Principles of Experimental Design and Data Analysis

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Principles of Experimental Design and Data Analysis

Attribution Non-Commercial (BY-NC)

Als PDF, TXT **herunterladen** oder online auf Scribd lesen

- RT Vol. 6, No. 4 A hybrid history
- Introduction to Gene Mapping
- Powerpoint presentation - Experimental Design Used in Rice Research
- Experimental Design Used in Rice Research
- RT Vol. 4, No. 2 A hybrid pioneer
- Intermediate R - Principal Component Analysis
- Exercises - Principles of Experimental Design and Data Analysis
- Powerpoint - Principles of Experimental Design and Data Analysis
- Regression and Correlation Analysis - Regression and Correlation Analysis
- Data Management and Statistical Analysis - Basic R Graphics
- Data Manipulation and Statistical analysis - Analysis of Variance
- Data Management and Statistical Analysis - Loading data
- R CropStat Introduction
- Intermediate R - Cluster Analysis
- Data Management and Statistical Analysis - Data Manipulation
- A few Basics about QTL Mapping
- Analysis of Variance (ANOVA)
- Intermediate R - Analysis of Count and Proportion Data
- Powerpoint - Regression and Correlation Analysis
- QTL Mapping

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Learning Objective

treatment effect and interaction effect;

• choose appropriate treatments to answer the objectives of the experiment and

formulate contrasts for each objective;

• choose dependent variables or responses to be measured to provide

information about the problem of the experiment; and

• describe the three essential components of an experimental design and explain

the importance of each component.

Introduction

The term experimental design refers to a plan for assigning experimental conditions to

subjects and the statistical analysis associated with the plan (Kirk, 1982). Its purpose is to

ensure that measurements taken from experimental units (say, plot, plant, leaf, cow, etc.)

are free from bias and give results as precise as practicable.

Experimental Error

Experimental error results from the natural variation that exists among the experimental

units and random variations in procedure. For example, if one harvests two equal areas

of rice from a field, grain yield from the two areas will seldom be equal; the weight of

fruit from adjacent trees in an orchard is seldom the same; rates of weight gain of any two

animals of the same species and breed nearly always differ. Such differences among crop

or animal units result from genetic and environmental differences beyond the control of

the experimenter and from variation in application of treatments. Although they are not

errors in the sense of being wrong, they result in variability among measurements of

experimental units which is referred to as experimental error.

Bias and Precision

Bias is defined as the difference of the expected value of an estimate from the true value.

Bias can result from many factors such as badly calibrated measuring instruments,

subjective allocation of treatments to units and subjective measurements as scoring.

Some sources of bias can be avoided through randomization which is the process of

randomly allocating the treatments to the experimental units.

Precision on the other hand, refers to the closeness with which the measurement

approaches the average. Precision is achieved by the following:

1. Wise choice of experimental units (before randomization) which should be as

similar as possible while still representing the range of material to which the

results should apply.

2. Careful conduct of all operations on units before and during the experiment.

This includes high standard of care, accuracy of treatment, protection from

damage and irrelevant sources of variability and trustworthy measurement and

recording.

3. Replication, that is, repeating one treatment more than once.

Treatment

crop variety, a fertilizer level or a management practice is a kind of treatment.

When the treatments of an experiment consist of several levels of one factor, say different

varieties of a rice cultivar, then the experiment is said to be a single factor experiment.

On the other hand, when the treatments are a combination of two or more factors, the

experiment is said to be a factorial experiment. For example, testing five varieties under

four nitrogen rates.

Experimental Unit

applied. It can be a single leaf, a whole plant, an area of land containing many plants, a

pot or a bench in the greenhouse, a single animal, several animals, or an entire herd.

response with the application of a particular treatment. On the other hand, an interaction

effect is the combined effect of two or more factors. That is, factors A and B interact if

the effect of A is different at different levels of B or equivalently if the effect of B is

different at different levels of A.

Preliminary Considerations when Planning an Experiment

The most important step in planning an experiment is to decide what the objectives of the

experiment are. It is often necessary not only to state what is to be tested, but also to

specify the population to which results are to apply. For example, it may be desired to

estimate the increase in the yield of rice resulting from a fertilizer application. The type

of fertilizer and time and method of application need first to be decided in specifying the

treatment. It is then necessary to decide to what population the results are to apply. The

widest possible population consists of all rice varieties and the whole range of conditions

under which they might be cultivated, i.e., all possible soil types, fertilizers and other

cultivation, weather conditions and so on. Practically we must be content with less

general, but still meaningful populations.

Choice of Treatment

The choice of treatments is basically dependent on the questions the experimenter wants

to be answered. For a simple question like, “Which is higher yielding, Variety A or

Variety B?” may have Varieties A and B as the treatment in an experiment. A simple

experiment such as this gives a simple conclusion like, “Variety A is higher yielding than

Variety B.” However, often times, conclusions are not as simple as this. More

realistically, one could say that Variety A is higher yielding if there is no tungro

infestation, otherwise Variety B is higher yielding because it is more resistant to tungro.

Or one could also say that Variety A is higher yielding if this amount of nitrogen is used.

There is no information as to which will give higher yield at different levels of nitrogen.

It is under this situation that an experimenter may include another factor, say level of

nitrogen, to widen the scope of his conclusions. If one is assessing the effect of weeds on

different rice varieties, he may have different varieties treated with different types of

herbicide. He may also wish to include a control treatment, such as no herbicide

application, against which to assess each herbicide. That is, a herbicide is said to be

effective if its use results in yield significantly higher than the yield of the control. One

may also wish to test different levels of a factor, say nitrogen, to assess the optimum level

that would optimize the response. One may choose the levels to be tested at random from

all possible levels of application or purposely selecting the levels to be included. One

may include more levels at the point where curvature is expected and less levels near

zero. For this type of experiment, one may or may not include a zero level. If it is known

that yield is very much lower with no nitrogen, then it may not be necessary to include a

zero level in the experiment. However one level is usually a reference point, such as the

level recommended for the current variety, this serves as the control treatment.

Contrasts

As mentioned above, the choice of treatments depends mostly on the questions the

experimenter wants to answer. Often these questions can be formulated statistically by

contrasts. Contrasts are combinations of the treatment means designed to have specified

expected values if there are no treatment effects and different values if there are effects.

Consider an experiment with four treatments, O, A, B and C where O is the control, say

no herbicide control and A, B and C are different herbicides. The experimenter may wish

to test the effectiveness of each herbicide by comparing each with the control. In such

case the contrasts are the following:

O A B C

1 -1 0 0

1 0 -1 0

1 0 0 -1

The numbers in the rows of table are the coefficients to be multiplied with the

corresponding means, the products are then added to form the contrast value.

Unfortunately the contrasts set up in this table will not lead to independent test so that the

researcher would have to be careful in interpreting the results. This is because the

coefficient sets are not orthogonal. One can easily test whether two contrasts are

orthogonal if their sum of products of corresponding coefficients will be zero. In order to

obtain independent tests this control-treatment comparison is usually formulated as

follows:

O A B C

3 -1 -1 -1

0 2 -1 -1

0 0 1 -1

The first contrast tests the control against the average of the three treatments A, B and C,

the second compares A to the average of B and C and the third compares B and C. Note

these contrasts are orthogonal.

Given k treatments it is only possible to construct sets with k-1 orthogonal contrasts

which are related to the df for the treatments SS.

Note that the sum of the coefficients of each contrast should be equal to zero. The first

contrast compares treatment A with the control O, ignoring treatments B and C. If A has

no effect compared to O, the value of this contrast should be near zero. If one wishes to

compare the average effect of A, B and C with O then the contrast coefficients are 3, -1, -

1, -1 or equivalently 1 -1/3 -1/3 -1/3.

92 Choice of Measurements

If the levels of the treatment to be tested are quantitative, say 0, 30, 60 and 90 units of

some application then comparisons between individual pairs of means are not usually of

interest. What is of interest is to know the relationship between the response variable and

the treatment. Two specific questions may be whether this relationship is linear or

quadratic over the range of values tested. In such case, the contrasts are the following:

Contrast 0 30 60 90

Linear -3 -1 1 3

Quadratic 1 -1 -1 1

Coefficients for orthogonal polynomials such as those given above are usually provided

in most experimental design books (e.g., Gomez & Gomez p. 641). On the other hand,

coefficients for treatment comparisons are constructed using the following rules:

1. If two groups of equal size are to be compared, simply assign coefficients of +1 to the

members of one group and -1 to members of the other group. It is immaterial which

group is assigned the positive coefficients.

2. Treatments not involved in the comparison are assigned a coefficient of zero.

3. In comparing groups containing different numbers of treatments, assign to the first

group coefficients equal to the number of treatments in the second group, and to the

second group coefficients of the opposite sign equal to the number of treatments in

the first group. Thus, if among five treatments, the first two were to be compared to

the last three, the coefficients would be +3, +3, -2, -2, -2.

4. Reduce coefficients to the smallest possible integers. For example, in comparing a

group of two treatments with a group of four, by rule 3, the coefficients are +4, +4, -2,

-2, -2, -2, but these can be reduced to +2, +2, -1, -1, -1, -1.

5. Interaction coefficients can always be found by multiplying the corresponding

coefficients of the main effects.

his experiment as early as possible so as to make adjustments in the experimental design

to give higher precision to more important contrasts. For example, in the case of the

herbicide trial given above, if C is a newly developed herbicide which is less expensive

than A and B, the recommendation for its usage will depend on its comparative

performance with A, B and O. Hence the experimenter may wish to give a higher

precision to the estimate of the mean of C. This could be done by having more plots

treated with herbicide C that is, C will have more replications than treatments A, B

and O.

Choice of Measurements to be Made

In choosing a response or dependent variable, the experimenter must be sure that the

response to be measured really provides information about the problem under study.

Measurements collected during an experiment can be classified into the following types:

comparisons are of utmost importance or are needed in the calculation of such

quantities. For example, in rice research the most common primary character is grain

yield. A treatment is said to be better that another treatment if its yield is higher.

obtain, it may be profitable to use a substitute observation that is easier to get. For

example, photosynthesis in rice is almost impossible to measure because of its highly

variable nature due to its dependence to environmental conditions. Besides, the

equipment needed to measure it is very expensive. A substitute character that can be

used to characterize photosynthesis or the amount of light intercepted by the plant is

the amount of nonstructural carbohydrates present in the plant.

effects found on the primary observations. For example, to explain the variation in

grain yield, number of tillers per hill may be measured to show that an increase in

tiller number coincides with increases in grain yield.

during the course of the experiment, the plants were infested by tungro, data on

percent infestation may be recorded. This variable may then be used as a covariate in

an analysis of covariance later.

5. Observations for checking the application of the treatments. For example if the

treatments correspond to different temperatures, it will be natural to check

independently that the appropriate temperature has in fact been achieved.

94 Choice of Measurements

Choice of Experimental Design

The choice of an experimental design is dependent upon the nature and variability in the

experimental area or material and on the number and nature of the treatments to be

applied. The chosen design must provide estimates of the most important contrasts, in

terms of the experimental objectives, with the greatest precision possible given the

resources available. It must also give reasonably good estimates of the variances of these

contrasts (Dyke, 1997).

By this we mean that in addition to requiring high precision (small variance), we would

like a reliable estimate of variance to provide high power for testing the contrasts.

This depends on the number of error degrees of freedom (DF) as can be seen from critical

t-values which decrease with the number of error DF, rapidly at first and then slowly to

the critical normal variate for large DF.

The statistical design of experiments refers to the process of planning the experiment so

that appropriate data will be collected and can be analyzed by statistical methods resulting

in valid and objective conclusions. A statistically valid experimental design is necessary

if meaningful conclusions are to be drawn from the data. There are three basic

requirements for a valid experimental design. It should be unbiased, precise enough to

detect differences of practical importance and it should provide a valid estimate of

experimental error so that the variability of estimates can be assessed and hypotheses can

be tested. Three principles which can ensure the validity of experimental designs are:

replication, randomization and error control.

Replication

Replication means that a treatment is repeated two or more times. Its function is to

provide an estimate of experimental error by directly measuring the variability between

similarly treated units and to provide a more precise measure of treatment effects by

averaging the individual observations. The number of replication that will be required in

a particular experiment depends on the magnitude of the differences the experimenter

wishes to detect and the variability of the data to be collected. Considering these two

things at the beginning of an experiment will save much frustration.

Randomization

considered have an equal chance of receiving a treatment. It guarantees unbiased

estimates of treatment means and of the experimental error. In most situations it is

required to assure the validity of the statistical theory underlying the tests of significance

in the analysis.

Error Control

reduce experimental error. For example, in the Randomized Complete Block Design,

treatments are grouped into blocks that are expected to perform differently, allowing a

block effect to be removed from the total variation in the trial.

References

Box, G.E.P., Hunter, W.G. & Hunter, J.S. (1978). Statistics for Experiments, John Wiley

& Sons, Inc.

Cochran, W.G. & Cox, G. M. (1950). Experimental Design, John Wiley & Sons, Inc.

Dyke, E. (1997). How to Avoid Bad Statistics. Field Crop Research, Vol. 51, 165-187.

Federer, W.T. (1955). Experimental Design, McMillan.

Gomez, K.A. & Gomez, A.A. (1984). Statistical Procedures for Agricultural Research,

John Wiley & Sons, Inc.

John, P.W.M. (1971). Statistical Design and Analysis of Experiments, The McMillan

Company.

Montgomery, D.C. (1984). Design and Analysis of Experiments, 2nd Ed., John Wiley &

Sons, Inc.

Ostle, B. & Mensing, R.W. (1975). Statistics in Research, 3rd. ed., The Iowa State

University Press.

96

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