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• Null and Alternative Hypotheses | Definitions & Examples

Null and Alternative Hypotheses | Definitions & Examples

Published on 5 October 2022 by Shaun Turney . Revised on 6 December 2022.

The null and alternative hypotheses are two competing claims that researchers weigh evidence for and against using a statistical test :

• Null hypothesis (H 0 ): There’s no effect in the population .
• Alternative hypothesis (H A ): There’s an effect in the population.

The effect is usually the effect of the independent variable on the dependent variable .

Table of contents

Answering your research question with hypotheses, what is a null hypothesis, what is an alternative hypothesis, differences between null and alternative hypotheses, how to write null and alternative hypotheses, frequently asked questions about null and alternative hypotheses.

The null and alternative hypotheses offer competing answers to your research question . When the research question asks “Does the independent variable affect the dependent variable?”, the null hypothesis (H 0 ) answers “No, there’s no effect in the population.” On the other hand, the alternative hypothesis (H A ) answers “Yes, there is an effect in the population.”

The null and alternative are always claims about the population. That’s because the goal of hypothesis testing is to make inferences about a population based on a sample . Often, we infer whether there’s an effect in the population by looking at differences between groups or relationships between variables in the sample.

You can use a statistical test to decide whether the evidence favors the null or alternative hypothesis. Each type of statistical test comes with a specific way of phrasing the null and alternative hypothesis. However, the hypotheses can also be phrased in a general way that applies to any test.

The null hypothesis is the claim that there’s no effect in the population.

If the sample provides enough evidence against the claim that there’s no effect in the population ( p ≤ α), then we can reject the null hypothesis . Otherwise, we fail to reject the null hypothesis.

Although “fail to reject” may sound awkward, it’s the only wording that statisticians accept. Be careful not to say you “prove” or “accept” the null hypothesis.

Null hypotheses often include phrases such as “no effect”, “no difference”, or “no relationship”. When written in mathematical terms, they always include an equality (usually =, but sometimes ≥ or ≤).

Examples of null hypotheses

The table below gives examples of research questions and null hypotheses. There’s always more than one way to answer a research question, but these null hypotheses can help you get started.

*Note that some researchers prefer to always write the null hypothesis in terms of “no effect” and “=”. It would be fine to say that daily meditation has no effect on the incidence of depression and p 1 = p 2 .

The alternative hypothesis (H A ) is the other answer to your research question . It claims that there’s an effect in the population.

Often, your alternative hypothesis is the same as your research hypothesis. In other words, it’s the claim that you expect or hope will be true.

The alternative hypothesis is the complement to the null hypothesis. Null and alternative hypotheses are exhaustive, meaning that together they cover every possible outcome. They are also mutually exclusive, meaning that only one can be true at a time.

Alternative hypotheses often include phrases such as “an effect”, “a difference”, or “a relationship”. When alternative hypotheses are written in mathematical terms, they always include an inequality (usually ≠, but sometimes > or <). As with null hypotheses, there are many acceptable ways to phrase an alternative hypothesis.

Examples of alternative hypotheses

The table below gives examples of research questions and alternative hypotheses to help you get started with formulating your own.

Null and alternative hypotheses are similar in some ways:

• They’re both answers to the research question
• They both make claims about the population
• They’re both evaluated by statistical tests.

However, there are important differences between the two types of hypotheses, summarized in the following table.

To help you write your hypotheses, you can use the template sentences below. If you know which statistical test you’re going to use, you can use the test-specific template sentences. Otherwise, you can use the general template sentences.

The only thing you need to know to use these general template sentences are your dependent and independent variables. To write your research question, null hypothesis, and alternative hypothesis, fill in the following sentences with your variables:

Does independent variable affect dependent variable ?

• Null hypothesis (H 0 ): Independent variable does not affect dependent variable .
• Alternative hypothesis (H A ): Independent variable affects dependent variable .

Test-specific

Once you know the statistical test you’ll be using, you can write your hypotheses in a more precise and mathematical way specific to the test you chose. The table below provides template sentences for common statistical tests.

Note: The template sentences above assume that you’re performing one-tailed tests . One-tailed tests are appropriate for most studies.

The null hypothesis is often abbreviated as H 0 . When the null hypothesis is written using mathematical symbols, it always includes an equality symbol (usually =, but sometimes ≥ or ≤).

The alternative hypothesis is often abbreviated as H a or H 1 . When the alternative hypothesis is written using mathematical symbols, it always includes an inequality symbol (usually ≠, but sometimes < or >).

A research hypothesis is your proposed answer to your research question. The research hypothesis usually includes an explanation (‘ x affects y because …’).

A statistical hypothesis, on the other hand, is a mathematical statement about a population parameter. Statistical hypotheses always come in pairs: the null and alternative hypotheses. In a well-designed study , the statistical hypotheses correspond logically to the research hypothesis.

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9.1 Null and Alternative Hypotheses

The actual test begins by considering two hypotheses . They are called the null hypothesis and the alternative hypothesis . These hypotheses contain opposing viewpoints.

H 0 , the — null hypothesis: a statement of no difference between sample means or proportions or no difference between a sample mean or proportion and a population mean or proportion. In other words, the difference equals 0.

H a —, the alternative hypothesis: a claim about the population that is contradictory to H 0 and what we conclude when we reject H 0 .

Since the null and alternative hypotheses are contradictory, you must examine evidence to decide if you have enough evidence to reject the null hypothesis or not. The evidence is in the form of sample data.

After you have determined which hypothesis the sample supports, you make a decision. There are two options for a decision. They are reject H 0 if the sample information favors the alternative hypothesis or do not reject H 0 or decline to reject H 0 if the sample information is insufficient to reject the null hypothesis.

Mathematical Symbols Used in H 0 and H a :

H 0 always has a symbol with an equal in it. H a never has a symbol with an equal in it. The choice of symbol depends on the wording of the hypothesis test. However, be aware that many researchers use = in the null hypothesis, even with > or < as the symbol in the alternative hypothesis. This practice is acceptable because we only make the decision to reject or not reject the null hypothesis.

Example 9.1

H 0 : No more than 30 percent of the registered voters in Santa Clara County voted in the primary election. p ≤ 30 H a : More than 30 percent of the registered voters in Santa Clara County voted in the primary election. p > 30

A medical trial is conducted to test whether or not a new medicine reduces cholesterol by 25 percent. State the null and alternative hypotheses.

Example 9.2

We want to test whether the mean GPA of students in American colleges is different from 2.0 (out of 4.0). The null and alternative hypotheses are the following: H 0 : μ = 2.0 H a : μ ≠ 2.0

We want to test whether the mean height of eighth graders is 66 inches. State the null and alternative hypotheses. Fill in the correct symbol (=, ≠, ≥, <, ≤, >) for the null and alternative hypotheses.

• H 0 : μ __ 66
• H a : μ __ 66

Example 9.3

We want to test if college students take fewer than five years to graduate from college, on the average. The null and alternative hypotheses are the following: H 0 : μ ≥ 5 H a : μ < 5

We want to test if it takes fewer than 45 minutes to teach a lesson plan. State the null and alternative hypotheses. Fill in the correct symbol ( =, ≠, ≥, <, ≤, >) for the null and alternative hypotheses.

• H 0 : μ __ 45
• H a : μ __ 45

Example 9.4

An article on school standards stated that about half of all students in France, Germany, and Israel take advanced placement exams and a third of the students pass. The same article stated that 6.6 percent of U.S. students take advanced placement exams and 4.4 percent pass. Test if the percentage of U.S. students who take advanced placement exams is more than 6.6 percent. State the null and alternative hypotheses. H 0 : p ≤ 0.066 H a : p > 0.066

On a state driver’s test, about 40 percent pass the test on the first try. We want to test if more than 40 percent pass on the first try. Fill in the correct symbol (=, ≠, ≥, <, ≤, >) for the null and alternative hypotheses.

• H 0 : p __ 0.40
• H a : p __ 0.40

Collaborative Exercise

Bring to class a newspaper, some news magazines, and some internet articles. In groups, find articles from which your group can write null and alternative hypotheses. Discuss your hypotheses with the rest of the class.

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• Knowledge Base

Methodology

• How to Write a Strong Hypothesis | Steps & Examples

How to Write a Strong Hypothesis | Steps & Examples

Published on May 6, 2022 by Shona McCombes . Revised on November 20, 2023.

A hypothesis is a statement that can be tested by scientific research. If you want to test a relationship between two or more variables, you need to write hypotheses before you start your experiment or data collection .

Example: Hypothesis

Daily apple consumption leads to fewer doctor’s visits.

Table of contents

What is a hypothesis, developing a hypothesis (with example), hypothesis examples, other interesting articles, frequently asked questions about writing hypotheses.

A hypothesis states your predictions about what your research will find. It is a tentative answer to your research question that has not yet been tested. For some research projects, you might have to write several hypotheses that address different aspects of your research question.

A hypothesis is not just a guess – it should be based on existing theories and knowledge. It also has to be testable, which means you can support or refute it through scientific research methods (such as experiments, observations and statistical analysis of data).

Variables in hypotheses

Hypotheses propose a relationship between two or more types of variables .

• An independent variable is something the researcher changes or controls.
• A dependent variable is something the researcher observes and measures.

If there are any control variables , extraneous variables , or confounding variables , be sure to jot those down as you go to minimize the chances that research bias  will affect your results.

In this example, the independent variable is exposure to the sun – the assumed cause . The dependent variable is the level of happiness – the assumed effect .

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Step 1. ask a question.

Writing a hypothesis begins with a research question that you want to answer. The question should be focused, specific, and researchable within the constraints of your project.

Step 2. Do some preliminary research

Your initial answer to the question should be based on what is already known about the topic. Look for theories and previous studies to help you form educated assumptions about what your research will find.

At this stage, you might construct a conceptual framework to ensure that you’re embarking on a relevant topic . This can also help you identify which variables you will study and what you think the relationships are between them. Sometimes, you’ll have to operationalize more complex constructs.

Step 3. Formulate your hypothesis

Now you should have some idea of what you expect to find. Write your initial answer to the question in a clear, concise sentence.

4. Refine your hypothesis

You need to make sure your hypothesis is specific and testable. There are various ways of phrasing a hypothesis, but all the terms you use should have clear definitions, and the hypothesis should contain:

• The relevant variables
• The specific group being studied
• The predicted outcome of the experiment or analysis

5. Phrase your hypothesis in three ways

To identify the variables, you can write a simple prediction in  if…then form. The first part of the sentence states the independent variable and the second part states the dependent variable.

In academic research, hypotheses are more commonly phrased in terms of correlations or effects, where you directly state the predicted relationship between variables.

If you are comparing two groups, the hypothesis can state what difference you expect to find between them.

6. Write a null hypothesis

If your research involves statistical hypothesis testing , you will also have to write a null hypothesis . The null hypothesis is the default position that there is no association between the variables. The null hypothesis is written as H 0 , while the alternative hypothesis is H 1 or H a .

• H 0 : The number of lectures attended by first-year students has no effect on their final exam scores.
• H 1 : The number of lectures attended by first-year students has a positive effect on their final exam scores.

If you want to know more about the research process , methodology , research bias , or statistics , make sure to check out some of our other articles with explanations and examples.

• Sampling methods
• Simple random sampling
• Stratified sampling
• Cluster sampling
• Likert scales
• Reproducibility

 Statistics

• Null hypothesis
• Statistical power
• Probability distribution
• Effect size
• Poisson distribution

Research bias

• Optimism bias
• Cognitive bias
• Implicit bias
• Hawthorne effect
• Anchoring bias
• Explicit bias

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A hypothesis is not just a guess — it should be based on existing theories and knowledge. It also has to be testable, which means you can support or refute it through scientific research methods (such as experiments, observations and statistical analysis of data).

Null and alternative hypotheses are used in statistical hypothesis testing . The null hypothesis of a test always predicts no effect or no relationship between variables, while the alternative hypothesis states your research prediction of an effect or relationship.

Hypothesis testing is a formal procedure for investigating our ideas about the world using statistics. It is used by scientists to test specific predictions, called hypotheses , by calculating how likely it is that a pattern or relationship between variables could have arisen by chance.

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The Craft of Writing a Strong Hypothesis

Table of Contents

Writing a hypothesis is one of the essential elements of a scientific research paper. It needs to be to the point, clearly communicating what your research is trying to accomplish. A blurry, drawn-out, or complexly-structured hypothesis can confuse your readers. Or worse, the editor and peer reviewers.

A captivating hypothesis is not too intricate. This blog will take you through the process so that, by the end of it, you have a better idea of how to convey your research paper's intent in just one sentence.

What is a Hypothesis?

The first step in your scientific endeavor, a hypothesis, is a strong, concise statement that forms the basis of your research. It is not the same as a thesis statement , which is a brief summary of your research paper .

The sole purpose of a hypothesis is to predict your paper's findings, data, and conclusion. It comes from a place of curiosity and intuition . When you write a hypothesis, you're essentially making an educated guess based on scientific prejudices and evidence, which is further proven or disproven through the scientific method.

The reason for undertaking research is to observe a specific phenomenon. A hypothesis, therefore, lays out what the said phenomenon is. And it does so through two variables, an independent and dependent variable.

The independent variable is the cause behind the observation, while the dependent variable is the effect of the cause. A good example of this is “mixing red and blue forms purple.” In this hypothesis, mixing red and blue is the independent variable as you're combining the two colors at your own will. The formation of purple is the dependent variable as, in this case, it is conditional to the independent variable.

Different Types of Hypotheses‌

Types of hypotheses

Some would stand by the notion that there are only two types of hypotheses: a Null hypothesis and an Alternative hypothesis. While that may have some truth to it, it would be better to fully distinguish the most common forms as these terms come up so often, which might leave you out of context.

Apart from Null and Alternative, there are Complex, Simple, Directional, Non-Directional, Statistical, and Associative and casual hypotheses. They don't necessarily have to be exclusive, as one hypothesis can tick many boxes, but knowing the distinctions between them will make it easier for you to construct your own.

1. Null hypothesis

A null hypothesis proposes no relationship between two variables. Denoted by H 0 , it is a negative statement like “Attending physiotherapy sessions does not affect athletes' on-field performance.” Here, the author claims physiotherapy sessions have no effect on on-field performances. Even if there is, it's only a coincidence.

2. Alternative hypothesis

Considered to be the opposite of a null hypothesis, an alternative hypothesis is donated as H1 or Ha. It explicitly states that the dependent variable affects the independent variable. A good  alternative hypothesis example is “Attending physiotherapy sessions improves athletes' on-field performance.” or “Water evaporates at 100 °C. ” The alternative hypothesis further branches into directional and non-directional.

• Directional hypothesis: A hypothesis that states the result would be either positive or negative is called directional hypothesis. It accompanies H1 with either the ‘<' or ‘>' sign.
• Non-directional hypothesis: A non-directional hypothesis only claims an effect on the dependent variable. It does not clarify whether the result would be positive or negative. The sign for a non-directional hypothesis is ‘≠.'

3. Simple hypothesis

A simple hypothesis is a statement made to reflect the relation between exactly two variables. One independent and one dependent. Consider the example, “Smoking is a prominent cause of lung cancer." The dependent variable, lung cancer, is dependent on the independent variable, smoking.

4. Complex hypothesis

In contrast to a simple hypothesis, a complex hypothesis implies the relationship between multiple independent and dependent variables. For instance, “Individuals who eat more fruits tend to have higher immunity, lesser cholesterol, and high metabolism.” The independent variable is eating more fruits, while the dependent variables are higher immunity, lesser cholesterol, and high metabolism.

5. Associative and casual hypothesis

Associative and casual hypotheses don't exhibit how many variables there will be. They define the relationship between the variables. In an associative hypothesis, changing any one variable, dependent or independent, affects others. In a casual hypothesis, the independent variable directly affects the dependent.

6. Empirical hypothesis

Also referred to as the working hypothesis, an empirical hypothesis claims a theory's validation via experiments and observation. This way, the statement appears justifiable and different from a wild guess.

Say, the hypothesis is “Women who take iron tablets face a lesser risk of anemia than those who take vitamin B12.” This is an example of an empirical hypothesis where the researcher  the statement after assessing a group of women who take iron tablets and charting the findings.

7. Statistical hypothesis

The point of a statistical hypothesis is to test an already existing hypothesis by studying a population sample. Hypothesis like “44% of the Indian population belong in the age group of 22-27.” leverage evidence to prove or disprove a particular statement.

Characteristics of a Good Hypothesis

Writing a hypothesis is essential as it can make or break your research for you. That includes your chances of getting published in a journal. So when you're designing one, keep an eye out for these pointers:

• A research hypothesis has to be simple yet clear to look justifiable enough.
• It has to be testable — your research would be rendered pointless if too far-fetched into reality or limited by technology.
• It has to be precise about the results —what you are trying to do and achieve through it should come out in your hypothesis.
• A research hypothesis should be self-explanatory, leaving no doubt in the reader's mind.
• If you are developing a relational hypothesis, you need to include the variables and establish an appropriate relationship among them.
• A hypothesis must keep and reflect the scope for further investigations and experiments.

Separating a Hypothesis from a Prediction

Outside of academia, hypothesis and prediction are often used interchangeably. In research writing, this is not only confusing but also incorrect. And although a hypothesis and prediction are guesses at their core, there are many differences between them.

A hypothesis is an educated guess or even a testable prediction validated through research. It aims to analyze the gathered evidence and facts to define a relationship between variables and put forth a logical explanation behind the nature of events.

Predictions are assumptions or expected outcomes made without any backing evidence. They are more fictionally inclined regardless of where they originate from.

For this reason, a hypothesis holds much more weight than a prediction. It sticks to the scientific method rather than pure guesswork. "Planets revolve around the Sun." is an example of a hypothesis as it is previous knowledge and observed trends. Additionally, we can test it through the scientific method.

Whereas "COVID-19 will be eradicated by 2030." is a prediction. Even though it results from past trends, we can't prove or disprove it. So, the only way this gets validated is to wait and watch if COVID-19 cases end by 2030.

Finally, How to Write a Hypothesis

Quick tips on writing a hypothesis

1.  Be clear about your research question

A hypothesis should instantly address the research question or the problem statement. To do so, you need to ask a question. Understand the constraints of your undertaken research topic and then formulate a simple and topic-centric problem. Only after that can you develop a hypothesis and further test for evidence.

2. Carry out a recce

Once you have your research's foundation laid out, it would be best to conduct preliminary research. Go through previous theories, academic papers, data, and experiments before you start curating your research hypothesis. It will give you an idea of your hypothesis's viability or originality.

Making use of references from relevant research papers helps draft a good research hypothesis. SciSpace Discover offers a repository of over 270 million research papers to browse through and gain a deeper understanding of related studies on a particular topic. Additionally, you can use SciSpace Copilot , your AI research assistant, for reading any lengthy research paper and getting a more summarized context of it. A hypothesis can be formed after evaluating many such summarized research papers. Copilot also offers explanations for theories and equations, explains paper in simplified version, allows you to highlight any text in the paper or clip math equations and tables and provides a deeper, clear understanding of what is being said. This can improve the hypothesis by helping you identify potential research gaps.

3. Create a 3-dimensional hypothesis

Variables are an essential part of any reasonable hypothesis. So, identify your independent and dependent variable(s) and form a correlation between them. The ideal way to do this is to write the hypothetical assumption in the ‘if-then' form. If you use this form, make sure that you state the predefined relationship between the variables.

In another way, you can choose to present your hypothesis as a comparison between two variables. Here, you must specify the difference you expect to observe in the results.

4. Write the first draft

Now that everything is in place, it's time to write your hypothesis. For starters, create the first draft. In this version, write what you expect to find from your research.

Clearly separate your independent and dependent variables and the link between them. Don't fixate on syntax at this stage. The goal is to ensure your hypothesis addresses the issue.

5. Proof your hypothesis

After preparing the first draft of your hypothesis, you need to inspect it thoroughly. It should tick all the boxes, like being concise, straightforward, relevant, and accurate. Your final hypothesis has to be well-structured as well.

Research projects are an exciting and crucial part of being a scholar. And once you have your research question, you need a great hypothesis to begin conducting research. Thus, knowing how to write a hypothesis is very important.

Now that you have a firmer grasp on what a good hypothesis constitutes, the different kinds there are, and what process to follow, you will find it much easier to write your hypothesis, which ultimately helps your research.

Now it's easier than ever to streamline your research workflow with SciSpace Discover . Its integrated, comprehensive end-to-end platform for research allows scholars to easily discover, write and publish their research and fosters collaboration.

It includes everything you need, including a repository of over 270 million research papers across disciplines, SEO-optimized summaries and public profiles to show your expertise and experience.

If you found these tips on writing a research hypothesis useful, head over to our blog on Statistical Hypothesis Testing to learn about the top researchers, papers, and institutions in this domain.

Frequently Asked Questions (FAQs)

1. what is the definition of hypothesis.

According to the Oxford dictionary, a hypothesis is defined as “An idea or explanation of something that is based on a few known facts, but that has not yet been proved to be true or correct”.

2. What is an example of hypothesis?

The hypothesis is a statement that proposes a relationship between two or more variables. An example: "If we increase the number of new users who join our platform by 25%, then we will see an increase in revenue."

3. What is an example of null hypothesis?

A null hypothesis is a statement that there is no relationship between two variables. The null hypothesis is written as H0. The null hypothesis states that there is no effect. For example, if you're studying whether or not a particular type of exercise increases strength, your null hypothesis will be "there is no difference in strength between people who exercise and people who don't."

4. What are the types of research?

• Fundamental research

• Applied research

• Qualitative research

• Quantitative research

• Mixed research

• Exploratory research

• Longitudinal research

• Cross-sectional research

• Field research

• Laboratory research

• Fixed research

• Flexible research

• Action research

• Policy research

• Classification research

• Comparative research

• Causal research

• Inductive research

• Deductive research

5. How to write a hypothesis?

• Your hypothesis should be able to predict the relationship and outcome.

• Avoid wordiness by keeping it simple and brief.

• Your hypothesis should contain observable and testable outcomes.

• Your hypothesis should be relevant to the research question.

6. What are the 2 types of hypothesis?

• Null hypotheses are used to test the claim that "there is no difference between two groups of data".

• Alternative hypotheses test the claim that "there is a difference between two data groups".

7. Difference between research question and research hypothesis?

A research question is a broad, open-ended question you will try to answer through your research. A hypothesis is a statement based on prior research or theory that you expect to be true due to your study. Example - Research question: What are the factors that influence the adoption of the new technology? Research hypothesis: There is a positive relationship between age, education and income level with the adoption of the new technology.

8. What is plural for hypothesis?

The plural of hypothesis is hypotheses. Here's an example of how it would be used in a statement, "Numerous well-considered hypotheses are presented in this part, and they are supported by tables and figures that are well-illustrated."

9. What is the red queen hypothesis?

The red queen hypothesis in evolutionary biology states that species must constantly evolve to avoid extinction because if they don't, they will be outcompeted by other species that are evolving. Leigh Van Valen first proposed it in 1973; since then, it has been tested and substantiated many times.

10. Who is known as the father of null hypothesis?

The father of the null hypothesis is Sir Ronald Fisher. He published a paper in 1925 that introduced the concept of null hypothesis testing, and he was also the first to use the term itself.

11. When to reject null hypothesis?

You need to find a significant difference between your two populations to reject the null hypothesis. You can determine that by running statistical tests such as an independent sample t-test or a dependent sample t-test. You should reject the null hypothesis if the p-value is less than 0.05.

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7.3: The Research Hypothesis and the Null Hypothesis

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• Michelle Oja
• Taft College

Hypotheses are predictions of expected findings.

The Research Hypothesis

A research hypothesis is a mathematical way of stating a research question.  A research hypothesis names the groups (we'll start with a sample and a population), what was measured, and which we think will have a higher mean.  The last one gives the research hypothesis a direction.  In other words, a research hypothesis should include:

• The name of the groups being compared.  This is sometimes considered the IV.
• What was measured.  This is the DV.
• Which group are we predicting will have the higher mean.  

There are two types of research hypotheses related to sample means and population means:  Directional Research Hypotheses and Non-Directional Research Hypotheses

Directional Research Hypothesis

If we expect our obtained sample mean to be above or below the other group's mean (the population mean, for example), we have a directional hypothesis. There are two options:

• Symbol:       \( \displaystyle \bar{X} > \mu \)
• (The mean of the sample is greater than than the mean of the population.)
• Symbol:     \( \displaystyle \bar{X} < \mu \)
• (The mean of the sample is less than than mean of the population.)

Example \(\PageIndex{1}\)

A study by Blackwell, Trzesniewski, and Dweck (2007) measured growth mindset and how long the junior high student participants spent on their math homework.  What’s a directional hypothesis for how scoring higher on growth mindset (compared to the population of junior high students) would be related to how long students spent on their homework?  Write this out in words and symbols.

Answer in Words:            Students who scored high on growth mindset would spend more time on their homework than the population of junior high students.

Answer in Symbols:         \( \displaystyle \bar{X} > \mu \) 

Non-Directional Research Hypothesis

A non-directional hypothesis states that the means will be different, but does not specify which will be higher.  In reality, there is rarely a situation in which we actually don't want one group to be higher than the other, so we will focus on directional research hypotheses.  There is only one option for a non-directional research hypothesis: "The sample mean differs from the population mean."  These types of research hypotheses don’t give a direction, the hypothesis doesn’t say which will be higher or lower.

A non-directional research hypothesis in symbols should look like this:    \( \displaystyle \bar{X} \neq \mu \) (The mean of the sample is not equal to the mean of the population).

Exercise \(\PageIndex{1}\)

What’s a non-directional hypothesis for how scoring higher on growth mindset higher on growth mindset (compared to the population of junior high students) would be related to how long students spent on their homework (Blackwell, Trzesniewski, & Dweck, 2007)?  Write this out in words and symbols.

Answer in Words:            Students who scored high on growth mindset would spend a different amount of time on their homework than the population of junior high students.

Answer in Symbols:        \( \displaystyle \bar{X} \neq \mu \) 

See how a non-directional research hypothesis doesn't really make sense?  The big issue is not if the two groups differ, but if one group seems to improve what was measured (if having a growth mindset leads to more time spent on math homework).  This textbook will only use directional research hypotheses because researchers almost always have a predicted direction (meaning that we almost always know which group we think will score higher).

The Null Hypothesis

The hypothesis that an apparent effect is due to chance is called the null hypothesis, written \(H_0\) (“H-naught”). We usually test this through comparing an experimental group to a comparison (control) group.  This null hypothesis can be written as:

\[\mathrm{H}_{0}: \bar{X} = \mu \nonumber \]

For most of this textbook, the null hypothesis is that the means of the two groups are similar.  Much later, the null hypothesis will be that there is no relationship between the two groups.  Either way, remember that a null hypothesis is always saying that nothing is different.  

This is where descriptive statistics diverge from inferential statistics.  We know what the value of \(\overline{\mathrm{X}}\) is – it’s not a mystery or a question, it is what we observed from the sample.  What we are using inferential statistics to do is infer whether this sample's descriptive statistics probably represents the population's descriptive statistics.  This is the null hypothesis, that the two groups are similar.  

Keep in mind that the null hypothesis is typically the opposite of the research hypothesis. A research hypothesis for the ESP example is that those in my sample who say that they have ESP would get more correct answers than the population would get correct, while the null hypothesis is that the average number correct for the two groups will be similar. 

In general, the null hypothesis is the idea that nothing is going on: there is no effect of our treatment, no relation between our variables, and no difference in our sample mean from what we expected about the population mean. This is always our baseline starting assumption, and it is what we seek to reject. If we are trying to treat depression, we want to find a difference in average symptoms between our treatment and control groups. If we are trying to predict job performance, we want to find a relation between conscientiousness and evaluation scores. However, until we have evidence against it, we must use the null hypothesis as our starting point.

In sum, the null hypothesis is always : There is no difference between the groups’ means OR There is no relationship between the variables .

In the next chapter, the null hypothesis is that there’s no difference between the sample mean   and population mean.  In other words:

• There is no mean difference between the sample and population.
• The mean of the sample is the same as the mean of a specific population.
• \(\mathrm{H}_{0}: \bar{X} = \mu \nonumber \)
• We expect our sample’s mean to be same as the population mean.

Exercise \(\PageIndex{2}\)

A study by Blackwell, Trzesniewski, and Dweck (2007) measured growth mindset and how long the junior high student participants spent on their math homework.  What’s the null hypothesis for scoring higher on growth mindset (compared to the population of junior high students) and how long students spent on their homework?  Write this out in words and symbols.

Answer in Words:            Students who scored high on growth mindset would spend a similar amount of time on their homework as the population of junior high students.

Answer in Symbols:    \( \bar{X} = \mu \)

Contributors and Attributions

Foster et al.  (University of Missouri-St. Louis, Rice University, & University of Houston, Downtown Campus)

Dr. MO ( Taft College )

What is The Null Hypothesis & When Do You Reject The Null Hypothesis

Julia Simkus

Editor at Simply Psychology

BA (Hons) Psychology, Princeton University

Julia Simkus is a graduate of Princeton University with a Bachelor of Arts in Psychology. She is currently studying for a Master's Degree in Counseling for Mental Health and Wellness in September 2023. Julia's research has been published in peer reviewed journals.

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On This Page:

A null hypothesis is a statistical concept suggesting no significant difference or relationship between measured variables. It’s the default assumption unless empirical evidence proves otherwise.

The null hypothesis states no relationship exists between the two variables being studied (i.e., one variable does not affect the other).

The null hypothesis is the statement that a researcher or an investigator wants to disprove.

Testing the null hypothesis can tell you whether your results are due to the effects of manipulating ​ the dependent variable or due to random chance. 

How to Write a Null Hypothesis

Null hypotheses (H0) start as research questions that the investigator rephrases as statements indicating no effect or relationship between the independent and dependent variables.

It is a default position that your research aims to challenge or confirm.

For example, if studying the impact of exercise on weight loss, your null hypothesis might be:

There is no significant difference in weight loss between individuals who exercise daily and those who do not.

Examples of Null Hypotheses

When do we reject the null hypothesis .

We reject the null hypothesis when the data provide strong enough evidence to conclude that it is likely incorrect. This often occurs when the p-value (probability of observing the data given the null hypothesis is true) is below a predetermined significance level.

If the collected data does not meet the expectation of the null hypothesis, a researcher can conclude that the data lacks sufficient evidence to back up the null hypothesis, and thus the null hypothesis is rejected. 

Rejecting the null hypothesis means that a relationship does exist between a set of variables and the effect is statistically significant ( p > 0.05).

If the data collected from the random sample is not statistically significance , then the null hypothesis will be accepted, and the researchers can conclude that there is no relationship between the variables. 

You need to perform a statistical test on your data in order to evaluate how consistent it is with the null hypothesis. A p-value is one statistical measurement used to validate a hypothesis against observed data.

Calculating the p-value is a critical part of null-hypothesis significance testing because it quantifies how strongly the sample data contradicts the null hypothesis.

The level of statistical significance is often expressed as a  p  -value between 0 and 1. The smaller the p-value, the stronger the evidence that you should reject the null hypothesis.

Usually, a researcher uses a confidence level of 95% or 99% (p-value of 0.05 or 0.01) as general guidelines to decide if you should reject or keep the null.

When your p-value is less than or equal to your significance level, you reject the null hypothesis.

In other words, smaller p-values are taken as stronger evidence against the null hypothesis. Conversely, when the p-value is greater than your significance level, you fail to reject the null hypothesis.

In this case, the sample data provides insufficient data to conclude that the effect exists in the population.

Because you can never know with complete certainty whether there is an effect in the population, your inferences about a population will sometimes be incorrect.

When you incorrectly reject the null hypothesis, it’s called a type I error. When you incorrectly fail to reject it, it’s called a type II error.

Why Do We Never Accept The Null Hypothesis?

The reason we do not say “accept the null” is because we are always assuming the null hypothesis is true and then conducting a study to see if there is evidence against it. And, even if we don’t find evidence against it, a null hypothesis is not accepted.

A lack of evidence only means that you haven’t proven that something exists. It does not prove that something doesn’t exist. 

It is risky to conclude that the null hypothesis is true merely because we did not find evidence to reject it. It is always possible that researchers elsewhere have disproved the null hypothesis, so we cannot accept it as true, but instead, we state that we failed to reject the null. 

One can either reject the null hypothesis, or fail to reject it, but can never accept it.

Why Do We Use The Null Hypothesis?

We can never prove with 100% certainty that a hypothesis is true; We can only collect evidence that supports a theory. However, testing a hypothesis can set the stage for rejecting or accepting this hypothesis within a certain confidence level.

The null hypothesis is useful because it can tell us whether the results of our study are due to random chance or the manipulation of a variable (with a certain level of confidence).

A null hypothesis is rejected if the measured data is significantly unlikely to have occurred and a null hypothesis is accepted if the observed outcome is consistent with the position held by the null hypothesis.

Rejecting the null hypothesis sets the stage for further experimentation to see if a relationship between two variables exists. 

Hypothesis testing is a critical part of the scientific method as it helps decide whether the results of a research study support a particular theory about a given population. Hypothesis testing is a systematic way of backing up researchers’ predictions with statistical analysis.

It helps provide sufficient statistical evidence that either favors or rejects a certain hypothesis about the population parameter. 

Purpose of a Null Hypothesis 

• The primary purpose of the null hypothesis is to disprove an assumption. 
• Whether rejected or accepted, the null hypothesis can help further progress a theory in many scientific cases.
• A null hypothesis can be used to ascertain how consistent the outcomes of multiple studies are.

Do you always need both a Null Hypothesis and an Alternative Hypothesis?

The null (H0) and alternative (Ha or H1) hypotheses are two competing claims that describe the effect of the independent variable on the dependent variable. They are mutually exclusive, which means that only one of the two hypotheses can be true. 

While the null hypothesis states that there is no effect in the population, an alternative hypothesis states that there is statistical significance between two variables. 

The goal of hypothesis testing is to make inferences about a population based on a sample. In order to undertake hypothesis testing, you must express your research hypothesis as a null and alternative hypothesis. Both hypotheses are required to cover every possible outcome of the study. 

What is the difference between a null hypothesis and an alternative hypothesis?

The alternative hypothesis is the complement to the null hypothesis. The null hypothesis states that there is no effect or no relationship between variables, while the alternative hypothesis claims that there is an effect or relationship in the population.

It is the claim that you expect or hope will be true. The null hypothesis and the alternative hypothesis are always mutually exclusive, meaning that only one can be true at a time.

What are some problems with the null hypothesis?

One major problem with the null hypothesis is that researchers typically will assume that accepting the null is a failure of the experiment. However, accepting or rejecting any hypothesis is a positive result. Even if the null is not refuted, the researchers will still learn something new.

Why can a null hypothesis not be accepted?

We can either reject or fail to reject a null hypothesis, but never accept it. If your test fails to detect an effect, this is not proof that the effect doesn’t exist. It just means that your sample did not have enough evidence to conclude that it exists.

We can’t accept a null hypothesis because a lack of evidence does not prove something that does not exist. Instead, we fail to reject it.

Failing to reject the null indicates that the sample did not provide sufficient enough evidence to conclude that an effect exists.

If the p-value is greater than the significance level, then you fail to reject the null hypothesis.

Is a null hypothesis directional or non-directional?

A hypothesis test can either contain an alternative directional hypothesis or a non-directional alternative hypothesis. A directional hypothesis is one that contains the less than (“<“) or greater than (“>”) sign.

A nondirectional hypothesis contains the not equal sign (“≠”).  However, a null hypothesis is neither directional nor non-directional.

A null hypothesis is a prediction that there will be no change, relationship, or difference between two variables.

The directional hypothesis or nondirectional hypothesis would then be considered alternative hypotheses to the null hypothesis.

Gill, J. (1999). The insignificance of null hypothesis significance testing.  Political research quarterly ,  52 (3), 647-674.

Krueger, J. (2001). Null hypothesis significance testing: On the survival of a flawed method.  American Psychologist ,  56 (1), 16.

Masson, M. E. (2011). A tutorial on a practical Bayesian alternative to null-hypothesis significance testing.  Behavior research methods ,  43 , 679-690.

Nickerson, R. S. (2000). Null hypothesis significance testing: a review of an old and continuing controversy.  Psychological methods ,  5 (2), 241.

Rozeboom, W. W. (1960). The fallacy of the null-hypothesis significance test.  Psychological bulletin ,  57 (5), 416.

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AP®︎/College Statistics

Course: ap®︎/college statistics   >   unit 10.

• Idea behind hypothesis testing

Examples of null and alternative hypotheses

• Writing null and alternative hypotheses
• P-values and significance tests
• Comparing P-values to different significance levels
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Video transcript

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Neag School of Education

Educational Research Basics by Del Siegle

Null and alternative hypotheses.

Converting research questions to hypothesis is a simple task. Take the questions and make it a positive statement that says a relationship exists (correlation studies) or a difference exists between the groups (experiment study) and you have the alternative hypothesis. Write the statement such that a relationship does not exist or a difference does not exist and you have the null hypothesis. You can reverse the process if you have a hypothesis and wish to write a research question.

When you are comparing two groups, the groups are the independent variable. When you are testing whether something affects something else, the cause is the independent variable. The independent variable is the one you manipulate.

Teachers given higher pay will have more positive attitudes toward children than teachers given lower pay. The first step is to ask yourself “Are there two or more groups being compared?” The answer is “Yes.” What are the groups? Teachers who are given higher pay and teachers who are given lower pay. The independent variable is teacher pay. The dependent variable (the outcome) is attitude towards school.

You could also approach is another way. “Is something causing something else?” The answer is “Yes.”  What is causing what? Teacher pay is causing attitude towards school. Therefore, teacher pay is the independent variable (cause) and attitude towards school is the dependent variable (outcome).

By tradition, we try to disprove (reject) the null hypothesis. We can never prove a null hypothesis, because it is impossible to prove something does not exist. We can disprove something does not exist by finding an example of it. Therefore, in research we try to disprove the null hypothesis. When we do find that a relationship (or difference) exists then we reject the null and accept the alternative. If we do not find that a relationship (or difference) exists, we fail to reject the null hypothesis (and go with it). We never say we accept the null hypothesis because it is never possible to prove something does not exist. That is why we say that we failed to reject the null hypothesis, rather than we accepted it.

Del Siegle, Ph.D. Neag School of Education – University of Connecticut [email protected] www.delsiegle.com

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Once you have developed a clear and focused research question or set of research questions, you’ll be ready to conduct further research, a literature review, on the topic to help you make an educated guess about the answer to your question(s). This educated guess is called a hypothesis.

In research, there are two types of hypotheses: null and alternative. They work as a complementary pair, each stating that the other is wrong.

• Null Hypothesis (H 0 ) – This can be thought of as the implied hypothesis. “Null” meaning “nothing.”  This hypothesis states that there is no difference between groups or no relationship between variables. The null hypothesis is a presumption of status quo or no change.
• Alternative Hypothesis (H a ) – This is also known as the claim. This hypothesis should state what you expect the data to show, based on your research on the topic. This is your answer to your research question.

Null Hypothesis:   H 0 : There is no difference in the salary of factory workers based on gender. Alternative Hypothesis :  H a : Male factory workers have a higher salary than female factory workers.

Null Hypothesis :  H 0 : There is no relationship between height and shoe size. Alternative Hypothesis :  H a : There is a positive relationship between height and shoe size.

Null Hypothesis :  H 0 : Experience on the job has no impact on the quality of a brick mason’s work. Alternative Hypothesis :  H a : The quality of a brick mason’s work is influenced by on-the-job experience.

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Statistics Made Easy

How to Write a Null Hypothesis (5 Examples)

A hypothesis test uses sample data to determine whether or not some claim about a population parameter is true.

Whenever we perform a hypothesis test, we always write a null hypothesis and an alternative hypothesis, which take the following forms:

H 0 (Null Hypothesis): Population parameter =,  ≤, ≥ some value

H A  (Alternative Hypothesis): Population parameter <, >, ≠ some value

Note that the null hypothesis always contains the equal sign .

We interpret the hypotheses as follows:

Null hypothesis: The sample data provides no evidence to support some claim being made by an individual.

Alternative hypothesis: The sample data  does provide sufficient evidence to support the claim being made by an individual.

For example, suppose it’s assumed that the average height of a certain species of plant is 20 inches tall. However, one botanist claims the true average height is greater than 20 inches.

To test this claim, she may go out and collect a random sample of plants. She can then use this sample data to perform a hypothesis test using the following two hypotheses:

H 0 : μ ≤ 20 (the true mean height of plants is equal to or even less than 20 inches)

H A : μ > 20 (the true mean height of plants is greater than 20 inches)

If the sample data gathered by the botanist shows that the mean height of this species of plants is significantly greater than 20 inches, she can reject the null hypothesis and conclude that the mean height is greater than 20 inches.

Read through the following examples to gain a better understanding of how to write a null hypothesis in different situations.

Example 1: Weight of Turtles

A biologist wants to test whether or not the true mean weight of a certain species of turtles is 300 pounds. To test this, he goes out and measures the weight of a random sample of 40 turtles.

Here is how to write the null and alternative hypotheses for this scenario:

H 0 : μ = 300 (the true mean weight is equal to 300 pounds)

H A : μ ≠ 300 (the true mean weight is not equal to 300 pounds)

Example 2: Height of Males

It’s assumed that the mean height of males in a certain city is 68 inches. However, an independent researcher believes the true mean height is greater than 68 inches. To test this, he goes out and collects the height of 50 males in the city.

H 0 : μ ≤ 68 (the true mean height is equal to or even less than 68 inches)

H A : μ > 68 (the true mean height is greater than 68 inches)

Example 3: Graduation Rates

A university states that 80% of all students graduate on time. However, an independent researcher believes that less than 80% of all students graduate on time. To test this, she collects data on the proportion of students who graduated on time last year at the university.

H 0 : p ≥ 0.80 (the true proportion of students who graduate on time is 80% or higher)

H A : μ < 0.80 (the true proportion of students who graduate on time is less than 80%)

Example 4: Burger Weights

A food researcher wants to test whether or not the true mean weight of a burger at a certain restaurant is 7 ounces. To test this, he goes out and measures the weight of a random sample of 20 burgers from this restaurant.

H 0 : μ = 7 (the true mean weight is equal to 7 ounces)

H A : μ ≠ 7 (the true mean weight is not equal to 7 ounces)

Example 5: Citizen Support

A politician claims that less than 30% of citizens in a certain town support a certain law. To test this, he goes out and surveys 200 citizens on whether or not they support the law.

H 0 : p ≥ .30 (the true proportion of citizens who support the law is greater than or equal to 30%)

H A : μ < 0.30 (the true proportion of citizens who support the law is less than 30%)

Additional Resources

Introduction to Hypothesis Testing Introduction to Confidence Intervals An Explanation of P-Values and Statistical Significance

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What Is A Research (Scientific) Hypothesis? A plain-language explainer + examples

By:  Derek Jansen (MBA)  | Reviewed By: Dr Eunice Rautenbach | June 2020

If you’re new to the world of research, or it’s your first time writing a dissertation or thesis, you’re probably noticing that the words “research hypothesis” and “scientific hypothesis” are used quite a bit, and you’re wondering what they mean in a research context .

“Hypothesis” is one of those words that people use loosely, thinking they understand what it means. However, it has a very specific meaning within academic research. So, it’s important to understand the exact meaning before you start hypothesizing. 

Research Hypothesis 101

• What is a hypothesis ?
• What is a research hypothesis (scientific hypothesis)?
• Requirements for a research hypothesis
• Definition of a research hypothesis
• The null hypothesis

What is a hypothesis?

Let’s start with the general definition of a hypothesis (not a research hypothesis or scientific hypothesis), according to the Cambridge Dictionary:

Hypothesis: an idea or explanation for something that is based on known facts but has not yet been proved.

In other words, it’s a statement that provides an explanation for why or how something works, based on facts (or some reasonable assumptions), but that has not yet been specifically tested . For example, a hypothesis might look something like this:

Hypothesis: sleep impacts academic performance.

This statement predicts that academic performance will be influenced by the amount and/or quality of sleep a student engages in – sounds reasonable, right? It’s based on reasonable assumptions , underpinned by what we currently know about sleep and health (from the existing literature). So, loosely speaking, we could call it a hypothesis, at least by the dictionary definition.

But that’s not good enough…

Unfortunately, that’s not quite sophisticated enough to describe a research hypothesis (also sometimes called a scientific hypothesis), and it wouldn’t be acceptable in a dissertation, thesis or research paper . In the world of academic research, a statement needs a few more criteria to constitute a true research hypothesis .

What is a research hypothesis?

A research hypothesis (also called a scientific hypothesis) is a statement about the expected outcome of a study (for example, a dissertation or thesis). To constitute a quality hypothesis, the statement needs to have three attributes – specificity , clarity and testability .

Let’s take a look at these more closely.

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Hypothesis Essential #1: Specificity & Clarity

A good research hypothesis needs to be extremely clear and articulate about both what’ s being assessed (who or what variables are involved ) and the expected outcome (for example, a difference between groups, a relationship between variables, etc.).

Let’s stick with our sleepy students example and look at how this statement could be more specific and clear.

Hypothesis: Students who sleep at least 8 hours per night will, on average, achieve higher grades in standardised tests than students who sleep less than 8 hours a night.

As you can see, the statement is very specific as it identifies the variables involved (sleep hours and test grades), the parties involved (two groups of students), as well as the predicted relationship type (a positive relationship). There’s no ambiguity or uncertainty about who or what is involved in the statement, and the expected outcome is clear.

Contrast that to the original hypothesis we looked at – “Sleep impacts academic performance” – and you can see the difference. “Sleep” and “academic performance” are both comparatively vague , and there’s no indication of what the expected relationship direction is (more sleep or less sleep). As you can see, specificity and clarity are key.

Hypothesis Essential #2: Testability (Provability)

A statement must be testable to qualify as a research hypothesis. In other words, there needs to be a way to prove (or disprove) the statement. If it’s not testable, it’s not a hypothesis – simple as that.

For example, consider the hypothesis we mentioned earlier:

Hypothesis: Students who sleep at least 8 hours per night will, on average, achieve higher grades in standardised tests than students who sleep less than 8 hours a night.  

We could test this statement by undertaking a quantitative study involving two groups of students, one that gets 8 or more hours of sleep per night for a fixed period, and one that gets less. We could then compare the standardised test results for both groups to see if there’s a statistically significant difference. 

Again, if you compare this to the original hypothesis we looked at – “Sleep impacts academic performance” – you can see that it would be quite difficult to test that statement, primarily because it isn’t specific enough. How much sleep? By who? What type of academic performance?

So, remember the mantra – if you can’t test it, it’s not a hypothesis 🙂

Defining A Research Hypothesis

You’re still with us? Great! Let’s recap and pin down a clear definition of a hypothesis.

A research hypothesis (or scientific hypothesis) is a statement about an expected relationship between variables, or explanation of an occurrence, that is clear, specific and testable.

So, when you write up hypotheses for your dissertation or thesis, make sure that they meet all these criteria. If you do, you’ll not only have rock-solid hypotheses but you’ll also ensure a clear focus for your entire research project.

What about the null hypothesis?

You may have also heard the terms null hypothesis , alternative hypothesis, or H-zero thrown around. At a simple level, the null hypothesis is the counter-proposal to the original hypothesis.

For example, if the hypothesis predicts that there is a relationship between two variables (for example, sleep and academic performance), the null hypothesis would predict that there is no relationship between those variables.

At a more technical level, the null hypothesis proposes that no statistical significance exists in a set of given observations and that any differences are due to chance alone.

And there you have it – hypotheses in a nutshell. 

If you have any questions, be sure to leave a comment below and we’ll do our best to help you. If you need hands-on help developing and testing your hypotheses, consider our private coaching service , where we hold your hand through the research journey.

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16 Comments

Very useful information. I benefit more from getting more information in this regard.

Very great insight,educative and informative. Please give meet deep critics on many research data of public international Law like human rights, environment, natural resources, law of the sea etc

In a book I read a distinction is made between null, research, and alternative hypothesis. As far as I understand, alternative and research hypotheses are the same. Can you please elaborate? Best Afshin

This is a self explanatory, easy going site. I will recommend this to my friends and colleagues.

Very good definition. How can I cite your definition in my thesis? Thank you. Is nul hypothesis compulsory in a research?

It’s a counter-proposal to be proven as a rejection

Please what is the difference between alternate hypothesis and research hypothesis?

It is a very good explanation. However, it limits hypotheses to statistically tasteable ideas. What about for qualitative researches or other researches that involve quantitative data that don’t need statistical tests?

In qualitative research, one typically uses propositions, not hypotheses.

could you please elaborate it more

I’ve benefited greatly from these notes, thank you.

This is very helpful

well articulated ideas are presented here, thank you for being reliable sources of information

Excellent. Thanks for being clear and sound about the research methodology and hypothesis (quantitative research)

I have only a simple question regarding the null hypothesis. – Is the null hypothesis (Ho) known as the reversible hypothesis of the alternative hypothesis (H1? – How to test it in academic research?

this is very important note help me much more

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A Practical Guide to Writing Quantitative and Qualitative Research Questions and Hypotheses in Scholarly Articles

Edward barroga.

1 Department of General Education, Graduate School of Nursing Science, St. Luke’s International University, Tokyo, Japan.

Glafera Janet Matanguihan

2 Department of Biological Sciences, Messiah University, Mechanicsburg, PA, USA.

The development of research questions and the subsequent hypotheses are prerequisites to defining the main research purpose and specific objectives of a study. Consequently, these objectives determine the study design and research outcome. The development of research questions is a process based on knowledge of current trends, cutting-edge studies, and technological advances in the research field. Excellent research questions are focused and require a comprehensive literature search and in-depth understanding of the problem being investigated. Initially, research questions may be written as descriptive questions which could be developed into inferential questions. These questions must be specific and concise to provide a clear foundation for developing hypotheses. Hypotheses are more formal predictions about the research outcomes. These specify the possible results that may or may not be expected regarding the relationship between groups. Thus, research questions and hypotheses clarify the main purpose and specific objectives of the study, which in turn dictate the design of the study, its direction, and outcome. Studies developed from good research questions and hypotheses will have trustworthy outcomes with wide-ranging social and health implications.

INTRODUCTION

Scientific research is usually initiated by posing evidenced-based research questions which are then explicitly restated as hypotheses. 1 , 2 The hypotheses provide directions to guide the study, solutions, explanations, and expected results. 3 , 4 Both research questions and hypotheses are essentially formulated based on conventional theories and real-world processes, which allow the inception of novel studies and the ethical testing of ideas. 5 , 6

It is crucial to have knowledge of both quantitative and qualitative research 2 as both types of research involve writing research questions and hypotheses. 7 However, these crucial elements of research are sometimes overlooked; if not overlooked, then framed without the forethought and meticulous attention it needs. Planning and careful consideration are needed when developing quantitative or qualitative research, particularly when conceptualizing research questions and hypotheses. 4

There is a continuing need to support researchers in the creation of innovative research questions and hypotheses, as well as for journal articles that carefully review these elements. 1 When research questions and hypotheses are not carefully thought of, unethical studies and poor outcomes usually ensue. Carefully formulated research questions and hypotheses define well-founded objectives, which in turn determine the appropriate design, course, and outcome of the study. This article then aims to discuss in detail the various aspects of crafting research questions and hypotheses, with the goal of guiding researchers as they develop their own. Examples from the authors and peer-reviewed scientific articles in the healthcare field are provided to illustrate key points.

DEFINITIONS AND RELATIONSHIP OF RESEARCH QUESTIONS AND HYPOTHESES

A research question is what a study aims to answer after data analysis and interpretation. The answer is written in length in the discussion section of the paper. Thus, the research question gives a preview of the different parts and variables of the study meant to address the problem posed in the research question. 1 An excellent research question clarifies the research writing while facilitating understanding of the research topic, objective, scope, and limitations of the study. 5

On the other hand, a research hypothesis is an educated statement of an expected outcome. This statement is based on background research and current knowledge. 8 , 9 The research hypothesis makes a specific prediction about a new phenomenon 10 or a formal statement on the expected relationship between an independent variable and a dependent variable. 3 , 11 It provides a tentative answer to the research question to be tested or explored. 4

Hypotheses employ reasoning to predict a theory-based outcome. 10 These can also be developed from theories by focusing on components of theories that have not yet been observed. 10 The validity of hypotheses is often based on the testability of the prediction made in a reproducible experiment. 8

Conversely, hypotheses can also be rephrased as research questions. Several hypotheses based on existing theories and knowledge may be needed to answer a research question. Developing ethical research questions and hypotheses creates a research design that has logical relationships among variables. These relationships serve as a solid foundation for the conduct of the study. 4 , 11 Haphazardly constructed research questions can result in poorly formulated hypotheses and improper study designs, leading to unreliable results. Thus, the formulations of relevant research questions and verifiable hypotheses are crucial when beginning research. 12

CHARACTERISTICS OF GOOD RESEARCH QUESTIONS AND HYPOTHESES

Excellent research questions are specific and focused. These integrate collective data and observations to confirm or refute the subsequent hypotheses. Well-constructed hypotheses are based on previous reports and verify the research context. These are realistic, in-depth, sufficiently complex, and reproducible. More importantly, these hypotheses can be addressed and tested. 13

There are several characteristics of well-developed hypotheses. Good hypotheses are 1) empirically testable 7 , 10 , 11 , 13 ; 2) backed by preliminary evidence 9 ; 3) testable by ethical research 7 , 9 ; 4) based on original ideas 9 ; 5) have evidenced-based logical reasoning 10 ; and 6) can be predicted. 11 Good hypotheses can infer ethical and positive implications, indicating the presence of a relationship or effect relevant to the research theme. 7 , 11 These are initially developed from a general theory and branch into specific hypotheses by deductive reasoning. In the absence of a theory to base the hypotheses, inductive reasoning based on specific observations or findings form more general hypotheses. 10

TYPES OF RESEARCH QUESTIONS AND HYPOTHESES

Research questions and hypotheses are developed according to the type of research, which can be broadly classified into quantitative and qualitative research. We provide a summary of the types of research questions and hypotheses under quantitative and qualitative research categories in Table 1 .

Research questions in quantitative research

In quantitative research, research questions inquire about the relationships among variables being investigated and are usually framed at the start of the study. These are precise and typically linked to the subject population, dependent and independent variables, and research design. 1 Research questions may also attempt to describe the behavior of a population in relation to one or more variables, or describe the characteristics of variables to be measured ( descriptive research questions ). 1 , 5 , 14 These questions may also aim to discover differences between groups within the context of an outcome variable ( comparative research questions ), 1 , 5 , 14 or elucidate trends and interactions among variables ( relationship research questions ). 1 , 5 We provide examples of descriptive, comparative, and relationship research questions in quantitative research in Table 2 .

Hypotheses in quantitative research

In quantitative research, hypotheses predict the expected relationships among variables. 15 Relationships among variables that can be predicted include 1) between a single dependent variable and a single independent variable ( simple hypothesis ) or 2) between two or more independent and dependent variables ( complex hypothesis ). 4 , 11 Hypotheses may also specify the expected direction to be followed and imply an intellectual commitment to a particular outcome ( directional hypothesis ) 4 . On the other hand, hypotheses may not predict the exact direction and are used in the absence of a theory, or when findings contradict previous studies ( non-directional hypothesis ). 4 In addition, hypotheses can 1) define interdependency between variables ( associative hypothesis ), 4 2) propose an effect on the dependent variable from manipulation of the independent variable ( causal hypothesis ), 4 3) state a negative relationship between two variables ( null hypothesis ), 4 , 11 , 15 4) replace the working hypothesis if rejected ( alternative hypothesis ), 15 explain the relationship of phenomena to possibly generate a theory ( working hypothesis ), 11 5) involve quantifiable variables that can be tested statistically ( statistical hypothesis ), 11 6) or express a relationship whose interlinks can be verified logically ( logical hypothesis ). 11 We provide examples of simple, complex, directional, non-directional, associative, causal, null, alternative, working, statistical, and logical hypotheses in quantitative research, as well as the definition of quantitative hypothesis-testing research in Table 3 .

Research questions in qualitative research

Unlike research questions in quantitative research, research questions in qualitative research are usually continuously reviewed and reformulated. The central question and associated subquestions are stated more than the hypotheses. 15 The central question broadly explores a complex set of factors surrounding the central phenomenon, aiming to present the varied perspectives of participants. 15

There are varied goals for which qualitative research questions are developed. These questions can function in several ways, such as to 1) identify and describe existing conditions ( contextual research question s); 2) describe a phenomenon ( descriptive research questions ); 3) assess the effectiveness of existing methods, protocols, theories, or procedures ( evaluation research questions ); 4) examine a phenomenon or analyze the reasons or relationships between subjects or phenomena ( explanatory research questions ); or 5) focus on unknown aspects of a particular topic ( exploratory research questions ). 5 In addition, some qualitative research questions provide new ideas for the development of theories and actions ( generative research questions ) or advance specific ideologies of a position ( ideological research questions ). 1 Other qualitative research questions may build on a body of existing literature and become working guidelines ( ethnographic research questions ). Research questions may also be broadly stated without specific reference to the existing literature or a typology of questions ( phenomenological research questions ), may be directed towards generating a theory of some process ( grounded theory questions ), or may address a description of the case and the emerging themes ( qualitative case study questions ). 15 We provide examples of contextual, descriptive, evaluation, explanatory, exploratory, generative, ideological, ethnographic, phenomenological, grounded theory, and qualitative case study research questions in qualitative research in Table 4 , and the definition of qualitative hypothesis-generating research in Table 5 .

Qualitative studies usually pose at least one central research question and several subquestions starting with How or What . These research questions use exploratory verbs such as explore or describe . These also focus on one central phenomenon of interest, and may mention the participants and research site. 15

Hypotheses in qualitative research

Hypotheses in qualitative research are stated in the form of a clear statement concerning the problem to be investigated. Unlike in quantitative research where hypotheses are usually developed to be tested, qualitative research can lead to both hypothesis-testing and hypothesis-generating outcomes. 2 When studies require both quantitative and qualitative research questions, this suggests an integrative process between both research methods wherein a single mixed-methods research question can be developed. 1

FRAMEWORKS FOR DEVELOPING RESEARCH QUESTIONS AND HYPOTHESES

Research questions followed by hypotheses should be developed before the start of the study. 1 , 12 , 14 It is crucial to develop feasible research questions on a topic that is interesting to both the researcher and the scientific community. This can be achieved by a meticulous review of previous and current studies to establish a novel topic. Specific areas are subsequently focused on to generate ethical research questions. The relevance of the research questions is evaluated in terms of clarity of the resulting data, specificity of the methodology, objectivity of the outcome, depth of the research, and impact of the study. 1 , 5 These aspects constitute the FINER criteria (i.e., Feasible, Interesting, Novel, Ethical, and Relevant). 1 Clarity and effectiveness are achieved if research questions meet the FINER criteria. In addition to the FINER criteria, Ratan et al. described focus, complexity, novelty, feasibility, and measurability for evaluating the effectiveness of research questions. 14

The PICOT and PEO frameworks are also used when developing research questions. 1 The following elements are addressed in these frameworks, PICOT: P-population/patients/problem, I-intervention or indicator being studied, C-comparison group, O-outcome of interest, and T-timeframe of the study; PEO: P-population being studied, E-exposure to preexisting conditions, and O-outcome of interest. 1 Research questions are also considered good if these meet the “FINERMAPS” framework: Feasible, Interesting, Novel, Ethical, Relevant, Manageable, Appropriate, Potential value/publishable, and Systematic. 14

As we indicated earlier, research questions and hypotheses that are not carefully formulated result in unethical studies or poor outcomes. To illustrate this, we provide some examples of ambiguous research question and hypotheses that result in unclear and weak research objectives in quantitative research ( Table 6 ) 16 and qualitative research ( Table 7 ) 17 , and how to transform these ambiguous research question(s) and hypothesis(es) into clear and good statements.

a These statements were composed for comparison and illustrative purposes only.

b These statements are direct quotes from Higashihara and Horiuchi. 16

a This statement is a direct quote from Shimoda et al. 17

The other statements were composed for comparison and illustrative purposes only.

CONSTRUCTING RESEARCH QUESTIONS AND HYPOTHESES

To construct effective research questions and hypotheses, it is very important to 1) clarify the background and 2) identify the research problem at the outset of the research, within a specific timeframe. 9 Then, 3) review or conduct preliminary research to collect all available knowledge about the possible research questions by studying theories and previous studies. 18 Afterwards, 4) construct research questions to investigate the research problem. Identify variables to be accessed from the research questions 4 and make operational definitions of constructs from the research problem and questions. Thereafter, 5) construct specific deductive or inductive predictions in the form of hypotheses. 4 Finally, 6) state the study aims . This general flow for constructing effective research questions and hypotheses prior to conducting research is shown in Fig. 1 .

Research questions are used more frequently in qualitative research than objectives or hypotheses. 3 These questions seek to discover, understand, explore or describe experiences by asking “What” or “How.” The questions are open-ended to elicit a description rather than to relate variables or compare groups. The questions are continually reviewed, reformulated, and changed during the qualitative study. 3 Research questions are also used more frequently in survey projects than hypotheses in experiments in quantitative research to compare variables and their relationships.

Hypotheses are constructed based on the variables identified and as an if-then statement, following the template, ‘If a specific action is taken, then a certain outcome is expected.’ At this stage, some ideas regarding expectations from the research to be conducted must be drawn. 18 Then, the variables to be manipulated (independent) and influenced (dependent) are defined. 4 Thereafter, the hypothesis is stated and refined, and reproducible data tailored to the hypothesis are identified, collected, and analyzed. 4 The hypotheses must be testable and specific, 18 and should describe the variables and their relationships, the specific group being studied, and the predicted research outcome. 18 Hypotheses construction involves a testable proposition to be deduced from theory, and independent and dependent variables to be separated and measured separately. 3 Therefore, good hypotheses must be based on good research questions constructed at the start of a study or trial. 12

In summary, research questions are constructed after establishing the background of the study. Hypotheses are then developed based on the research questions. Thus, it is crucial to have excellent research questions to generate superior hypotheses. In turn, these would determine the research objectives and the design of the study, and ultimately, the outcome of the research. 12 Algorithms for building research questions and hypotheses are shown in Fig. 2 for quantitative research and in Fig. 3 for qualitative research.

EXAMPLES OF RESEARCH QUESTIONS FROM PUBLISHED ARTICLES

• EXAMPLE 1. Descriptive research question (quantitative research)
• - Presents research variables to be assessed (distinct phenotypes and subphenotypes)
• “BACKGROUND: Since COVID-19 was identified, its clinical and biological heterogeneity has been recognized. Identifying COVID-19 phenotypes might help guide basic, clinical, and translational research efforts.
• RESEARCH QUESTION: Does the clinical spectrum of patients with COVID-19 contain distinct phenotypes and subphenotypes? ” 19
• EXAMPLE 2. Relationship research question (quantitative research)
• - Shows interactions between dependent variable (static postural control) and independent variable (peripheral visual field loss)
• “Background: Integration of visual, vestibular, and proprioceptive sensations contributes to postural control. People with peripheral visual field loss have serious postural instability. However, the directional specificity of postural stability and sensory reweighting caused by gradual peripheral visual field loss remain unclear.
• Research question: What are the effects of peripheral visual field loss on static postural control ?” 20
• EXAMPLE 3. Comparative research question (quantitative research)
• - Clarifies the difference among groups with an outcome variable (patients enrolled in COMPERA with moderate PH or severe PH in COPD) and another group without the outcome variable (patients with idiopathic pulmonary arterial hypertension (IPAH))
• “BACKGROUND: Pulmonary hypertension (PH) in COPD is a poorly investigated clinical condition.
• RESEARCH QUESTION: Which factors determine the outcome of PH in COPD?
• STUDY DESIGN AND METHODS: We analyzed the characteristics and outcome of patients enrolled in the Comparative, Prospective Registry of Newly Initiated Therapies for Pulmonary Hypertension (COMPERA) with moderate or severe PH in COPD as defined during the 6th PH World Symposium who received medical therapy for PH and compared them with patients with idiopathic pulmonary arterial hypertension (IPAH) .” 21
• EXAMPLE 4. Exploratory research question (qualitative research)
• - Explores areas that have not been fully investigated (perspectives of families and children who receive care in clinic-based child obesity treatment) to have a deeper understanding of the research problem
• “Problem: Interventions for children with obesity lead to only modest improvements in BMI and long-term outcomes, and data are limited on the perspectives of families of children with obesity in clinic-based treatment. This scoping review seeks to answer the question: What is known about the perspectives of families and children who receive care in clinic-based child obesity treatment? This review aims to explore the scope of perspectives reported by families of children with obesity who have received individualized outpatient clinic-based obesity treatment.” 22
• EXAMPLE 5. Relationship research question (quantitative research)
• - Defines interactions between dependent variable (use of ankle strategies) and independent variable (changes in muscle tone)
• “Background: To maintain an upright standing posture against external disturbances, the human body mainly employs two types of postural control strategies: “ankle strategy” and “hip strategy.” While it has been reported that the magnitude of the disturbance alters the use of postural control strategies, it has not been elucidated how the level of muscle tone, one of the crucial parameters of bodily function, determines the use of each strategy. We have previously confirmed using forward dynamics simulations of human musculoskeletal models that an increased muscle tone promotes the use of ankle strategies. The objective of the present study was to experimentally evaluate a hypothesis: an increased muscle tone promotes the use of ankle strategies. Research question: Do changes in the muscle tone affect the use of ankle strategies ?” 23

EXAMPLES OF HYPOTHESES IN PUBLISHED ARTICLES

• EXAMPLE 1. Working hypothesis (quantitative research)
• - A hypothesis that is initially accepted for further research to produce a feasible theory
• “As fever may have benefit in shortening the duration of viral illness, it is plausible to hypothesize that the antipyretic efficacy of ibuprofen may be hindering the benefits of a fever response when taken during the early stages of COVID-19 illness .” 24
• “In conclusion, it is plausible to hypothesize that the antipyretic efficacy of ibuprofen may be hindering the benefits of a fever response . The difference in perceived safety of these agents in COVID-19 illness could be related to the more potent efficacy to reduce fever with ibuprofen compared to acetaminophen. Compelling data on the benefit of fever warrant further research and review to determine when to treat or withhold ibuprofen for early stage fever for COVID-19 and other related viral illnesses .” 24
• EXAMPLE 2. Exploratory hypothesis (qualitative research)
• - Explores particular areas deeper to clarify subjective experience and develop a formal hypothesis potentially testable in a future quantitative approach
• “We hypothesized that when thinking about a past experience of help-seeking, a self distancing prompt would cause increased help-seeking intentions and more favorable help-seeking outcome expectations .” 25
• “Conclusion
• Although a priori hypotheses were not supported, further research is warranted as results indicate the potential for using self-distancing approaches to increasing help-seeking among some people with depressive symptomatology.” 25
• EXAMPLE 3. Hypothesis-generating research to establish a framework for hypothesis testing (qualitative research)
• “We hypothesize that compassionate care is beneficial for patients (better outcomes), healthcare systems and payers (lower costs), and healthcare providers (lower burnout). ” 26
• Compassionomics is the branch of knowledge and scientific study of the effects of compassionate healthcare. Our main hypotheses are that compassionate healthcare is beneficial for (1) patients, by improving clinical outcomes, (2) healthcare systems and payers, by supporting financial sustainability, and (3) HCPs, by lowering burnout and promoting resilience and well-being. The purpose of this paper is to establish a scientific framework for testing the hypotheses above . If these hypotheses are confirmed through rigorous research, compassionomics will belong in the science of evidence-based medicine, with major implications for all healthcare domains.” 26
• EXAMPLE 4. Statistical hypothesis (quantitative research)
• - An assumption is made about the relationship among several population characteristics ( gender differences in sociodemographic and clinical characteristics of adults with ADHD ). Validity is tested by statistical experiment or analysis ( chi-square test, Students t-test, and logistic regression analysis)
• “Our research investigated gender differences in sociodemographic and clinical characteristics of adults with ADHD in a Japanese clinical sample. Due to unique Japanese cultural ideals and expectations of women's behavior that are in opposition to ADHD symptoms, we hypothesized that women with ADHD experience more difficulties and present more dysfunctions than men . We tested the following hypotheses: first, women with ADHD have more comorbidities than men with ADHD; second, women with ADHD experience more social hardships than men, such as having less full-time employment and being more likely to be divorced.” 27
• “Statistical Analysis
• ( text omitted ) Between-gender comparisons were made using the chi-squared test for categorical variables and Students t-test for continuous variables…( text omitted ). A logistic regression analysis was performed for employment status, marital status, and comorbidity to evaluate the independent effects of gender on these dependent variables.” 27

EXAMPLES OF HYPOTHESIS AS WRITTEN IN PUBLISHED ARTICLES IN RELATION TO OTHER PARTS

• EXAMPLE 1. Background, hypotheses, and aims are provided
• “Pregnant women need skilled care during pregnancy and childbirth, but that skilled care is often delayed in some countries …( text omitted ). The focused antenatal care (FANC) model of WHO recommends that nurses provide information or counseling to all pregnant women …( text omitted ). Job aids are visual support materials that provide the right kind of information using graphics and words in a simple and yet effective manner. When nurses are not highly trained or have many work details to attend to, these job aids can serve as a content reminder for the nurses and can be used for educating their patients (Jennings, Yebadokpo, Affo, & Agbogbe, 2010) ( text omitted ). Importantly, additional evidence is needed to confirm how job aids can further improve the quality of ANC counseling by health workers in maternal care …( text omitted )” 28
• “ This has led us to hypothesize that the quality of ANC counseling would be better if supported by job aids. Consequently, a better quality of ANC counseling is expected to produce higher levels of awareness concerning the danger signs of pregnancy and a more favorable impression of the caring behavior of nurses .” 28
• “This study aimed to examine the differences in the responses of pregnant women to a job aid-supported intervention during ANC visit in terms of 1) their understanding of the danger signs of pregnancy and 2) their impression of the caring behaviors of nurses to pregnant women in rural Tanzania.” 28
• EXAMPLE 2. Background, hypotheses, and aims are provided
• “We conducted a two-arm randomized controlled trial (RCT) to evaluate and compare changes in salivary cortisol and oxytocin levels of first-time pregnant women between experimental and control groups. The women in the experimental group touched and held an infant for 30 min (experimental intervention protocol), whereas those in the control group watched a DVD movie of an infant (control intervention protocol). The primary outcome was salivary cortisol level and the secondary outcome was salivary oxytocin level.” 29
• “ We hypothesize that at 30 min after touching and holding an infant, the salivary cortisol level will significantly decrease and the salivary oxytocin level will increase in the experimental group compared with the control group .” 29
• EXAMPLE 3. Background, aim, and hypothesis are provided
• “In countries where the maternal mortality ratio remains high, antenatal education to increase Birth Preparedness and Complication Readiness (BPCR) is considered one of the top priorities [1]. BPCR includes birth plans during the antenatal period, such as the birthplace, birth attendant, transportation, health facility for complications, expenses, and birth materials, as well as family coordination to achieve such birth plans. In Tanzania, although increasing, only about half of all pregnant women attend an antenatal clinic more than four times [4]. Moreover, the information provided during antenatal care (ANC) is insufficient. In the resource-poor settings, antenatal group education is a potential approach because of the limited time for individual counseling at antenatal clinics.” 30
• “This study aimed to evaluate an antenatal group education program among pregnant women and their families with respect to birth-preparedness and maternal and infant outcomes in rural villages of Tanzania.” 30
• “ The study hypothesis was if Tanzanian pregnant women and their families received a family-oriented antenatal group education, they would (1) have a higher level of BPCR, (2) attend antenatal clinic four or more times, (3) give birth in a health facility, (4) have less complications of women at birth, and (5) have less complications and deaths of infants than those who did not receive the education .” 30

Research questions and hypotheses are crucial components to any type of research, whether quantitative or qualitative. These questions should be developed at the very beginning of the study. Excellent research questions lead to superior hypotheses, which, like a compass, set the direction of research, and can often determine the successful conduct of the study. Many research studies have floundered because the development of research questions and subsequent hypotheses was not given the thought and meticulous attention needed. The development of research questions and hypotheses is an iterative process based on extensive knowledge of the literature and insightful grasp of the knowledge gap. Focused, concise, and specific research questions provide a strong foundation for constructing hypotheses which serve as formal predictions about the research outcomes. Research questions and hypotheses are crucial elements of research that should not be overlooked. They should be carefully thought of and constructed when planning research. This avoids unethical studies and poor outcomes by defining well-founded objectives that determine the design, course, and outcome of the study.

Disclosure: The authors have no potential conflicts of interest to disclose.

Author Contributions:

• Conceptualization: Barroga E, Matanguihan GJ.
• Methodology: Barroga E, Matanguihan GJ.
• Writing - original draft: Barroga E, Matanguihan GJ.
• Writing - review & editing: Barroga E, Matanguihan GJ.

What is Null Hypothesis? What Is Its Importance in Research?

Scientists begin their research with a hypothesis that a relationship of some kind exists between variables. The null hypothesis is the opposite stating that no such relationship exists. Null hypothesis may seem unexciting, but it is a very important aspect of research. In this article, we discuss what null hypothesis is, how to make use of it, and why you should use it to improve your statistical analyses.

What is the Null Hypothesis?

The null hypothesis can be tested using statistical analysis  and is often written as H 0 (read as “H-naught”). Once you determine how likely the sample relationship would be if the H 0   were true, you can run your analysis. Researchers use a significance test to determine the likelihood that the results supporting the H 0 are not due to chance.

The null hypothesis is not the same as an alternative hypothesis. An alternative hypothesis states, that there is a relationship between two variables, while H 0 posits the opposite. Let us consider the following example.

A researcher wants to discover the relationship between exercise frequency and appetite. She asks:

Q: Does increased exercise frequency lead to increased appetite? Alternative hypothesis: Increased exercise frequency leads to increased appetite. H 0 assumes that there is no relationship between the two variables: Increased exercise frequency does not lead to increased appetite.

Let us look at another example of how to state the null hypothesis:

Q: Does insufficient sleep lead to an increased risk of heart attack among men over age 50? H 0 : The amount of sleep men over age 50 get does not increase their risk of heart attack.

Why is Null Hypothesis Important?

Many scientists often neglect null hypothesis in their testing. As shown in the above examples, H 0 is often assumed to be the opposite of the hypothesis being tested. However, it is good practice to include H 0 and ensure it is carefully worded. To understand why, let us return to our previous example. In this case,

Alternative hypothesis: Getting too little sleep leads to an increased risk of heart attack among men over age 50.

H 0 : The amount of sleep men over age 50 get has no effect on their risk of heart attack.

Note that this H 0 is different than the one in our first example. What if we were to conduct this experiment and find that neither H 0 nor the alternative hypothesis was supported? The experiment would be considered invalid . Take our original H 0 in this case, “the amount of sleep men over age 50 get, does not increase their risk of heart attack”. If this H 0 is found to be untrue, and so is the alternative, we can still consider a third hypothesis. Perhaps getting insufficient sleep actually decreases the risk of a heart attack among men over age 50. Because we have tested H 0 , we have more information that we would not have if we had neglected it.

Do I Really Need to Test It?

The biggest problem with the null hypothesis is that many scientists see accepting it as a failure of the experiment. They consider that they have not proven anything of value. However, as we have learned from the replication crisis , negative results are just as important as positive ones. While they may seem less appealing to publishers, they can tell the scientific community important information about correlations that do or do not exist. In this way, they can drive science forward and prevent the wastage of resources.

Do you test for the null hypothesis? Why or why not? Let us know your thoughts in the comments below.

The following null hypotheses were formulated for this study: Ho1. There are no significant differences in the factors that influence urban gardening when respondents are grouped according to age, sex, household size, social status and average combined monthly income.

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Home » Research Hypothesis Vs Null Hypothesis

Research Hypothesis Vs Null Hypothesis

Table of Contents

The difference between Research Hypothesis Vs Null Hypothesis is as follows:

Research Hypothesis

A Research Hypothesis is a tentative statement that proposes a relationship between two or more variables. It is based on a theoretical or conceptual framework and is typically tested through empirical research.

Null Hypothesis

A Null Hypothesis is a statement that proposes that there is no relationship between two or more variables. It is the opposite of the research hypothesis and is used as a comparison in statistical analysis.

Comparison Table:

In summary, a research hypothesis proposes a relationship between variables, while a null hypothesis proposes no relationship between variables. The research hypothesis guides empirical research, while the null hypothesis is used as a comparison in statistical analysis. The research hypothesis is supported if statistical analysis provides evidence to reject the null hypothesis, while the null hypothesis is rejected if statistical analysis provides evidence to support the research hypothesis.

About the author

Muhammad Hassan

Researcher, Academic Writer, Web developer

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• Open access
• Published: 11 April 2024

Control of false discoveries in grouped hypothesis testing for eQTL data

• Pratyaydipta Rudra 1 ,
• Yi-Hui Zhou 2 ,
• Andrew Nobel 3 &
• Fred A. Wright 2  

BMC Bioinformatics volume  25 , Article number:  147 ( 2024 ) Cite this article

Metrics details

Expression quantitative trait locus (eQTL) analysis aims to detect the genetic variants that influence the expression of one or more genes. Gene-level eQTL testing forms a natural grouped-hypothesis testing strategy with clear biological importance. Methods to control family-wise error rate or false discovery rate for group testing have been proposed earlier, but may not be powerful or easily apply to eQTL data, for which certain structured alternatives may be defensible and may enable the researcher to avoid overly conservative approaches.

In an empirical Bayesian setting, we propose a new method to control the false discovery rate (FDR) for grouped hypotheses. Here, each gene forms a group, with SNPs annotated to the gene corresponding to individual hypotheses. The heterogeneity of effect sizes in different groups is considered by the introduction of a random effects component. Our method, entitled Random Effects model and testing procedure for Group-level FDR control (REG-FDR), assumes a model for alternative hypotheses for the eQTL data and controls the FDR by adaptive thresholding. As a convenient alternate approach, we also propose Z-REG-FDR, an approximate version of REG-FDR, that uses only Z-statistics of association between genotype and expression for each gene-SNP pair. The performance of Z-REG-FDR is evaluated using both simulated and real data. Simulations demonstrate that Z-REG-FDR performs similarly to REG-FDR, but with much improved computational speed.

Our results demonstrate that the Z-REG-FDR method performs favorably compared to other methods in terms of statistical power and control of FDR. It can be of great practical use for grouped hypothesis testing for eQTL analysis or similar problems in statistical genomics due to its fast computation and ability to be fit using only summary data.

Peer Review reports

Expression quantitative trait locus (eQTL) analysis aims to detect genetic loci that are associated with the expression of one or more genes [ 1 ]. For each gene, expression can be considered as a quantitative trait potentially associated with the genotypes at different sites in the genome, typically single nucleotide polymorphisms (SNPs) [ 2 ]. Although there is a substantial literature on both eQTL mapping [ 3 , 4 , 5 ] and grouped hypothesis testing [ 6 , 7 , 8 ], consideration of the natural gene-level grouping of the SNPs, e.g., SNPs local to a gene for a cis-eQTL problem, is comparatively unexplored or requires permutation methods or approximations [ 9 , 10 ]. Analysis of gene-level eQTLs and meaningful consideration of causal SNPs is an important biological problem [ 11 ]. Testing whether there is any eQTL (local SNP) for an entire gene while controlling the false discovery rate (FDR) across the set of all genes may be interesting for various reasons, which has been imperfectly addressed in the “e-Gene” concept employed by the GTEx Consortium [ 12 ].

Local ( cis ) eQTL testing includes tests of individual SNPs nearby a gene, which leads to summaries at the gene level [ 12 ]. The natural hierarchical organization would suggest standard methods for group-level testing [ 6 , 13 ]. However, local eQTL testing can include additional structure to be exploited: (i) the number of cis-eQTLs is typically large, so that explicit consideration of the proportion and “strength” of alternatives is possible; (ii) the conditional analyses of discovered eQTLs suggest that, to a first approximation, most local eQTLs can be considered unique within the region [ 14 ]; (iii) correlation of test statistics is driven by regional SNP correlation.

In the following sections, we discuss the structure of eQTL data and how the grouped nature can be effectively modeled using a random effects model. We consider the case of cis -eQTLs, i.e. local to the gene [ 14 , 15 ], where the variant affecting the gene expression is in the immediate neighborhood of the gene. Our proposed method, entitled Random Effects model and testing procedure for Group-level FDR control ( REG-FDR ), uses an empirical Bayes framework to model the eQTL data and controls the FDR by adaptive thresholding. We also propose an alternate approach Z-REG-FDR , an approximate version of REG-FDR , that uses only the summary measures given by the Z-statistics of association between genotype and expression for each gene-SNP pair. We demonstrate using simulations and real data analysis that this approximate version performs well compared to other possible approaches while having a much faster computation time.

Structure of the eQTL data and the hypotheses

eQTL data can typically be expressed in the form of an expression matrix, consisting of N genes along with a genotype matrix which has genotypes ( m SNPs) for the same n sample units. We denote the expression matrix as \(Y_{N \times n}\) and the genotype submatrix corresponding to the i th gene as \(X^{(i)}_{m_i \times n}\) , \(i=1,2,\ldots ,N\) , where \(m_i\) is the number of SNPs local to the i th gene. Linear modeling of eQTLs typically includes additional covariates, such as expression cofactors [ 12 , 16 ]. The t -statistics for the partial correlations between Y and \(X^{(i)}_{m_i \times n}\) , after both are adjusted for covariates, are equivalent to the Wald statistics for the \(X^{(i)}_{m_i \times n}\) when conducting the full linear regression in which Y is modeled as a function of \(X^{(i)}_{m_i \times n}\) and the additional covariates [ 17 , 18 ]. We assume that the sample size n is large enough that the residual degrees of freedom for the t statistic is sufficient to use a standard normal approximation. Thus we can can directly work with z -statistics for Y and \(X^{(i)}_{m_i \times n}\) , where we consider each of these matrices to have been covariate-residualized.

Let \(H_{0ij}\) denote the gene-SNP level null hypothesis that there is no eQTL at the j th SNP local to the i th gene, \(j=1,2,\ldots ,m_i, i=1,2,\ldots ,N\) . Therefore there are \(\sum _{i=1}^N{m_i}\) gene-SNP level tests. These tests can be grouped into N groups corresponding to the N genes with \(m_i\) tests in the i th group. Define \(H_{0i}\) to be the gene-level null hypothesis for the i th gene that there is no eQTL for the i th gene. Therefore the gene-level null hypothesis \(H_{0i}\) can be written as

i.e. the gene-level null requires that all of the corresponding \(m_i\) gene-SNP level hypotheses be null.

An empirical Bayes model

We adopt an empirical Bayes approach for controlling the gene-level FDR. Empirical Bayes approaches have been used in many genetic applications, and indeed these applications have been a prime motivator for the methods [ 19 , 20 ]. The advantages of using an empirical Bayes approach based on the local false discovery rate (lfdr), instead of p -value-based FDR-controlling approaches, has been discussed in [ 21 ] and [ 22 ]. The lfdr corresponding to the gene-level null hypothesis \(H_{0i}\) is

Here \(Y_i\) denotes the i th row of the matrix Y . If we obtain the lfdr \(\lambda _{i}\) for each of the gene-level hypotheses, we can control the FDR at target level \(\alpha \) for gene-level testing, using the following adaptive thresholding procedure, which has been used extensively in the literature [ 7 , 23 , 24 , 25 ].

Enumerate the index \(i_1,i_2,\ldots ,i_N\) of the genes such that \(\lambda _{i_1} \le \lambda _{i_2} \le \cdots \le \lambda _{i_N}\) .

Reject hypotheses \(H_{0i_1},\ldots ,H_{0i_L}\) where L is the largest integer such that

[ 24 ] and subsequently [ 7 ] showed that the adaptive thresholding procedure is valid in the sense that it controls the FDR at target level \(\alpha \) for an ‘oracle’ procedure where the true parameters of the model are assumed to be known. It is asymptotically valid for a ‘data-driven’ procedure when the parameters are consistently estimated from the data. [ 25 ] proved its validity under further relaxed conditions. The proof makes use of the following result (Averaging Theorem, [ 19 ]).

Let \(\text {lfdr}(z)=P(H_0|z)\) denote the lfdr for observed data z . Then, for a rejection region \({\mathcal {R}}\) , the FDR will be given by

The adaptive thresholding procedure can be used to control the FDR for testing the gene-level hypotheses \(H_{0i}\) ’s and a similar procedure can be used to test the gene-SNP level hypotheses \(H_{0ij}\) ’s. However, obtaining the gene-level lfdr’s is a non-trivial problem. In the next section, we propose a model which enables us to calculate the lfdr’s.

The random effects model and testing procedure for group-level FDR control ( REG-FDR )

Here we propose a model to obtain the gene-level lfdr values, that can be subsequently used to test the gene-level hypotheses while controlling the FDR using the adaptive thresholding method. The model is based on the following assumptions.

For any gene i , under the gene-level alternative hypothesis \(H_{0i}^c\) , there exists a single causal SNP that influences its expression.

Each of the \(m_i\) SNPs has equal probability to be the causal SNP.

First, we note that Assumption (A1) is at best a simplification, but very large eQTL studies have supported the view that most genes with eQTLs have a primary local eQTL [ 26 ], with other loci having much smaller effect sizes. We therefore treat A1 as a ‘workable condition’ [ 27 , 28 , 29 ].

Assumption (A2) could easily be relaxed, and one might use a distributional assumption over the SNPs as a modest modification of our method below (see the Discussion section). We note that it is trivial to enforce Assumption (A2) by, for example, randomizing the SNP identities within gene i prior to analysis.

Under these assumptions, the gene-level lfdr for the i th gene has the following form:

where \(\pi _0=P(H_{0i})\) is the prior probability of \(H_{0i}\) , \(f_0(Y_i)\) is the density of \(Y_i\) under the null, and \(f_1(Y_i|X_j^{(i)},\beta _{ij})\) is the conditional density under the alternative given that the j th SNP is causal. Here \(\beta _{ij}\) is correlation between the expression of the i th gene and the causal SNP j . Note that the marginal density \(p(X^{(i)})\) cancels from numerator and denominator. Importantly, this cancellation allows us to bypass the modeling of the dependence structure of the SNPs, which otherwise might have been difficult to estimate accurately.

We assume that \(f_0(.)\) is the density of the \(N_n(0,I_n)\) distribution (noting that expression data can be normalized), and that \(f_1(.|X_j^{(i)},\beta _{ij})\) is the density of the \(N_n(\beta _{ij} X_j^{(i)}, (1-\beta _{ij}^2)I_n)\) distribution, where \(\beta _{ij}\) is the correlation between \(Y_i\) and \(X_j^{(i)}\) . This choice of \(f_1\) ensures that the unconditional variance of \(Y_i\) is free of \(\beta _{ij}\) . To account for variability across genes, we assume \(\beta _{ij}\) to be a random effect such that \(\sqrt{n-3} \ tanh^{-1}(\beta _{ij})\) follows a \(N(0,\sigma ^2)\) distribution. As \(\beta _{ij}\) is a correlation coefficient, the Fisher transformation is used to ensure that the variance does not depend on the mean. Moreover, \(\sigma \) will be estimated from the data, and so the apparent dependence on n is not important to the procedure.

Our procedure treats the genotype values as fixed, and assuming the expression of genes to be independent, given genotypes, we can estimate \(\pi _0\) and \(\sigma \) using a maximum likelihood approach and follow with plug-in estimates to obtain estimates of \(\lambda _i(Y_i,X^{(i)})\) from Eq.  5 . The assumption that the expression of different genes are independent is violated in general, but our approach can be viewed as employing a composite likelihood [ 30 ], and thus consistent for \(\pi _0\) and \(\sigma \) even under independence violations [ 31 ]. An EM algorithm is used (see Additional file 1 : Section 1) for the maximum likelihood estimation. The procedure enables us to use the adaptive thresholding procedure to provide proper gene-level control of the FDR.

The Z-REG-FDR model

One computational challenge presented by the REG-FDR model is that the density \(f_1(Y_i|X_j^{(i)})\) does not have a closed form expression. While it can be expressed as the following integral

numeric maximum likelihood estimation is computationally burdensome. We propose an alternative model, termed Z-REG-FDR , which avoids this problem. In this approach, we consider the Fisher transformed and scaled z -statistics as our data. Thus, for each gene i , we have a vector of z -statistics

\(z^{(i)} = (z_{1}^{(i)},z_{2}^{(i)},\ldots ,z_{m_i}^{(i)}) , \ i=1,2,\ldots ,N,\)

where \(z_{j}^{(i)}=\sqrt{n-3} \ tanh^{-1}(r_{j}^{(i)})\) and \(r_{j}^{(i)}\) is the sample correlation of \(Y_i\) and \(X_j^{(i)}\) .

Fisher transformation and scaling ensures that \(z^{(i)}\) is approximately normal and that the variance of each component is approximately 1 under both null and alternative. Under the null, the mean of \(z^{(i)}\) is zero. We treat the component \(z^{(i)}\) as if they are independent across different genes, again relying on approximate conditional independence (given genotypes) and a compositie likelihood interpretation.

The Z-REG-FDR procedure is based on an additional assumption to (A1) and (A2) above. If the k th SNP is causal, we assume (Assumption (A3)) that the distribution of \((z_{1}^{(i)},\ldots ,z_{k-1}^{(i)},z_{k+1}^{(i)},\ldots ,z_{m_i}^{(i)})\) given \(z_{k}^{(i)}\) under the alternative is same as that under the null. In particular, we note that this assumption is true if the components of \(z^{(i)}\) have a Markov dependence structure with the same serial correlation under null and alternative, which is true in the special case that the successive marker correlations are zero. In general, this assumption can be violated, but as shown in “ Simulations: performance of Z-REG-FDR as an approximate maximum likelihood estimation ” section, the resultant procedure appears to work well in many circumstances as an approximate maximum likelihood method even when Assumption (A3) is not satisfied.

Under the above assumptions, we can write the joint distribution of the random vector \(z^{(i)}=(z_{1}^{(i)},z_{2}^{(i)},\ldots ,z_{m_i}^{(i)})\) as

under the null, and

under the alternative. We assume \(p_0(.)\) to be N (0, 1) and \(p_1(.)\) to be \(N(\mu ,1)\) , where \(\mu \) is assumed to be random with a \(N(0,\sigma ^2)\) distribution. We do not assume anything about the form of \(f_{0|k}\) except that it does not involve the parameters \(\pi _0\) and \(\sigma \) . Under these assumptions, the gene-level lfdr for this model reduces to

This follows from the cancellation of \(f_{0|k}(z_{1}^{(i)},\ldots ,z_{k-1}^{(i)},z_{k+1}^{(i)},\ldots ,z_{m_i}^{(i)})\) in the numerator and denominator. While estimating \(\pi _0\) and \(\sigma \) , a similar cancellation helps us bypass maximizing the full (approximate) likelihood

Instead, we maximize

This is equivalent to the maximum likelihood estimation under the assumption that \(f_{0|k}\) does not involve the parameters \(\pi _0\) and \(\sigma \) . Note that we need to estimate only the parameters \(\pi _0\) and \(\sigma \) to obtain the gene-level lfdr using Eq.  9 .

When the required assumptions are not satisfied, this method still has value as an approximate maximum likelihood approach. For instance, when the \(X_j^{(i)}\) ’s are related by an AR(1) structure, it can be shown that the correlation between the z -statistics depends on the effect size, i.e. the correlation between \(Y_i\) and the causal SNP, hence violating Assumption (A3). Additional file 1 : Lemma 1 and Additional file 1 : Figures 1 and 2 show the extent to which the conditional distribution \(f_{0|k}\) might depend on the effect size for any correlation structure among normally distributed SNPs. However, our results in “ Simulations: performance of Z-REG-FDR as an approximate maximum likelihood estimation ” section demonstrate that it does not have a significant adverse effect on the performance of the estimation and control of false discovery.

Simulations: performance of Z-REG-FDR when all assumptions are satisfied

First, we conducted a simulation study to explore the performance of Z-REG-FDR under the ideal situation where all assumptions are satisfied. Table  1 shows the results for simulated datasets (1000 simulations of datasets with 10,000 genes and 200 samples) where z ’s are directly simulated from an autoregressive structure, and therefore Assumption (A3) is also satisfied. The estimates are accurate to within about 15% when the true \(\sigma \) is at least 2.0. The control of the FDR is also satisfactory for \(\sigma > 2\) . However, the performance is not as good for small \(\sigma \) , which is due to the fact that it is difficult to separate the null and alternative cases when the effect sizes are small; this is true even when all the assumptions are satisfied. This is a property of the two group mixture model in the empirical Bayes set up, and not a limitation due to the approximate nature of Z-REG-FDR .

Simulations: performance of Z-REG-FDR as an approximate maximum likelihood estimation

We wished to study the accuracy of the estimation under the approximations employed and for a relatively small sample size, in order to ensure that the approach can work in this challenging situation. Accordingly, we simulated data that uses the covariate adjusted genotype matrix of a real dataset from the GTEx project (V3) [ 12 ]. The genotype matrix corresponding to the tissue ‘heart’, which had 83 samples, was selected for analysis. For computational purposes, 10,000 genes were chosen randomly from 28,991 genes. Use of genotype matrices from real data ensures that we are not enforcing Assumption (A3) while simulating, and our choice of \(f_{0|k}\) for the simulation is obtained from the data. We simulate the \(Y_i\) ’s (1,000 simulations) using the following scheme.

For each gene, decide whether it has an eQTL using a Bernoulli( \(\pi _0\) ) distribution.

If the gene has an eQTL, pick a causal SNP using a discrete uniform distribution over the \(m_i\) SNPs. Let it be the k th SNP.

If the gene has an eQTL, simulate each element of \(Y_i\) from \(N(\beta _{ij} X_k^{(i)}, 1-\beta _{ij}^2)\) with \(\sqrt{n-3} \ tanh^{-1}(\beta _{ij})\) simulated from a \(N(0,\sigma ^2)\) distribution. If the gene doesn’t have an eQTL, simulate each element of \(Y_i\) from N (0, 1).

Table  2 shows the results for this data, indicating that the estimates are still accurate and control of FDR is satisfactory unless \(\sigma \) is very small. Large eQTL studies have observed large effect sizes for cis-eQTL analysis [ 15 , 32 ] which implies that \(\sigma \) is not expected to be very small. Thus our numerical results indicate that the Z-REG-FDR method has valid applications for eQTL data.

Figure  1 shows the plot of REG-FDR estimates against the Z-REG-FDR estimates for 500 simulated datasets using the simulation scheme described above. It is clear from the plot that the two methods agree with each other (with correlations 0.906 and 0.952 for \(\pi _0\) and \(\sigma \) , respectively) and largely fall near the unit line. These results suggest that the approximate maximum likelihood method in Z-REG-FDR is quite effective in controlling the FDR, with a much improved computation speed—a few minutes on a single computer for a dataset with 10,000 genes and 100–200 samples as opposed to more than a day for REG-FDR . A comparison of the estimated lfdr and estimated FDR of the two methods is shown in Fig.  2 . It is evident that the slight over-estimation of \(\pi _0\) and the slight underestimation of \(\sigma \) by Z-REG-FDR work in opposite directions, which leads to similar lfdr values when compared to REG-FDR . The correlation between the estimated FDR based on the true values of the parameters and that based on REG-FDR or Z-REG-FDR are also very high (see Additional file 1 : Figure 3).

Comparison of the parameter estimates using REG-FDR and Z-REG-FDR . Except a small number of cases, the two estimates agree with each other. The blue lines show the true values of the parameters

A. Estimated lfdr and B. estimated FDR for REG-FDR and Z-REG-FDR

Behavior of the expected pseudo-log-likelihood of Z-REG-FDR

It is a standard result that the expected log-likelihood is maximized at the true value of the parameter under standard regularity conditions [ 33 ]. Since REG-FDR is the true maximum likelihood method for the proposed model, it is expected to satisfy this property. If Assumption (A3) is not satisfied then Z-REG-FDR is an approximate maximum likelihood method, and as such, its pseudo-log-likelihood need not be maximized at the true value of the parameter. We explored several realistic combinations of the true parameters and observed that the pseudo-log-likelihood of Z-REG-FDR is maximized very near the true parameter value. It is a difficult task to analytically compute the expected pseudo-log-likelihood, and so Monte-Carlo integration was used for this task. Figure  3 shows the expected pseudo-log-likelihood surface of Z-REG-FDR for \(\pi _0=0.2\) and \(\sigma =3\) . A contour plot also confirms the fact the surface peaks near the true values of the parameters.

Demonstration of the optimization of log-likelihood properties using Z-REG-FDR method. A. Surface plot and B. Contour plot of expected pseudo-log-likelihood surface for the Z-REG-FDR method. True \(\pi _0\) and \(\sigma \) are 0.2 and 3 respectively

Simulations: comparison of Z-REG-FDR with other methods

It is possible to use other methodologies to control the FDR in grouped hypothesis testing problem for eQTL data. A conservative approach is to obtain the Bonferroni adjusted p -values for each gene, where the p -value for each gene-SNP pair is computed based on the usual t -test or z -test, and then use an FDR controlling approach [eg 34 , 35 , 36 ] to assess the conservative p -values. [ 29 ] used a permutation based (“eGene”) approach in their analysis of the GTEx data. The method uses the smallest gene-SNP p -value for a gene as the test statistic and computes its distribution by permuting the expression values. Such a distribution can be used to obtain p -values for each gene, which can subsequently be used to control the FDR by using methods such as Storey’s q-value method [ 35 ].

The Bonferroni method is typically conservative and hence less powerful. The permutation method, while correctly controlling false positives, can suffer from lack of power to detect genes having an eQTL since it uses an extreme value statistic (not based on likelihood). Our model, on the other hand, utilizes more information through its use of approximate likelihood. We carried out a simulation study to compare the performance of the methods in terms of their power. The simulations were performed using the simulation scheme described in “ Simulations: performance of Z-REG-FDR as an approximate maximum likelihood estimation ” section and statistical power was obtained using an FDR threshold of 0.05. The results are shown in Fig.  4 . As expected, the Bonferroni method turned out to have very low power and is not shown in Fig.  4 . The permutation approach with Storey’s q -value method [ 35 ] was conservative and less powerful in comparison with Z-REG-FDR . To address the possible concern that Z-REG-FDR can be slightly anti-conservative, and therefore the comparison with the permutation method is unfair, we also included an adjusted version of the Z-REG-FDR method where a slightly lower FDR threshold was chosen based on the simulations in such a way that the estimated FDR was exactly 0.05. This adjusted version had slightly less power compared to unadjusted Z-REG-FDR , but was more powerful than the permutation method.

Power curves of different methods for varying combinations of the true parameter values

Analysis of real data

Finally, we also applied the Z-REG-FDR on a real dataset obtained from GTEx (V6) [ 12 ]. Besides Z-REG-FDR , we also used the permutation method and Simes method [ 37 ], which is expected to be more powerful than the Bonferroni method although it may not control the FDR for all types of correlation structures. We applied each method on the GTEx data for 44 tissues, separately for each tissue.

For each tissue, the normalized gene expression data and SNP genotype data were separately residualized after adjusting for covariates provided by GTEx. We fit a linear regression model with individuals’ gene expression or SNP genotype as the response variable and covariates as the explanatory variables. Then we extracted the model residuals to obtain “covariate-corrected” gene expression and SNP genotypes.

Figure  5 shows a comparison of the number of significant genes found by Z-REG-FDR and the permutation method employed by [ 12 ]. A complete list of the sample sizes and the number of significant genes discovered for the 44 tissues is provided in Additional file 1 : Table 2. The methods agree with each other to some extent in terms of number of discoveries. The Z-REG-FDR method has higher number of discoveries compared to the Permutation method and the Simes method in most cases. The parameter estimates for each tissue using Z-REG-FDR are shown in Fig.  6 .

Comparison of Z-REG-FDR and the permutation method for GTEx data

Parameter estimates using Z-REG-FDR for the GTEx data

We have introduced a principled procedure to perform gene-level FDR test, most appropriate and useful in the eQTL setting. A major advantage of Z-REG-FDR is its computational efficiency. While other methods such as the permutation method or our REG-FDR method can take days on a single PC to complete the analysis of a real eQTL dataset, Z-REG-FDR can do the same in a few minutes. For instance, it takes approximately two minutes to fit the model and find significant genes by Z-REG-FDR for a data set with 4.5 million SNPs grouped as local SNPs for 10, 000 genes. REG-FDR takes about a day, and the permutation method (for 10, 000 permutations) takes about 6 hours to analyze the same data. Since there are thousands of simultaneous tests, even 10, 000 permutations may not be enough to provide sufficient p -value resolution. While the Bonferroni method is very fast, it has little power to detect the genes having true eQTLs.

Z-REG-FDR has additional advantages. One important feature of the method is that it does not require access to the full data. In fact, the symmetry of the distributions involved in the Z-REG-FDR pseudo-likelihood ensure that only the gene-SNP level p -values (or equivalently the absolute z -values) are needed to fit the model. Z-REG-FDR does not model the correlation structure of the SNPs, and therefore does not require access to that data. This might be very useful since, in many genetic applications, data are found in the form of summary measures.

Z-REG-FDR can be slightly anti-conservative depending on the true values of the parameters. Various simulations show that if \(\sigma \) is large, which appears to often be the case for eQTL data, the control of FDR is satisfactory. The fact that Assumption (A3) is not satisfied does not significantly affect the FDR control. Therefore the assumption can be thought of as a means to reduce computational burden, rather than a necessary assumption for the practical workability of the model.

Assumptions (A1) and (A2) also have the potential to be relaxed, although we consider that to be beyond the scope of this paper. For example, the method can be extended by relaxing Assumption (A2) and incorporating a non-uniform prior for the causal location. If a well-grounded prior exists, then it can be incorporated into our method in a straightforward manner using weighted versions of our statistics. We have included an example in the Additional file 1 to demonstrate empirical evidence that the method remains valid even for more than one causal SNPs under certain conditions.

Our use of the lfdr statistics, while valid, does not utilize gene-level local correlation structures [ 38 , 39 , 40 , 41 ] that might provide additional power. Implementation of such methods would require sensitive estimation of gene-level correlations, and a possible direction of future effort.

With the continuous increase in the size of genomic data sets, and with the possibility of further extensions of our approach, we strongly believe that the approximate likelihood approach of the Z-REG-FDR method can be of great practical use for grouped hypothesis testing for eQTL analysis or similar problems in statistical genomics.

Availability of data and materials

Supplementary material is available in the file Supplementary.pdf. Software in the form of R code and documentation is available at https://doi.org/10.5281/zenodo.8331734 .

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Supported in part by R01ES033243 and R01ES029911.

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Pratyaydipta Rudra

Bioinformatics Research Center, Departments of Statistics and Biological Sciences, North Carolina State University, Raleigh, NC, USA

Yi-Hui Zhou & Fred A. Wright

Department of Statistics and Operations Research, University of North Carolina, Chapel Hill, NC, USA

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PR constructed the models and performed the statistical analyses of simulated and real data. YZ conducted pre-processing and covariate adjustment for the real data. FAW and AN supervised the modeling and analysis. All authors have read and approved the final version of this manuscript.

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Rudra, P., Zhou, YH., Nobel, A. et al. Control of false discoveries in grouped hypothesis testing for eQTL data. BMC Bioinformatics 25 , 147 (2024). https://doi.org/10.1186/s12859-024-05736-3

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Received : 18 August 2023

Accepted : 08 March 2024

Published : 11 April 2024

DOI : https://doi.org/10.1186/s12859-024-05736-3

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