional remedies or by serendipitous discovery...
- Z-factorZ-factorThe Z-factor is a measure of statistical effect size. It has been proposed for use in high-throughput screening to judge whether the response in a particular assay is large enough to warrant further attention.-Background:...
- SSMD
- Dual-flashlight plotDual-flashlight plotIn statistics, a dual-flashlight plot is a type of scatter-plot in which the standardized mean is plotted against the mean of a contrast variable representing a comparison of interest...
- Compound managementCompound managementDrug discovery depends on methods by which many different chemicals are assayed for their activity. These chemicals are stored as physical quantities in a chemical library or libraries which are often assembled from both outside vendors and internal chemical synthesis efforts. These chemical...
- Synthetic genetic arraySynthetic genetic arraySynthetic Genetic Array analysis is a high-throughput technique for exploring synthetic lethal and synthetic sick genetic interactions . SGA allows for the systematic construction of double mutants using a combination of recombinant genetic techniques, mating and selection steps...
- Yeast two-hybrid screening
- DNA-encoded chemical library
Further reading
- Zhang XHD (2011) "Optimal High-Throughput Screening: Practical Experimental Design and Data Analysis for Genome-scale RNAi Research, Cambridge University Press"
External links
- Molecular Screening Shared Resources (MSSR, UCLA)
- Advanced Cell Classifier (ACC) project for high-throughput screen evaluation (ETH, Zurich)
- Yale Center for High Throughput Cell Biology
- Biohts: HTS resources and links- Biohts
- Society for Biomolecular Sciences - links (SBS)
- Open Screening Environment
- Setting up High-Throughput Screening Laboratory (Koppal, Lab Manager Magazine)
- Assay Guidance Manual (NIH, NCGC)
- http://www.aurorabiomed.com/icr_intro.htm (Aurora Biomed High-Throughput Screening]