Guide to Communication Research Methodologies: Quantitative, Qualitative and Rhetorical Research

research methods for communication

Overview of Communication

Communication research methods, quantitative research, qualitative research, rhetorical research, mixed methodology.

Students interested in earning a graduate degree in communication should have at least some interest in understanding communication theories and/or conducting communication research. As students advance from undergraduate to graduate programs, an interesting change takes place — the student is no longer just a repository for knowledge. Rather, the student is expected to learn while also creating knowledge. This new knowledge is largely generated through the development and completion of research in communication studies. Before exploring the different methodologies used to conduct communication research, it is important to have a foundational understanding of the field of communication.

Defining communication is much harder than it sounds. Indeed, scholars have argued about the topic for years, typically differing on the following topics:

  • Breadth : How many behaviors and actions should or should not be considered communication.
  • Intentionality : Whether the definition includes an intention to communicate.
  • Success : Whether someone was able to effectively communicate a message, or merely attempted to without it being received or understood.

However, most definitions discuss five main components, which include: sender, receiver, context/environment, medium, and message. Broadly speaking, communication research examines these components, asking questions about each of them and seeking to answer those questions.

As students seek to answer their own questions, they follow an approach similar to most other researchers. This approach proceeds in five steps: conceptualize, plan and design, implement a methodology, analyze and interpret, reconceptualize.

  • Conceptualize : In the conceptualization process, students develop their area of interest and determine if their specific questions and hypotheses are worth investigating. If the research has already been completed, or there is no practical reason to research the topic, students may need to find a different research topic.
  • Plan and Design : During planning and design students will select their methods of evaluation and decide how they plan to define their variables in a measurable way.
  • Implement a Methodology : When implementing a methodology, students collect the data and information they require. They may, for example, have decided to conduct a survey study. This is the step when they would use their survey to collect data. If students chose to conduct a rhetorical criticism, this is when they would analyze their text.
  • Analyze and Interpret : As students analyze and interpret their data or evidence, they transform the raw findings into meaningful insights. If they chose to conduct interviews, this would be the point in the process where they would evaluate the results of the interviews to find meaning as it relates to the communication phenomena of interest.
  • Reconceptualize : During reconceptualization, students ask how their findings speak to a larger body of research — studies related to theirs that have already been completed and research they should execute in the future to continue answering new questions.

This final step is crucial, and speaks to an important tenet of communication research: All research contributes to a better overall understanding of communication and moves the field forward by enabling the development of new theories.

In the field of communication, there are three main research methodologies: quantitative, qualitative, and rhetorical. As communication students progress in their careers, they will likely find themselves using one of these far more often than the others.

Quantitative research seeks to establish knowledge through the use of numbers and measurement. Within the overarching area of quantitative research, there are a variety of different methodologies. The most commonly used methodologies are experiments, surveys, content analysis, and meta-analysis. To better understand these research methods, you can explore the following examples:

Experiments : Experiments are an empirical form of research that enable the researcher to study communication in a controlled environment. For example, a researcher might know that there are typical responses people use when they are interrupted during a conversation. However, it might be unknown as to how frequency of interruption provokes those different responses (e.g., do communicators use different responses when interrupted once every 10 minutes versus once per minute?). An experiment would allow a researcher to create these two environments to test a hypothesis or answer a specific research question. As you can imagine, it would be very time consuming — and probably impossible — to view this and measure it in the real world. For that reason, an experiment would be perfect for this research inquiry.

Surveys : Surveys are often used to collect information from large groups of people using scales that have been tested for validity and reliability. A researcher might be curious about how a supervisor sharing personal information with his or her subordinate affects way the subordinate perceives his or her supervisor. The researcher could create a survey where respondents answer questions about a) the information their supervisors self-disclose and b) their perceptions of their supervisors. The data collected about these two variables could offer interesting insights about this communication. As you would guess, an experiment would not work in this case because the researcher needs to assess a real relationship and they need insight into the mind of the respondent.

Content Analysis : Content analysis is used to count the number of occurrences of a phenomenon within a source of media (e.g., books, magazines, commercials, movies, etc.). For example, a researcher might be interested in finding out if people of certain races are underrepresented on television. They might explore this area of research by counting the number of times people of different races appear in prime time television and comparing that to the actual proportions in society.

Meta-Analysis : In this technique, a researcher takes a collection of quantitative studies and analyzes the data as a whole to get a better understanding of a communication phenomenon. For example, a researcher might be curious about how video games affect aggression. This researcher might find that many studies have been done on the topic, sometimes with conflicting results. In their meta-analysis, they could analyze the existing statistics as a whole to get a better understanding of the relationship between the two variables.

Qualitative research is interested in exploring subjects’ perceptions and understandings as they relate to communication. Imagine two researchers who want to understand student perceptions of the basic communication course at a university. The first researcher, a quantitative researcher, might measure absences to understand student perception. The second researcher, a qualitative researcher, might interview students to find out what they like and dislike about a course. The former is based on hard numbers, while the latter is based on human experience and perception.

Qualitative researchers employ a variety of different methodologies. Some of the most popular are interviews, focus groups, and participant observation. To better understand these research methods, you can explore the following examples:

Interviews : This typically consists of a researcher having a discussion with a participant based on questions developed by the researcher. For example, a researcher might be interested in how parents exert power over the lives of their children while the children are away at college. The researcher could spend time having conversations with college students about this topic, transcribe the conversations and then seek to find themes across the different discussions.

Focus Groups : A researcher using this method gathers a group of people with intimate knowledge of a communication phenomenon. For example, if a researcher wanted to understand the experience of couples who are childless by choice, he or she might choose to run a series of focus groups. This format is helpful because it allows participants to build on one another’s experiences, remembering information they may otherwise have forgotten. Focus groups also tend to produce useful information at a higher rate than interviews. That said, some issues are too sensitive for focus groups and lend themselves better to interviews.

Participant Observation : As the name indicates, this method involves the researcher watching participants in their natural environment. In some cases, the participants may not know they are being studied, as the researcher fully immerses his or herself as a member of the environment. To illustrate participant observation, imagine a researcher curious about how humor is used in healthcare. This researcher might immerse his or herself in a long-term care facility to observe how humor is used by healthcare workers interacting with patients.

Rhetorical research (or rhetorical criticism) is a form of textual analysis wherein the researcher systematically analyzes, interprets, and critiques the persuasive power of messages within a text. This takes on many forms, but all of them involve similar steps: selecting a text, choosing a rhetorical method, analyzing the text, and writing the criticism.

To illustrate, a researcher could be interested in how mass media portrays “good degrees” to prospective college students. To understand this communication, a rhetorical researcher could take 30 articles on the topic from the last year and write a rhetorical essay about the criteria used and the core message argued by the media.

Likewise, a researcher could be interested in how women in management roles are portrayed in television. They could select a group of popular shows and analyze that as the text. This might result in a rhetorical essay about the behaviors displayed by these women and what the text says about women in management roles.

As a final example, one might be interested in how persuasion is used by the president during the White House Correspondent’s Dinner. A researcher could select several recent presidents and write a rhetorical essay about their speeches and how they employed persuasion during their delivery.

Taking a mixed methods approach results in a research study that uses two or more techniques discussed above. Often, researchers will pair two methods together in the same study examining the same phenomenon. Other times, researchers will use qualitative methods to develop quantitative research, such as a researcher who uses a focus group to discuss the validity of a survey before it is finalized.

The benefit of mixed methods is that it offers a richer picture of a communication phenomenon by gathering data and information in multiple ways. If we explore some of the earlier examples, we can see how mixed methods might result in a better understanding of the communication being studied.

Example 1 : In surveys, we discussed a researcher interested in understanding how a supervisor sharing personal information with his or her subordinate affects the way the subordinate perceives his or her supervisor. While a survey could give us some insight into this communication, we could also add interviews with subordinates. Exploring their experiences intimately could give us a better understanding of how they navigate self-disclosure in a relationship based on power differences.

Example 2 : In content analysis, we discussed measuring representation of different races during prime time television. While we can count the appearances of members of different races and compare that to the composition of the general population, that doesn’t tell us anything about their portrayal. Adding rhetorical criticism, we could talk about how underrepresented groups are portrayed in either a positive or negative light, supporting or defying commonly held stereotypes.

Example 3 : In interviews, we saw a researcher who explored how power could be exerted by parents over their college-age children who are away at school. After determining the tactics used by parents, this interview study could have a phase two. In this phase, the researcher could develop scales to measure each tactic and then use those scales to understand how the tactics affect other communication constructs. One could argue, for example, that student anxiety would increase as a parent exerts greater power over that student. A researcher could conduct a hierarchical regression to see how each power tactic effects the levels of stress experienced by a student.

As you can see, each methodology has its own merits, and they often work well together. As students advance in their study of communication, it is worthwhile to learn various research methods. This allows them to study their interests in greater depth and breadth. Ultimately, they will be able to assemble stronger research studies and answer their questions about communication more effectively.

Note : For more information about research in the field of communication, check out our Guide to Communication Research and Scholarship .

research methods for communication

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  • Communication Research Methods II: A Sourcebook An updated version of the "bible" of how to do research in communication and media studies.
  • Handbook of Media and Communication Research: Qualitative and Quantitative Methodologies A great overview of both qualitative and quantitative approaches to content analysis and media studies research.
  • Qualitative Media Analysis The authors of this work show readers how to obtain, categorize, and analyze different media documents. They look at traditional primary documents such as newspapers and magazines but also at more recent forms–television newscasts and cyberspace.
  • Mass Communications Research Methods Originally published in 1988. Step-by-step, this book leads students from problem identification, through the mazes of surveys, experimentation, historical/qualitative studies, statistical analysis, and computer data processing to the final submission and publication in scientific or popular publications.
  • Communication Research Methods in Postmodern Culture: A Revisionist Approach The second edition of Communication Research Methods in Postmodern Culture continues to explore research from a postmodern perspective. Typical qualitative and quantitative research methods are adjusted to fit the needs of contemporary culture.
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Three Types of Communication Research Methods: Quantitative, Qualitative, and Participatory

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research methods for communication

  • Jan Servaes 2 , 3  

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This chapter presents a brief overview of the three types of communication research methods being applied in development communication settings: quantitative, qualitative, and participatory. This chapter attempts to outline the relative characteristics and merits of these approaches to research and to emphasize some of the philosophical issues which underpin them. It discusses the strengths and weaknesses of each and highlights the benefits of a more normative approach focused on the “poor” in society.

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Servaes, J. (2020). Three Types of Communication Research Methods: Quantitative, Qualitative, and Participatory. In: Servaes, J. (eds) Handbook of Communication for Development and Social Change. Springer, Singapore. https://doi.org/10.1007/978-981-15-2014-3_112

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Article contents

Methods for intercultural communication research.

  • John Oetzel , John Oetzel Waikato Management School, The University of Waikato
  • Saumya Pant Saumya Pant Mudra Institute of Communications, Ahmedabad (MICA)
  •  and  Nagesh Rao Nagesh Rao Partner, Siya Consulting
  • https://doi.org/10.1093/acrefore/9780190228613.013.202
  • Published online: 09 May 2016

Research on intercultural communication is conducted using primarily three different methodological approaches: social scientific, interpretive, and critical. Each of these approaches reflects different philosophical assumptions about the world and how we come to know it. Social scientific methods often involve quantitative data collection and research approaches such as surveys and experiments. From this perspective, intercultural communication is seen as patterns of interaction, and we seek to explain and understand these patterns through clear measurement and identification of key independent variables. Interpretive methods often involve qualitative data collection and research approaches such as interviews and ethnographic observation. From this perspective, intercultural communication and meaning is created through interaction, and we seek to understand these meanings by exploring the perspectives of people who participate as members of cultural communities. Critical methods often involve qualitative data collection and research approaches such as interviews and textual critique. From this perspective, intercultural communication involves inequalities that can be attributed to power and distortions created from (mis)use of this power. Critical scholars seek to unmask domination and inequality. Most scholars utilize one of these primary approaches given the consistency with their world views, theories, and research training. However, there are creative possibilities for combining these approaches that have potential for fuller understanding of intercultural communication.

  • social science methods
  • interpretive methods
  • critical methods
  • quantitative
  • qualitative
  • intercultural communication

Introduction

Our worldview shapes what is “interesting” to a particular audience, what is considered a problem, what problem is interesting to study, and whether the goal of studying a problem is to analyze the problem, to analyze and solve the problem, or to analyze, solve, and implement the solution. Our worldview defines if an issue is a problem or not and if we need to come up with a solution. For example, behaviors associated with attention deficit hyperactivity disorder (ADHD) are seen as a problem in the United States, and there are medications to solve the problem. In India, the same set of behaviors among children is seen as what children tend to do, as normal and not as a problem.

Our worldview not only shapes what we see as an interesting problem to study but also the methodology we use to study the problem. The purpose of this article is to describe, and explore integration of, the three main methodological perspectives in studying intercultural communication issues: social scientific, interpretive, and critical. First, the ontological, epistemological, and axiological assumptions underlying each of these methodological perspectives are explored. Then, for each methodological perspective, common methods and types of data collected and some exemplars are identified. Finally, we offer traditional integration of the three approaches and also alternate methodological perspectives to study intercultural issues from a non-Western lens.

Ontology, Epistemology, and Axiology

Ontology is the study of the researcher’s orientation to reality. In the social scientific perspective, the researcher views the world objectively in that there is a world outside of us that can be systematically studied. Researchers from this perspective use a deductive approach and are keen to explain and predict phenomena. Social scientific ontology provides clarity and direction due to its rigorous questioning of plausibility and reduction of subjectivity. In contrast and as a reaction to the social scientific perspective, interpretive researchers argue that the observer and the observed are subjective and the most important lessons are in how they co-create meaning. If the social scientists take a deterministic view of human behavior, interpretivists thrive in a person’s free will. Critical theorists focus particularly on social injustices and inequalities in life. Researchers in this area explore how social structures create power inequalities and injustices. Thus, they believe that power differences are at the base of social transactions (Scotland, 2012 ). Any ontological investigation for a critical theorist will thus have to help unearth these inequities.

Epistemology looks at how we come to know a chosen phenomenon and thus how researchers study this phenomenon. Social scientists, interested in assessing objective reality (or at least reduced subjectivity), use a scientific method to collect empirical evidence. They focus particularly on causal relationships between phenomena and generally use quantitative approaches to collect data. The basis of their assessment and data collection is the premise that objects have an existence independent of the knower (Cohen et al., 2007 ). Interpretivists, who are interested in situational and contextual meaning, generally use qualitative methods to assess participants’ sense of reality. They are not exploring one truth, but the play of multiple truths simultaneously. They do so by studying individual interactions and the historical and cultural contexts in which these individuals interact. Critical researchers use a variety of qualitative methods to explore, for example, how language is used to create power imbalances or how mass media is used to avoid critical thinking. Critical scholars are particularly sensitive to the overdependence on empirical and social scientific evidence. They do so as critical investigations are premised on the fact that social/positional power determines what is considered knowledge (Cohen et al., 2007 ).

Axiology explores the values that guide a researcher’s questions, the methods used to collect and analyze data, the interpretation of the data, and the implications of the findings. Social scientists study phenomena to find the truth, which, in turn, guides specific types of action. They are focused on exploring what is referred to as the value axiom, or how much a phenomenon being studied fulfills the requirements of the concept to which it belongs (Kelleher, 2013 ). Both interpretivists and critical theorists are interested in describing what exists, how the participants in the community interpret phenomena, with critical theorists particularly interested in reducing class imbalances and other forms of oppression. Interpretivists are axiologically determined to encourage the fact that observations drawn can always be disagreed upon and reopened to interpretation. With respect to control, social scientists wish to control as many variables as possible, narrowing down the causal pattern to the variables under study. Interpretivists seek active participation in the study to understand how they view reality. Critical theorists are particularly aware of the community members’ need to take control of their own situations. With this brief overview in mind, we now explain the methodological approaches of the social scientific, interpretive, and critical perspectives; the types of data collected; some exemplars for each perspective; and some general concerns about each of the methods.

Social Science Methods

Social science research methods address questions related to both cross-cultural and intercultural communication. Much of the foundational work on intercultural communication research is based on comparisons of two or more cultures. Both forms of communication research try to enhance the comprehension of communication that are mediated by and through cultural context (Sponcil & Gitimu, 2010 ). These comparisons helped to identify how the normative and subjective aspects of culture vary across cultures and presumably provided information about what to expect when interacting with members from different cultures. This type of research is classified as cross-cultural. In contrast, intercultural communication is the exchange of messages between people from different cultural groups (Gudykunst, 2003a ). Regardless of the interest in cross-cultural or intercultural communication, the social scientific perspective seeks to understand and predict the effect of culture on communication variables and the subsequent effect of communication on various outcomes. Thus, the methods of study are similar. This section reviews the three most prominent social scientific methods providing an example of each. Additionally, the types of data generated and methodological concerns are discussed.

There are three methods used by most social scientific researchers to study cross-cultural and intercultural research: (a) survey questionnaire, (b) experimental design, and (c) content analysis. The survey questionnaire is by far the most frequently used research method (e.g., Oetzel & Ting-Toomey, 2003 ; Rao, Singhal, Ren, & Zhang, 2001 ). It is typically a self-administered and self-report instrument that is distributed to large samples in multiple cultures. Most cross-cultural comparisons utilize self-report questionnaires because of the difficulty of collecting data from large samples in multiple cultures using other methods. Finally, self-report questionnaires are relatively easy to construct. Numerous cross-culturally valid scales exist, and methodological difficulties have been clearly identified (Gudykunst, 2003b ). While not easy to overcome, methodological difficulties of survey questionnaires are manageable (see below for more detail). Survey questionnaires provide detailed description of cultural associations of communication behavior and outcomes and allow for comparisons to other cultures.

Hanasono, Chen, and Wilson’s ( 2014 ) study of perceived discrimination, social support, and coping among racial minority university students is an example of survey research. The authors surveyed 345 students, half international students and half U.S. students, about their acculturation, experiences with discrimination, support, and coping needs. They found that the level of acculturation helped to explain students’ need for support and how they coped with discrimination.

Experimental designs are highly regarded social scientific research because of the control of variables, which enables causal relationships to be examined. Culture is not a variable that lends itself well to experimental manipulation, and thus experimental designs are relatively rare in this line of research. Rather than experimental controlling culture, researchers typically use quasi-experimental designs manipulating the composition of groups or dyads to be intra- or intercultural (e.g., Cai, Wilson, & Drake, 2000 ; Oetzel, 1998 ). These experiments collect a combination of self-report information (e.g., cultural and individual variables) as well as videotaped interaction. Additionally, some researchers have used experimental conditions on survey questionnaires (e.g., Han & Cai, 2010 ). These studies utilize stimulus variables (e.g., contextual features) that ask participants to respond to specific situations.

Brinson and Stohl’s ( 2012 ) study of media framing on attitudes toward Muslims, civil liberties, and counterterrorism policies is an example of experimental design. They used a Solomon four-group design involving 371 U.S. adults to compare the media framing of “domestic homegrown” and “international” terrorism of the London bombings in 2005 . The authors used video segments from actual broadcasts on July 7, 2005 , and edited them together to create an approximately 10-minute video for each of the two conditions. The authors found that media frames of homegrown terrorism produced greater fear than the international framing. Fear resulted in greater support for restricting civil liberties of Muslims and, under certain conditions, general negative feelings toward Muslims.

A third method used in social scientific research is content analysis of media sources. This method is utilized to identify patterns prevalent in the media (e.g., Dixon & Azocar, 2006 ; Klein & Shiffman, 2006 ). Additionally, some researchers survey participants for their reactions about media patterns. Content analysis, while time consuming, is convenient and inexpensive since the only access needed is a recording or transcript of the artifact of study. It involves the use of a coding scheme to provide an “objective” description of the media and thus insights into cultural values and behaviors. The categorizations are then compared across cultures. When these categorizations are compared, it is done on the basis of frames, which are defined as a “schema of interpretation, collection of anecdotes, and stereotypes” (Cissel, 2014 , p. 67). Once these frames are determined, the way in which individuals deal with their realities within and across cultures can be studied.

An example of such content analysis was the study of the coverage of the Fukushima nuclear accident in Japan in two Belgian newspapers: Le Soir and De Standard (Perko et al., 2011 ). The time period of the study was from March 11, 2011, to May 11, 2011 . Every article was coded by two independent coders. The authors had begun their study with a question as to how the framing of the question of nuclear power would appear in the two Belgian newspapers. They arrived at the conclusion that the reporting was mostly neutral. Further, since the Fukushima nuclear accident was in a country quite remote, the articles did not frame the issue as an example of a possible threat to their own country from nuclear power plants.

Data Analysis and Methodological Concerns

Data from these three methods are quantified to allow for statistical analysis. All forms of data must be reduced to categories that are independent from one another (exhaustive and exclusive categories). These can include frequency counts of behaviors, sequence of behaviors, and self-report information on numerical scales. Data are then analyzed with statistical software to determine associations between cultural (independent) and communication (dependent) variables (outcomes are dependent variables with culture and/or communication as independent). The nature of analysis depends on the numerical measurement of the variables, but frequent tests include t -tests, analysis of variance, correlation, and regression. Additionally, complex modeling of dependent variables can be undertaken using, for example, structural equation modeling and hierarchical linear modeling. The key concern with the statistical tests is accounting for variance in the dependent variables. The more variance explained means the “more important” a cultural factor is for communication behavior. Because of the vast number of factors that explain human behavior, intercultural researchers believe that as little as 5–10% of variance explained is meaningful.

There are four concerns for data analysis in social scientific research: (a) reliability, (b) measurement validity, (c) internal validity, and (d) external validity. Reliability is reproducibility. For the aforementioned methods, two types of reliability are relevant. First, internal consistency of measures is usually measured with Cronbach’s alpha. Second, when completing content or interaction analysis, intercoder reliability (agreement between two or more coders) is important and measured with Cohen’s K or Scott’s pi (or the like). Reliability means a researcher has consistent measures, whereas validity focuses on accurate information.

Validity is a combination of measurement, internal, and external validity (depending on the goals in the study). Measurement validity focuses on the accuracy with which a scale (or coding scheme) is measuring what is supposed to be measured. Internal validity is the strength to which a researcher can conclude that the independent variable is associated with the dependent variable as hypothesized. Internal validity is established by eliminating rival explanations for statistical associations through statistical or experimental control of confounding (or nuisance) variables. External validity is the degree to which a study’s results can be generalized to the larger populations from which a sample was drawn. In intercultural research, researchers are more concerned with measurement and internal validity than external validity.

While these general methodological concerns are true for all social science research, there are also unique concerns with cross-cultural/intercultural communication research (Gudykunst, 2003b ; Levine, Park, & Kim, 2007 ). Gudykunst ( 2003b ) outlined a number of concerns with cross-cultural research, but chief among the methodological issues is establishing equivalence. In order to make cross-cultural comparisons (and have valid measures for intercultural research), researchers need to ensure that the constructs and measures are equivalent on five levels. First, constructs must be functionally equivalent; that is, the construct must work the same way in the cultures under study. Second, constructs must be conceptually equivalent; that is, the construct must have the same meaning within the cognitive system of the members of cultures being examined. Third, linguistic equivalence for constructs refers having language that is equivalent. Linguistic equivalence is often established through translating and backtranslating of measures. Fourth, metric equivalence is established by ensuring that participants in different cultures do not respond to numerical scales in different ways (e.g., one cultural group may not use the extreme scores in a scale). Finally, researchers need to take care and establish that there is sample equivalence in the two cultural groups. The samples need to be comparable (e.g., similar age, gender, education, etc.). Fletcher and colleagues ( 2014 ) explore the steps needed to statistically ensure equivalence in measurement across multiple cultures. Establishing equivalence on these issues helps to eliminate rival explanations and further ensures that differences found are due to cultural differences. In addition to such methodological rigor, scholars from other orientations argue that it is also imperative for the researcher to be reflexive and aware of theoretical and methodological centeredness that can come from such systematic rigor (Asante, Miike, & Yin, 2008 ).

Interpretive Methods

Interpretive scholars are interested in unearthing multiple simultaneous truths, believe in a person’s free will, acknowledge that the known and the knower cannot be separated, and believe that interpretation is based on one’s persuasive abilities. Striving for meaning, interpretive scholars generally use a variety of qualitative methods to study specific intercultural phenomena. As a result of this, interpretivists examine theoretical limits by comparing results from multiple forms of research about the same phenomenon (Szabo, 2007 ). For this article, we focus on ethnography of communication and interpretive interviews as these are two common approaches. We then discuss the general methodological issues in collecting and analyzing interpretive data.

Ethnography of communication (EOC) is a method to study the relationship between language and culture through extensive field experience. The concept of the ethnography of communication was developed by Dell Hymes (Hall, 2002 ). It can be defined as the discovery and explication of the rules for contextually appropriate behavior in a community or group or what the individual needs to know to be a functional member of the community. EOC applies ethnographic methods to understand the communication patterns of a speech community (Philipsen, 1975 ). A speech community is a group of speakers who share common speech codes and use these codes based on a specific situation. From the presence or absence of certain speech codes, one can interpret the culture of a community with its shared values, beliefs, and attitudes. In his classic study, Philipsen ( 1975 ) explored the communication patterns of white males in a predominantly blue-collar neighborhood called “Teamsterville” in South Chicago. Philipsen lived in the community for several years and worked and interacted as a member of the community while also conducting his research. Results from this study explained when talk was appropriate, at what levels, and when action was more appropriate than talk. When two men were of similar backgrounds, of more or less equal status, and were close friends, they could talk to each other. There was less talk when the relationship was asymmetrical (e.g., father–son and husband–wife). The least amount of talk occurred when a “Teamsterville” was responding to an insult or trying to assert his power over someone. It is in these instances that action was more appropriate than words. If a man did talk during this interaction, he was seen as not masculine enough. Another interesting study was conducted by Radford et al. ( 2011 ). The study focused on applying EOC to the case of virtual reference context. Here, the researchers focused on the interactions that constitute the context in which the participants make verbal statements and coordinate them with other statements in order to closely analyze the relational barriers and relational facilitators. The interactions spanned a 23-month time period ( July 2004–May 2006 ), and the transcripts of 746 live chats of this period were studied. The researchers were able to conclude from their study that when professional librarians chatted, they were more formal, less free with accepted online abbreviations, whereas students were more comfortable with using abbreviations and other turns of phrases. One of the conclusions the researchers drew was that if the librarians used more informal language they would appear more friendly and approachable.

Interpretive interviews are a second common approach. The purpose of the interpretive interview is to uncover insider meanings and understandings from the perspective of the participants (Denzin, 2001 ). According to Denzin ( 2001 ), the characteristic of interpretive interviews is that they allow us to understand the society in which we live, which is referred to as an interview society. Typically, these interviews are one-on-one and face-to-face interviews designed to elicit in-depth information. The interviews can focus on narratives, topics, perspectives, and opinions and often are conducted in a semi-structured manner (although unstructured interviews are sometimes conducted). One of the reasons why the semi-structured and/or narrative form is used is to allow for deeper and embedded meanings that might elude a more inquiry-based approach. An example of interpretive interviews is Baig, Ting-Toomey, and Dorjee’s ( 2014 ) study of meaning construction of the South Asian Indian term izzat (face) in intergenerational contexts. The authors interviewed six younger (aged 31–40) and six older (aged 55–72) South Asian Indian American women about face concerns in their intergenerational family communication situations. The authors found that family izzat is of primary importance in these contexts and that the motif of respect is central to the meaning of izzat . They also identified differences in the younger and older facework strategies.

The primary focus of analyzing interpretive research data is rather nicely summarized by Carbaugh ( 2007 ):

It is important to emphasize the interpretive task before the analyst: while engaging in a communication practice, an analyst seeks to understand what range of meanings is active in that practice, when it is getting done. The analyst sets out to interpret this practice, what is being presumed by participants for it to be what it is, that is, to understand the meta-cultural commentary imminent in it. What all does this practice have to say? (p. 174)

Thus, the interpretive scholar analyzes data in order to describe and interpret.

Carbaugh ( 2007 ) identified two concerns in analyzing interpretive data—the framework used to analyze the semantic content of cultural discourse and the vocabulary used to formulate these contents. A researcher’s analysis of the content of the communication exchange also includes a meta-analysis of the subject, the object, the context, the history, and the stories revolving around the exchange. Carbaugh ( 2007 ) noted that “these cultural meanings—about personhood, relationships, action, emotion, and dwelling, respectively—are formulated in cultural discourse analyses as radiants of cultural meaning” (p. 174). These radiants of cultural meanings focus on personhood and identity, relating and relationships, meanings about acting, action and practice, meanings about emotions and feelings, and meanings about place or context.

Reliability and validity are explicated differently in interpretive research compared to social science research. If social scientific scholars are interested in consistency for reliability, interpretive scholars see reliability as the quality of the information obtained; does the data give us a richer, clearer understanding of the phenomena (Golafshani, 2003 )? Lincoln and Guba ( 1985 ) used the term “dependability” in place of reliability to assess the quality of a research project. For validity, it is important to assess the quality based on the specific paradigm used to conduct the qualitative research. Further, while many scholars argue that validity is not a critical concept for interpretive research, Lincoln and Guba ( 1985 ) explained that the “trustworthiness” of the data is similar to validity in social science research. Do the community of scholars conducting interpretive research view the data as meaningful, useful, and following the research protocols appropriately?

After having considered these general considerations, we now consider three specific data analysis approaches using in interpretive intercultural communication research including grounded theory, constant comparative analysis, and thematic analysis. Other data analytic approaches for data analysis include narrative analysis, conversational analysis, EOC, and interpretive phenomenological analysis. Grounded theory is a continuum of practices that are inductive and iterative aimed at recognizing categories and concepts in texts in order to integrate them to formal theoretical models (Corbin & Strauss, 2008 ). They begin with the observations, experiences, and stories, and through a process of coding, analysts identify a theoretical model to fit the data. Another important approach that interpretive scholars use is that of constant comparative analysis (CCA). CCA has often been used as a part of grounded theory, but it is now being used separately to analyze cross-cultural and intercultural communication. CCA is used to balance the etic perspective (participant as outsider) with the emic perspective (participant as insider) to ensure balance between cultural readings and theoretical frameworks. CCA ensures that all data in the relevant set are compared with all other data in the same set to make sure that no data are dismissed on thematic grounds (O’Connor et al., 2008 ). Further, CCA tries to accommodate the most relevant theories though they may appear disparate. A final prominent approach is thematic analysis. Thematic analysis is a flexible and yet rigorous approach of identifying and analyzing patterns or themes of meaning from data. Braun and Clarke ( 2006 ) identify a six-step process for conducting thematic analysis.

Critical Methods

From the critical perspective, relationships between cultural groups are often characterized by dominance and resistance. Communication between groups is based on certain understanding of culture and ethnicity that is fixed, reified, and essentialized and is informed by certain cultural assumptions that tend to be rooted in Euro-American traditions and worldviews (Asante et al., 2008 ). Hermans and Kempen ( 1998 ) argued that dominant approaches to knowledge favor static conceptualizations of culture. It is the creation of these static categories in which the Western understanding of the rest of the world dominates the intercultural relations that results in the reification of culturally homogeneous “ethnic” and racial groups. Consequently, this orientation undermines ways in which the self is understood in different cultures.

Critical and feminist scholars have consistently raised questions about power imbalance between researchers and researched in the field, suggesting that if researchers fail to explore how their personal, professional, and structural positions frame social scientific investigations, researchers may inevitably reproduce dominant gender, race, and class biases (Fairclough, 1995 ; Lazar, 2005 ). This section illustrates postcolonial ethnography and critical discourse analysis as approaches for intercultural discovery from the critical lens. Additionally, we introduce the role of self-reflexivity and consciousness-raising in the context of methodological concerns from the critical perspective.

A variety of approaches to critical issues exist such as critical race theory, decolonizing and indigenous methodologies, engaged methodologies, and performative methodologies (Willink, Gutierrez-Perez, Shukri, & Stein, 2014 ). In this article, we explore two prominent methods to illustrate some of the key elements to critical approaches given that we cannot cover all of them: postcolonial ethnography and critical discourse analysis.

Postcolonial ethnography seeks to disrupt and restructure established academic practices and modes of knowledge development and dissemination (Pathak, 2010 ). It attempt to do this by pointing out that gender roles, academic institutions, racial binaries, and other power structures are not apolitical. Postcolonial ethnography seeks to question the reification and valorization of supposed objective, scientific, and disembodied knowledge formations. Instead they seek to find alternate and embodied knowledge forms that accommodate the subjective and the personal.

While postcolonial and third world feminist scholars point to myriad ways in which relations of domination infuse ethnography, they also offer some guidance for negotiating power inherent in the practice of fieldwork (Spivak, 1999 ). This guidance takes the form of feminist geopolitics, which involves not only questioning hegemonic structures and dominant power structures but also offering alternatives to those structures (Koopman, 2011 ). Postcolonial scholars argue that the practice of ethnography among marginalized groups is historically tainted by ethnocentric biases in traditional ethnographic practice and research (Collins, 1990 ). Further, as philosopher Sandra Harding ( 1998 ) emphasized, ethnocentricism is structured into the institutional and academic practices so as to produce relationships oppressive to indigenous cultures in the so-called first world as well as third world countries.

An example of postcolonial inquiry is that of an ethnographic encounter (Irani et al., 2010 ). As a part of this inquiry, the company that the researchers studied, Ddesign, had to develop prototypical home water purification filters (Irani et al., 2010 ). The site of their study was various villages in India where they were supposed to study the feasibility of home water purifiers among the economically deprived households of the villages. The researchers later were told that when Ddesign first started their study, they had notions of privations in the lives of the householders. During their study, they found that the reality was quite different from their preconceptions. They realized that the definitions of privations that the company personnel had were not applicable to the people or to their living conditions. In fact, the researchers were told by the company personnel that the villagers had a very different worldview from that of the personnel. Thus, the researchers and the company personnel realized that one group’s notions of well-being and happiness were not necessarily applicable to another group no matter how universal those notions might be.

A second approach is critical discourse analysis (CDA). The creative combining of different approaches of lived experience, texts or discourses, and the social and political structures of power has resulted in popularity of cultural studies as a critical site for different modes of enquiry. According to Fairclough ( 1995 ), “many analysts are becoming increasingly hesitant in their use of basic theoretical concepts such as power, ideology, class, and even truth/falsity” (p. 15). In recent social scientific research, there has been a turn toward language or, more specifically, toward discourse. According to the feminist critical scholar Michelle M. Lazar ( 2005 ), discourse is a “site of struggle, where forces of social (re)production and contestation are played out” (p. 4). Critical discourse analysis is known for its overtly political stance and deals with all forms of social inequality and injustice. It includes the study of processes premised on the acts and discursive interactions of individuals and groups on which both the local and international contexts bring to bear their limits in the production of legislation, news making, and other such products of discursive interactions (van Dijk, 2008 ).

An example of critical discourse analysis in intercultural communication research is Chen, Simmons, and Kang’s ( 2015 ) study of identity construction of college students. The authors contextualize their study in an era of “postracial” utopia resulting during the Obama administration. They coin the term “Multicultural/multiracial Obama-ism (MMO)” to reflect this era and the prominent frame of colorblindness and multiculturalism prominent in media discourse. They examined 65 student essays about three cultural identities that stood out in a particular context. They analyzed the essays using CDA and found three frames that support this construction of postracial utopia: meritocracy, identity as self-chosen, and equality of opportunity despite privilege. They critique these frames and identify implications for teaching about intercultural communication and identity in the classroom.

Key methodological issues in the critical approach are the role of reflexivity, consciousness-raising, and limitations/possibilities of the reflective approach. A sociology-of-knowledge approach to critical scholarship reveals the role of reflexivity as a source of insight (Cook & Fonow, 1984 ). Reflexivity means the tendency of critical scholars to reflect upon, examine critically, and explore analytically the nature of the research process. To some extent, this tendency toward reflection is part of a tradition of attention to what Kaplan ( 1964 ) referred to as “logic-in-use” or the actual occurrences that arise in the inquiry, idealized and unreconstructed. Feminist and critical epistemology carries this tradition of reflection further by using it to gain insight into the assumptions about gender and intercultural relations underlying the conduct of inquiry. This is often accomplished by a thoroughgoing review of the research setting and its participants, including an exploration of the investigator’s reactions to doing the research.

One of the ways in which reflexivity is employed involves the concept of consciousness-raising, a process of self-awareness familiar to those involved with the women’s movement. Underlying much of the reflexivity found in feminist scholarship is the notion found in the earlier work of scholars such as W. E. B. DuBois ( 1969 ) and Paulo Freire ( 1970 ) that consciousness of oppression can lead to a creative insight that is generated by experiencing contradictions. Under ideal circumstances, transformation occurs, during which something hidden is revealed about the formerly taken-for-granted aspects of intercultural relations.

Consciousness-raising is employed in various ways by the critical scholar. The first way is through attention to the consciousness-raising effects of research on the researcher. Consciousness-raising is also involved in discussions of ways in which the research process influences subjects of the inquiry. Some authors view the research act as an explicit attempt to reduce the distance between the researcher and subjects (Collins, 1990 ). These approaches have provided critical and feminist researchers with a way to tap collective consciousness as a source of data and have provided participants in the research process with a way to confirm the experiences that have often been denied as real in the past. The applications of critical consciousness-raising and reflexivity can be seen in discourses surrounding terrorism and counterterrorism. This application can be seen in the study by Schmid ( 2013 ) about radicalization, deradicalization, and counter-radicalization. Schmid has observed that the usual causes such as poverty, social inequality, oppression, and neglect by the West have not been empirically tested satisfactorily, yet they are believed to be the primary causes of radicalization. The study provides three levels of analysis that can be used to understand how “radicals” are born and how that complex construction can be interrogated: the micro level, dealing with the individual level in terms of identity and self-reflection; the meso level, which deals with the socio-political milieu surrounding the individual; and the macro level, which refers to the larger society and governance that affect the individual. Further, these three levels of analysis can also be used to see how the continuum from radical to political undesirable and terrorist can be studied.

Finally, there are limitations and possibilities of reflective practice. Critical researchers use self-reflection about power as a tool to deepen ethnographic analysis and to highlight the dilemmas in fieldwork. The call for reflective practice has also been informed by critiques of postcolonial theorists who argue for self-reflexive understanding of the epistemological investments that shape the politics of method (Mohanty, 1991 ). Cultural studies scholars have also questioned the call to reflective practice, arguing that taken to the extreme, “constant reflexivity” can make “social interaction extremely cumbersome” (Hurtado, 1996 , p. 29). In contrast, the call to “accountability” is said to offer a more collective approach than the “individual self assessment of one’s perspective” that the term “reflexivity implies” (Hurtado, 1996 , p. 29). However, from point of ethnographic practice, it is seldom clear to whom one should be “accountable,” and therefore the term reflective practice seems to be appropriate.

Reflective practice indicates both individual self-assessment and collective assessment of research strategies. Hurtado ( 1996 ) emphasized that a “reflexive mechanism for understanding how we are all involved in the dirty process of racializing and gendering others, limiting who they are and who they can become” (p. 124) is a necessary strategy to help dismantle domination. Such reflective strategies can also help ethnographers bring to the surface “their own privilege and possible bias” as well as “addressing the difference between different constituencies” (Hurtado, 1996 , p. 160) within the communities they study.

Integrating Social Science, Interpretive, and Critical Research Methods

Each set of methods presented in this chapter has strengths and limitations. They address specific purposes that collectively are all important for the field of intercultural communication. Moreover, integrating the research methods provides richer insights than using any method by itself. However, these integrations still may have limitations in exploring non-Western contexts. Thus, this section explores integrations of the methods and alternative methods for intercultural inquiry.

Integrations of Methods

The integration of research methods involves using different types of methods at different phases (Cresswell & Plano Clark, 2011 ). In this manner, the methods are used one after another (or concurrently) depending on the research question associated with the larger research program. Four phasic designs are most prevalent: a) qualitative/interpretive methods used to create a quantitative (social science measure); b) qualitative (interpretive and critical) methods used to embellish quantitative findings (Big Quant, Little Qual); c) quantitative methods used to embellish qualitative findings (Big Qual, Little Quant); and d) social science, interpretive, critical methods used conjointly. Space limitations prohibit us from providing examples of all of these approaches, so we detail two of them.

Zhang and colleagues (Zhang & Oetzel, 2006 ; Zhang, Oetzel, Gao, Wilcox, & Takai, 2007 ) provide an example of how to create a cross-culturally valid measure of a construct. Their purpose was to measure teacher immediacy. Teacher immediacy is the psychology closeness that is communicated from a teacher to a student. There exist different measures of immediacy, but Zhang and Oetzel ( 2006 ) argued that prior Western measures were not applicable in Chinese classrooms (i.e., they did not have conceptual equivalence). To address this issue, they first conducted open-ended interviews with Chinese students to identify themes associated with the meanings of immediacy. This phase of the research involved interpretive research methods as they put primacy on emic meanings. In the second phase, they used the emic meanings to create an operational measure of three dimensions of teacher immediacy (instructional, relational, and personal). This measure was administered to college students, and the data were analyzed with confirmatory factor analysis. The results dimensions were found to be internally consistent and had construct validity as they correlated with existing scales in expected directions. Zhang et al. ( 2007 ) then continued the development of the scale by administering the scale to college students in four national cultures: China, Japan, Germany, and the United States. With these data, the authors used confirmatory factor analysis to see if the three-dimensional model of teacher immediacy held up in each culture. They found cross-cultural support for the model and also the construct validity of the scales. Thus, their thorough testing from the interpretive phase to the social scientific phase led to the development of a teacher immediacy scale that has valid dimensions in at least four national cultures.

An example of integrating critical, social scientific, and interpretive methods into the same research program can be seen in the work on whiteness ideology (Nakayama & Martin, 1999 ). The project culminated in an edited book that included chapters using the various research methods. Whiteness ideology is the worldview that certain groups have privilege over others. It is labeled whiteness because whites tend to be the privileged groups in most societies. This research group’s work primarily focused on ethnic groups in the United States, but some international contexts were examined and other scholars have since examined international contexts we well (e.g., Collier, 2005 ). One part of the project examined the labels that white people in the United States prefer through a survey (Martin, Krizek, Nakayama, & Bradford, 1999 ). Another part of the project involved two of the team members’ integrated interpretive and critical methods to understand how whiteness is used as strategic rhetoric (Nakayama & Krizek, 1999 ). The volume included other scholars writing from different perspectives as well, and the editors attempted to bring together these various perspectives into a “coherent” picture about whiteness ideology. These scholars asked different questions and used different methods to investigate the same phenomena. Collectively, the research program told a richer and fuller story than any single study could have told. This example illustrates how different research methods can be used concurrently to advance understanding about intercultural phenomena.

Alternative Approaches to Studying Intercultural Communication

Intercultural research using the social scientific, interpretive, and critical methods have offered remarkable insights on a variety of intercultural phenomena. Each of these traditional approaches, however, uses a Euro-Western lens that is predominantly textocentric, privileging text, writing, and the lettered word in comparison to oral stories and visuals (Conquergood, 2002 ; Kim, 2002 ). We offer here two participatory approaches that, in some sense, hand over the power of the data to the participants. From these approaches, the ontology, epistemology, and axiology of the participants are more important than those of the researchers. Singhal, Harter, Chitnis, and Sharma ( 2007 ) explained that participation-based methodology allows for lateral communication between participants, creates a space for dialogue, focuses on the people’s needs, enables collective empowerment, and offers cultural-specific content. In contrast, they note that nonparticipatory methods allow top-down vertical communication, generally focus on individual behavior change, consider the donors’ and researchers’ goals of greater importance than community needs, and offer cultural-general information. This section discusses three participatory approaches: theater, photography, and community-based participatory research.

Participatory Theater

Based on the dialogic theorizing of Brazilian educator Paulo Freire ( 1970 ) and its application by Augusto Boal in his performative intervention, “Theater of the Oppressed” ( 1995 ), participatory theater can offer researchers an epistemology different from other research methods that rely on data from interviews and focus groups. This approach provides different kinds of data, discursive narratives that can be used to highlight some of the significant generative themes of the research participants.

The Theater of the Oppressed was developed in an effort to transform theater from the “monologue” of traditional performance into a “dialogue” between audience and stage. Boal ( 1995 ) experimented with many kinds of interactive theater. His explorations were based on the assumption that dialogue is the common, healthy dynamic between all humans and when a dialogue becomes a monologue, oppression ensues. Participatory theater is a research tool that produces generative and local knowledge, starting with the use of the body, the container of memory, emotions, and culture (Kaptani & Yuval-Davis, 2008 ). Theater has the ability to provide a useful connection to specific places as well as people. The encounter between the researcher and the researched in the theater space is outside the redundancy of everyday life. As a result, the researcher can see herself and her interactions between and with the researched in a way that is more distant than in everyday life, thus possibly making it easier to become reflexive.

Boal ( 1995 ) developed various forms of theater workshops and performances which aimed to meet the needs of all people for interaction, dialogue, critical thinking, action, and fun. For example, Forum Theater constitutes a series of workshops in which the participants are transformed from a passive audience into the double roles of actors and active audience. They construct dramatic scenes involving conflictual oppressive situations in small groups and show them to the other participants, who intervene by taking the place of the protagonists and suggesting better strategies for achieving their goals. One of the popular research tools used in Forum Theater is role-playing. Role-playing serves as a vehicle for analyzing power, stimulating public debate, and searching for solutions. Participants explore the complexity of the human condition and situate this knowledge in its cultural moment. The aim of the forum is not to find an ideal solution but to invent new ways of confronting problems. A second key tool is discussion. Following each intervention, audience members discuss the solution offered. A skilled facilitator encourages an in-depth discussion with the participants to generate ideas that will help to address issues under investigation.

Participatory Photography

Similarly, Paolo Friere is a pioneer in participatory photography. In 1973 , Freire and his team asked people living in a slum in Lima, Peru, to visualize “exploitation” by taking photographs (Singhal et al., 2007 ). One child took a photograph of a nail on a wall. While the photograph did not resonate with adults, many of the children strongly supported it. When asked to explain, it was learned that many of the boys in the neighborhood were shoeshine boys in the city. Since the shoeshine box was heavy and they could not carry it to the city, they rented a nail on the wall in one of the city shops. These shop owners charged the boys more than half of each day’s earnings as rent. The children expressed that the photo of the nail was the strongest symbol of exploitation. Friere and his team then used this photo to generate a discussion about exploitation and how the community members wished to address it.

Participatory photography, otherwise known as “photo voice” or “shooting back,” gives power to the participants, through photographs, to shape their own stories (Wang, 1999 ). Participatory photography has been used in a variety of contexts (slums, hospitals, schools, villages, etc.) and in different parts of the world (Singhal et al., 2007 ). For example, Briski and Kauffman ( 2004 ), in their Oscar-winning film, Born into Brothels: Calcutta’s Red Light Kids , taught the children of commercial sex workers how to take photographs. These children, then, took photos to depict their harsh reality. These powerful images became the foundation of this moving film. Another example is the work of Loignon et al. ( 2014 ) in Canada about the relation between impoverishment and lack of access to primary health care. The researchers recruited four family medicine residents and two medical supervisors to pursue their study. There were eight participants who came from economically underprivileged backgrounds trained in photographic techniques and photo voice philosophy. The researchers were able to realize the importance of primary health care professionals developing greater interpersonal and social acuity. They also realized that their patients were co-participants in the processes of diagnosis, prognosis, and medication. Finally, the researchers were also able to realize that they would be able to develop a greater competency by actually investing a part of their training time in the socioeconomic milieu of the patients they are to serve.

The implications of using participatory photography are significant. This method works best when the participants are given general directions and allowed to play with ideas. It is important for the participants to share their visual stories with the researchers. It is, however, critical for fellow participants in a community to share their stories with each other. The challenge of using photography is that it is, by nature, an intrusive process (Singhal et al., 2007 ). With terminology like “aim,” “shoot,” and “capture,” there can be a colonizing mentality in photography. It is particularly important that the participants be sensitive and reflective about how they take photographs of people and objects. While this may be difficult to accomplish across cultures, it is important to seek the permission of the participants before taking their photographs.

Community-Based Participatory Research

Community-based participatory research (CBPR) is a collaborative process where researchers and community members work together at all stages of the research process to address issues that are of importance to the community (Wallerstein et al., 2008 ). Rather than a top-down approach to health and social issues, CBPR focuses on a collaborative and bottom-up approach to identifying and defining problems and developing and implementing solutions (i.e., research “with” rather than “for” or “on”). CPBR is a preferred approach for researchers working with indigenous communities, other communities of color, or other communities facing disparities, which experience similar issues of mistrust for past research issues and social/health inequities. CBPR has goals of developing culturally centered research and interventions, building trust and synergy among partners, building the capacity of all members of the research team, changing power relations among communities and outside entities, developing sustainable change, and improving the social and health conditions of the community (Wallerstein et al., 2008 ).

CBPR is not a method, rather a philosophy of research. CBPR projects can include social scientific, interpretive, and critical approaches and often involve mixed methods. The specific methods meet the needs of the community and the research problem being addressed. The methods should follow key principles of CBPR, including: a) the project fits local/cultural beliefs, norms, and practices; b) the project emphasizes what is important in the community; c) the project builds on strengths in the community; d) the project balances research and social action; and e) the project disseminates findings to all partners and involves all partners in the dissemination process (Israel et al., 2008 ).

An example of CBPR is a project in Mysore, South India, addressing stigma and discrimination among men who have sex with men (MSM), many of whom were sex workers (Lorway et al., 2013 ). The project involved a collaboration of researchers and a sex workers collective in a long-term systematic process of knowledge production and action. The research approach involved training 10 community members as researchers who conducted interviews with MSM to understand their experiences. There were 70 in-depth interviews conducted in four days. Data analysis was completed with thematic analysis. The results provided a rallying point against stigma as the community cultivated its understanding of this concept and they mobilized to increase access to sexual health services.

The purpose of this article was to explore multidisciplinary methodological approaches to intercultural communication research. If our worldview shapes our reality, what we study and how we study phenomena is greatly influenced by our cultural frameworks. We described the traditional approaches to studying intercultural communication, namely, social scientific, interpretive, and critical perspectives. We identified the key ontological, epistemological, and axiological assumptions of each of these perspectives, offered an exemplar for each kind of perspective, types of data collected, and the methodological concerns in each framework. We then explained traditional ways to integrate the social scientific, interpretive, and critical perspectives, offered examples, and explicated the strengths of such integrations. We finally offered three alternative methodological approaches (participatory theater, participatory photography, and community-based participatory research) where the participants shape the scope of the study, interpret the meaning of the data, and offer practical implications for the study.

Historiography

The early history of intercultural communication, including some discussion of research methods, has been covered well by Leeds-Hurwitz ( 1990 ) and Moon ( 1996 ). Leeds-Hurwitz reviewed the early foundation of intercultural communication, which can be traced to the work of Edward T. Hall in the Foreign Service Institute in the 1950s and 1960s. The focus in the earlier years was on descriptive linguistic analysis of micro communication practices (e.g., proxemics, kinesics, and verbal practices) of multiple cultures. These early roots of intercultural communication were influenced by anthropological study of culture (i.e., ethnography).

The 1970s saw the development of the field of intercultural communication, with a focus on culture as race, gender, nationality, and socioeconomic status (Moon, 1996 ). The research at this time also reflected the social issues of the 1970s. Methods of research were diverse but predominantly included social scientific and interpretive methods.

The late 1970s and the 1980s saw a change where the focus of culture became nationality and a large emphasis was placed on cross-cultural comparisons. There was a pursuit to develop and apply Western theories to non-Western contexts. Methodologically, the 1980s was dominated by social scientific approaches.

The 1990s brought some backlash against social scientific approaches from interpretive scholars. There was also a rise of critical scholarship which critiqued the social scientific research methods. A number of critical approaches were identified and were especially used to develop theoretical approaches for understanding intercultural communications.

The 2000s brought more balance and integration of the research approaches. The Journal of International and Intercultural Communication was founded in 2008 . The three editors of this journal to date (Tom Nakayama, Shiv Ganesh, and Rona Halualani) issued editorial statements about the scope of the journal respecting and including diverse methodological approaches.

Further Reading

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The Sage Handbook of Qualitative Research in Organizational Communication

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The Sage Handbook of Qualitative Research in Organizational Communication  is a state-of-the-art resource for scholars, students, and practitioners seeking to deepen their understanding and expertise in this dynamic field. Written by a global team of established and emerging experts, this Handbook provides a comprehensive exploration of the field’s foundational traditions of epistemology and theory, as well as its latest methodologies, methods, issues, and debates.

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The Sage Handbook of Qualitative Research in Organizational Communication is an essential resource for management scholars, practitioners, and students alike. This groundbreaking handbook is a comprehensive guide that navigates the vast landscape of qualitative research within the realm of organizational communication. With contributions from leading experts in the field, it provides invaluable insights, methodologies, and theoretical frameworks helping to understand the complex dynamics of communication in organizational settings. It offers a diverse range of perspectives and approaches, inviting readers to explore the intricacies of organizational communication through a qualitative lens. This handbook is an indispensable companion for anyone seeking to delve deeper into this fascinating field.

Communication scholars have developed many rich and rigorous qualitative methods for capturing, analyzing, and interpreting the communicative aspects of organizing. It is exciting to see these methods brought together in a comprehensive handbook that features both classical approaches and reflections on new forms of data and analysis. Boris Brummans, Bryan Taylor, and Anu Sivunen have put together a truly wonderful resource that I will return to again and again.

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Dr. Wrench is an Associate Professor and former chair of the Department of Communication at the State University of New York at New Paltz.

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Dr. Wrench has published over 35 peer-reviewed research articles. You can find full text copies of all but his most recent publications here on his website. To ones that do not have full-text, links to the publishers' websites are provided.

Directory of Communication Measures

The Directory of Communication Mental Measures

In 2010 the National Communication Association published The Directory of Communication Related Mental Measures: A Comprehensive Index of Research Scales, Questionnaires, Indices, Measures, and Instruments by Jason Wrench, Doreen Jowi, and Alan Goodboy. Although this book is no longer in print, the content is now available as a searchable Wiki. Furthermore, new measures are always being added.

Quantitative Research Methods For Communication: A Hands-On Approach

Quantitative Research Methods for Communication: A Hands-On Approach

Quantitative Research Methods for Communication: A Hands-On Approach by Jason Wrench, Candice Thomas-Maddox, Virginia Richmond, and James McCroskey is currently in the 3rd edition and the 4th edition is arriving in 2019. The textbook's website contains a number of helpful video tutorials and links that help new researchers explore the world of quantitative communication research.

Training and Development: The Intersection of Communication and Talent Development in the Modern Workplace

Training and Development: The Intersection of Communication and Talent Development in the Modern Workplace The second edition will be available in Spring 2023.

This comprehensive book on the field of Training and Development was authored by Jason Wrench, Danette Johnson, and Maryalice Citera. The book walks learners through the process of creating both face-to-face and eLearning programs. Other features include information on human performance improvement, training management, and project management. The corresponding website has a number of online tools discussed in the textbook itself to provide learners a full toolkit to get them started in the world of Talent Development

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Dr. Jason S. Wrench is always conducting research related to his interests in intercultural, interpersonal, instructional, and organizational communication. To learn about some of his past and current projects click here:

Jason Wrench

Introduction to Dr. Jason S. Wrench

Jason S. Wrench, Ed.D. is a Professor at SUNY New Paltz.

About Current Publications

As a researcher, I am constantly engaged in a wide range of different research topics related to areas related to communication and technology, communication education, communication and religion, instructional communication, organizational communication, and quantitative research methods. 

Currently, my primary research partner, Dr. Narissra M. Punyanunt-Carter, have been working on a number of projects related communication and religion and communication and technology. We’ve been lucky to know each other for over 20 years and publish both research articles and books during that time.

Investigating the relationships among college students’ satisfaction, addiction, needs, communication apprehension, motives, and uses & gratifications with Snapchat

“i am spiritual, not religious”: examination of the religious receiver apprehension scale, affective learning: evolving from values and planned behaviors to internalization and pervasive behavioral change.

JASON S. WRENCH (Ed.D., West Virginia University) is an associate professor and chair of the Department of Communication at the State University of New York at New Paltz. Dr. Wrench specializes in workplace learning and performance, or the intersection of i

EdD in Curriculum & Instruction and Communication Studies

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What is Communication Research?

Bryn Farnsworth

Bryn Farnsworth

Table of Contents

Let’s start things off with a hopefully rather non-controversial assumption: you’re reading this text. Now, don’t worry, the rest of the post won’t continue to list such inanely clear truths, but this statement serves to highlight what’s occurring right now – communication.

It’s a rather one-sided process currently, with me doing the writing and you doing the reading, but the process of sending a message and that message being understood is occurring. There are endless ways of looking further at this – would you still be reading if you were on a different website? What about if you’re tired, or angry? How would whatever else you’ve read today – or ever – affect how you react to this information? This in essence is what communication research is about – how messages are sent, and how they are received.

At its broadest, communication research is concerned with identifying, exploring, and measuring the factors that surround communication, in any form and regarding any topic . Often from a theory-driven perspective, but increasingly with empirically-grounded methods. Want to know how to make political messaging more effective? Increase the appeal of advertising ? Make people adhere to a health campaign? Communication research these answers.

Below, we will discuss and define communication research further, the research that has shaped the field, and where the field is going.

Definition of Communication Research

As a field of study, communication research dates back either 2000 years or 100 years, depending on your level of pedantry. The study of rhetoric was a hot topic in ancient Greece, and shares some commonalities with the modern form, yet clearly much has changed. The field now focuses on gathering empirical data, and builds theories that help understand the complexity of communication on many levels. In a sense it has less interest in the linguistic style of debating philosophers, and more interest in the groups of people that might be listening.

Socrates death painting

History of Communication Research

One of the most influential books that helped give rise to modern communication research was “ Social Organization: a Study of the Larger Mind ” by Charles Cooley, published in 1909 [1]. Described by one reviewer as “a series of essays on fundamental sociological problems, written in delightful literary style, and with keen and sound psychological insight” and that “Professor Cooley gives, for the first time in sociological literature, strange as it may seem, full and adequate recognition of ‘communication’ as a fundamental fact in the social life” [2].

This book, with a delightful literary style , would set the stage for the work of other academics with an interest in communication, and ultimately the creation of the first academic departments with a clear focus on the field.

In 1952, Bernard Berelson released “ Content Analysis in Communication Research ” – a book that proved pivotal not only to communication researchers of the time, but also had a broader impact [3, 4]. Written in a way that was – according to one reviewer at the time – “unusually lucid for a social science publication”, the book describes the ways in which media and communication are compared, and explores the methods that are used to carry out those comparisons. The book ultimately helped shift the field towards a more quantitative, scientific approach.

In the 1960s and 1970s, social unrest brought about social change, and communication researchers looked more closely at the surrounding language. They explored the systems of thought and discourse that had traditionally been in place, how they were changing, and what that might mean for the future of communication [5]. This occurred alongside the continual expansion of mass communication methods – TV and radio continued their dominance of message-spreading in the western world.

The shift into empirical methodology continued. While theoretical discussions of communication remained (and remain) central to the field, the introduction of data-driven, quantified assessments became an increasingly routine aspect of communication research. The book “ Mass Communication Research Methods ”, released in 1998, helped cement this as standard, defining the experimental methods of the day [6].

These research methods – focus groups , observations, and surveys – have now long been central to the field, yet the next step in empirical quantification is already emerging. Continuing with the steps towards quantification and more thoroughly empirical approaches, new unbiased tools are now being used as a way to incisively measure the processes surrounding communication, to test theories, and to advance understanding further. But what does this look like?

New Methods for Communication Research

Eye tracking has become one of the most widely used technologies within communication research, largely as it “gives communication scholars the opportunity to examine more precisely how much visual attention has been paid to information” [7].

participant using the eye tracking webcam in front of the screen

In 2016, researchers from the University of Amsterdam carried out the first retrospective study examining the use of eye tracking technology within communication research [7]. They found that the majority of studies within communication using eye tracking had focused on advertising research , yet public health, language, and computer-mediated communication were also areas that had been looked at. They also conclude that “ that eye tracking has much more potential in communication research”.

research methods for communication

One example of this potential being seized upon is found in research by researchers from Ohio State University and the University of Illinois Urbana–Champaign, who developed eye tracking metrics to assess automatic stereotyping [8]. By using a gaze-contingency task , they were able to show that stereotype-congruent fixations were decreased for those with a higher political knowledge score.

Political Communication Research

The research showed that participants who are categorized as knowledgeable about politics were more capable of “moderating automatic responses” – adding a new layer of understanding to how political communication can impact reported and actual responses. The researchers go on to state that this “implies that the influence of automatic processes on political thinking is conditional” – meaning that our response to political communication may be less automatic than previously thought.

Responsive Media Messages

While these studies have used eye tracking to measure attention, other communication research has used a combination of methods. Researchers from Texas Tech University used facial electromyography (fEMG), electrocardiography (ECG) , and electrodermal activity (EDA) in order to assess affect in response to media messages [9].

They found that fEMG data provided reliable data regarding emotional state, while heart rate data collected from ECG indicated that negative messages received more attention than positive messages. The skin conductance data collected from EDA provided data that, together with a memory test, showed that the arousal level experienced was a greater predictor of memory retention for the media exposure, as compared to the valence experienced.

Increasing Engagement

Other research has also used arousal in order to understand the response to communication (for a review of some of these studies, see [10]). For example, researchers from Indiana University and the University of Wisconsin-Madison investigated responses to the number of edits within media using EDA and ECG [11]. They find that an increase of edits within the media can increase the encoding of the message without causing too much cognitive load , suggesting that media should feature a larger number of edits (where appropriate) to increase engagement.

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research methods for communication

[1] Cooley, C. H. (1962). Social Organization: A Study of the Larger Mind . New York: Schocken (first published 1909).

[2] Ellwood, C. A. (1910). Social Organization: A Study of the Larger Mind. Charles Horton Cooley. The International Journal of Ethics , 20 : 2 ,  228-230.

[3] Berelson, B. (1952). Content analysis in communication research . Glencoe, IL: Free Press.

[4] Bauer, M. (2000) “Classical Content Analysis: A Review,” in M. Bauer and G. Gaskell (eds.), Qualitative Researching with Text, Image and Sound — A Handbook . London: SAGE. pp. 131—150.

[5] Park, D. W., & Pooley, J. (2008). The history of media and communication research: Contested memories . New York: Peter Lang.

[6] Hansen, A., Cottle, S., Negrine, R. and Newbold, C. (1998). Mass Communication Research Methods . London: Macmillan.

[7] Bol, N., Boerman, S. C., Romano Bergstrom, J. C., & Kruikemeier, S. (2016). An overview of how eye tracking is used in communication research. In M. Antona & C. Stephanidis (Eds.), International conference on universal access in human-computer interaction . Proceedings HCII 2016, Part I, LNCS 9737 ed. (pp. 421–429). Switzerland: Springer International Publishing.

[8] Coronel, J. C., & Federmeier, K. D. (2016). The Effects of Gender Cues and Political Sophistication on Candidate Evaluation: A Comparison of Self-Report and Eye Movement Measures of Stereotyping. Communication Research , 43(7), 922-944. doi:10.1177/0093650215604024.

[9] Bolls, P.D., Lang, A., & Potter, R.F. (2001). The effects of message valence and listener arousal on attention, memory, and facial muscular responses to radio advertisements. Communication Research , 28, 627-651.

[10] Ravaja, N. (2004). Contributions of psychophysiology to media research: Review and recommendations. Media Psychology , 6, 193-235.

[11] Lang, A., Zhou, S., Schwartz, N., Bolls, P. D., & Potter, R. F. (2000). The effects of edits on arousal, attention, and memory for television messages: When an edit is an edit can an edit be too much? Journal of Broadcasting & Electronic Media , 44(1), 94-109.

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HeteroTCR: A heterogeneous graph neural network-based method for predicting peptide-TCR interaction

  • Zilan Yu   ORCID: orcid.org/0000-0002-9460-0984 1 , 2   na1 ,
  • Mengnan Jiang 1   na1 &
  • Xun Lan   ORCID: orcid.org/0000-0002-6523-046X 1 , 2 , 3 , 4  

Communications Biology volume  7 , Article number:  684 ( 2024 ) Cite this article

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Identifying interactions between T-cell receptors (TCRs) and immunogenic peptides holds profound implications across diverse research domains and clinical scenarios. Unsupervised clustering models (UCMs) cannot predict peptide-TCR binding directly, while supervised predictive models (SPMs) often face challenges in identifying antigens previously unencountered by the immune system or possessing limited TCR binding repertoires. Therefore, we propose HeteroTCR , an SPM based on Heterogeneous Graph Neural Network (GNN), to accurately predict peptide-TCR binding probabilities. HeteroTCR captures within-type (TCR-TCR or peptide-peptide) similarity information and between-type (peptide-TCR) interaction insights for predictions on unseen peptides and TCRs, surpassing limitations of existing SPMs. Our evaluation shows HeteroTCR outperforms state-of-the-art models on independent datasets. Ablation studies and visual interpretation underscore the Heterogeneous GNN module’s critical role in enhancing HeteroTCR’s performance by capturing pivotal binding process features. We further demonstrate the robustness and reliability of HeteroTCR through validation using single-cell datasets, aligning with the expectation that pMHC-TCR complexes with higher predicted binding probabilities correspond to increased binding fractions.

Introduction

T cells are a vital component of the adaptive immune system. T-cell receptors (TCRs) present on the surface of T cells have the ability to recognize peptides presented by Major Histocompatibility Complex (MHC) derived from various sources such as host proteins, pathogens, or tumors 1 . A majority of TCRs consist of a pair of α - and β -chains. During the maturation of T cells, TCR undergoes random rearrangement of variable ( V ), diversity ( D ), and joining ( J ) gene segments 2 . The interaction between TCR and peptide-MHC (pMHC) complex is mainly defined by three complementarity-determining regions (CDRs) for each chain 3 . Since the β -chain contains V -, D -, and J genes with higher diversity and the CDR3 regions are the determinant of the specific recognition of peptide 4 , most studies have focused on CDR3 regions of TCR β -chain 5 .

Reliably predicting peptide-TCR binding solely from sequences is of great significance for cancer immunotherapy and vaccine development 6 . Such predictions also provide valuable insights into patients’ immunological history, including responses to immunization, infections, and vaccines, thus paving the way for personalized healthcare 7 . Biological experiments to screen numerous unfiltered candidate immunogenic peptides are expensive and time-consuming 8 . Therefore, developing an effective computational method to predict peptide-TCR interactions is pressing due to the intricate nature of the molecular-level process. Notably, a peptide can bind to different TCRs, and conversely, a TCR is able to recognize multiple peptides 9 .

Existing models for predicting peptide-TCR binding can be divided into two categories 10 : (1) Unsupervised clustering models (UCMs) group similar TCR sequences together into clusters based on their shared features, including GLIPH 11 , TCRdist 12 , GIANA 13 , and iSMART 14 ; (2) Supervised predictive models (SPMs) can be further classified into categorical epitope models and generic models. Categorical epitope models are designed to learn TCR patterns that are specific to a particular peptide by using antigen-specific labels 15 . Examples of such models are TCR-classifier 16 , TCRex 17 , TCRGP 18 , and DeepTCR 19 . In contrast, generic models, such as NetTCR-1.0 20 , NetTCR-2.0 5 , ERGO Long Short Term Memory (LSTM) and Auto-encoder (AE) 21 , DLpTCR 22 , ImRex 23 , and TITAN 7 , can predict interactions between any TCR and peptide, without being restricted to a specific peptide.

Though UCMs have shown promise in various applications, they are not directly applicable to recognizing peptide-TCR binding 24 . Additionally, categorical epitope models require training for each peptide or set of peptides in multiclass classification, demanding ample TCR training data binding to the same peptide 23 . Furthermore, these models are inherently incapable of predicting the interactions of peptides not included in the training set (unseen epitopes) 7 . Existing generic models predict interactions between any TCR and peptide, without being restricted to a specific peptide, but rely on concatenating two interacting sequences encoded with a BLOSUM matrix or physicochemical properties 23 . These approaches might not directly focus on the interaction problem, instead learning embeddings for individual interactors, resulting in diminished model performance 23 . Moreover, although these studies perform well on the testing set that includes the training set peptides or TCRs, they cannot reliably predict interactions for those not present in the training set 6 . Additionally, they lack generalizability to new data from diverse databases 22 , due to the extensive variation in TCR and peptide sequences 23 .

Graph neural networks (GNNs) have made remarkable strides in recent years, establishing themselves as essential tools for a range of graph-based applications. Notably, they have demonstrated success in predicting chemical stability 25 , forecasting protein solubility 26 , modeling protein–protein interaction prediction 27 , and exploring drug–target interactions 28 . Therefore, motivated by these successes in the use of GNNs, we introduce HeteroTCR (Fig.  1 ), a Heterogeneous Graph Neural Network based SPM that utilizes only CDR3β sequence information from TCRs and peptides to improve predictive accuracy for peptide-TCR recognition across various datasets. Firstly, we extract the max-pooling layers of the pre-trained Convolutional Neural Network (CNN) module as numeric embeddings of TCRs and peptides to input the model. Then, we employ Heterogeneous GNN module as the backbone of our model, allowing HeteroTCR to extract information on between-type (peptide-TCR) interaction and within-type (TCR-TCR or peptide-peptide) similarity from input embeddings, thereby learning the probability of binding for classification. Existing SPMs mainly rely on the interaction information extracted from first-order neighborhoods while omitting insights originating from higher-order neighborhoods. An inherent advantage for HeteroTCR is that it can integrate information from neighborhoods of different orders, including both interaction information and similarity information. Our evaluation demonstrates that HeteroTCR outperforms state-of-the-art models on multiple independent datasets in terms of area under the curve (AUC) of the receiver operating characteristic (ROC). Ablation studies and visualization substantiate the essential role played by the Heterogeneous GNN module in enhancing the performance of HeteroTCR. This module adeptly captures critical features that underlie the binding process, showcasing its capacity for generalization across datasets with varying spatial distributions. Furthermore, we demonstrate HeteroTCR’s robustness and reliability in single-cell datasets by correlating predicted probabilities with experimentally derived pMHC-T cell binding fractions.

figure 1

To begin, the amino acid sequences of TCRs and peptides are encoded into numeric embeddings by a pre-trained CNN module. Subsequently, the Heterogeneous GNN module is employed to capture information pertaining to interactions between different types (peptide-TCR) and similarities within the same type (TCR-TCR or peptide-peptide). A circle node represents a TCR, while a triangle node represents a peptide. The edge of the solid line represents an interaction between a TCR and a peptide, while the dotted line represents no interaction, and no connection represents unknown. It is important to note that when initializing the graph, the nature of the relationship between peptides and TCRs is unknown, and we can only rely on the input training pairs to identify the existence of a relationship. We get the weight matrix by iterating 1000 epochs of training to learn and adjust the nature of the relationship between the given TCR and peptide, whether it is a solid line or a dotted line. We use a set of aggregator functions to learn aggregate feature information from K th -order neighborhoods of a node, with a default K value of 3. As shown in the graphs with green and blue shading, k  = 1, k  = 2, and k  = 3 represent the node information aggregated from the first, second, and third-order neighborhoods of the circle and triangle nodes. In other word, a given peptide/TCR can learn information from its 1 st -order binding TCRs/peptides, while the 1 st -order TCR/peptide can learn information from its 1 st -order binding peptides/TCRs which are 2 nd -order neighborhoods of given peptide/TCR. Therefore, a given peptide/TCR can extract between-type (peptide-TCR) interaction information from the 1 st - and 3 rd -order TCRs/peptides, and obtain within-type (TCR-TCR or peptide-peptide) similarity information from the 2 nd -order peptides/TCRs. This is because TCRs/peptides with shared features tend to interact with the same peptide/TCR. This process continues until order K is reached. Finally, an MLP is constructed utilizing these two numeric vectors to ascertain the presence of an interaction between a TCR and a peptide. Overall, HeteroTCR is composed of three main components: a pre-trained CNN, a Heterogeneous GNN, and an MLP classifier. The numeric embeddings extracted from the pre-trained CNN module are utilized as inputs for the Heterogeneous GNN module, which enables the extraction of information from the sequences. Specifically, the Heterogeneous GNN initially creates a global graph based on the entire dataset, and then trains on each pair of peptide-TCR inputs.

Model architecture

Conceptually, HeteroTCR divides the prediction of peptide-TCR interactions into three steps (Fig.  1 ). Firstly, a pre-training strategy is adopted, wherein the encoding vectors of the max-pooling layers before classification in CNN module are extracted as numeric embeddings of TCRs and peptides. Secondly, Heterogeneous GNN module is utilized to extract information on between-type (peptide-TCR) interaction and within-type (TCR-TCR or peptide-peptide) similarity. This results in TCR vectors containing information about interacting peptides and similar TCRs, and peptide vectors containing information about interacting TCRs and similar peptides. Finally, a Multi-Layer Perceptron (MLP) is created on top of these two numeric vectors to identify whether an interaction exists between a TCR and a peptide.

To numerically embed TCR and peptide sequences, we adopt a pre-training strategy inspired by Montemurro et al 5 who propose that the ability of classification in CNN-based prediction models is driven by the representation in the max-pooled CNN layer. Therefore, we extract the numeric embeddings of TCRs and peptides from the max-pooled CNN layer of the pre-trained CNN module for subsequent module. First, we encode amino acid (AA) sequences of TCRs and peptides using the BLOSUM50 matrix 29 , representing each AA as the score for substituting it with all 20 AAs. Then, TCR sequences and peptide sequences are individually processed using different convolution filters with kernel sizes {1, 3, 5, 7, 9}. Each kernel size’s convolutional output is max-pooled, yielding a single vector with 160 entries (80 for each input sequence) concatenated from multiple feature vectors. This 160-dimensional vector is fed into an MLP, producing the probability of peptide-TCR binding (Methods). Finally, the max-pooling layers of the pre-trained CNN module are extracted as numeric embeddings of TCRs and peptides, ready for the subsequent module.

For interaction information and similarity information on TCRs and peptides, existing algorithms for predicting peptide-TCR interactions typically extract sequence information from peptides and TCRs separately and simply concatenate them for classification prediction. However, this approach may not directly focus on the interaction problem, but learn an embedding for the individual interactors, resulting in reduced model performance 23 . Moreover, a single TCR can bind to multiple peptides, and a single peptide can bind to numerous TCRs. TCRs (peptides) associated with the same peptide (TCR) may have similarities to some extent. These established conditions consequently enable the capture of within-type (TCR-TCR or peptide-peptide) similarity using sequence information. Therefore, we adopt Heterogeneous GNN, a feature extraction module that stores information about different types of entities in nodes and their different types of relations in edges, to capture the within-type similarity information. We consider the complete dataset as a global graph, featuring two node types: TCR and peptide, and two edge types: TCR binding to peptide and peptide binding to TCR. It is worth noting that the two edge types are set to be equal, ensuring a balanced interaction between TCRs and peptides.

The features of nodes in Heterogeneous GNN are numeric embeddings of TCRs and peptides obtained from the max-pooling layers of the pre-trained CNN module. A solid line represents an interaction between a TCR and a peptide, while the dotted line represents no interaction, and no connection represents an unknown status (Fig.  1 ). Messages are transmitted on the edges through the network, and the weight matrix is obtained through backpropagation from the final MLP classifier (Methods). We use a set of aggregator functions to learn aggregate feature information from K th -order neighborhoods of a node, with a default K value of 3. In other words, a given peptide/TCR can learn information from its 1 st -order binding TCRs/peptides, while the 1 st -order TCR/peptide can learn information from its 1st-order binding peptides/TCRs which are 2 nd -order neighborhoods of given peptide/TCR. Therefore, a given peptide/TCR can extract between-type (peptide-TCR) interaction information from the 1st- and 3rd-order TCRs/peptides, and obtain within-type (TCR-TCR or peptide-peptide) similarity information from the 2 nd -order peptides/TCRs. This is because TCRs/peptides with shared features tends to interact with the same peptide/TCR. This process continues until order K is reached. Therefore, Heterogeneous GNN can extract information on between-type (peptide-TCR) interaction and within-type (TCR-TCR or peptide-peptide) similarity, allowing the vectors to learn about the probability of a binding event.

Finally, we utilize the numeric vectors of TCRs and peptides to learn their pairing. We construct an MLP (Methods) based on the Heterogeneous GNN module’s outputs. The final MLP layer is a single neuron for predicting the interaction between a TCR and a peptide. HeteroTCR outputs a continuous variable between 0 and 1, reflecting the predicted binding strength. A value greater than or equal to 0.5 indicates that there is an interaction between a peptide and a TCR, while a value below 0.5 predicts no interaction.

Comparisons with published methods on the independent testing dataset

We assessed the generalizability of HeteroTCR and other published methods on pair-based data sets (Methods). Specifically, the five models under comparison include NetTCR-1.0, NetTCR-2.0, ERGO LSTM, ERGO AE, and DLpTCR. NetTCR-1.0 20 is based on a simple 1D convolutional neural network (CNN), integrating peptide and CDR3 β sequence information encoded with BLOSUM50 matrix for the prediction of peptide-TCR specificity. NetTCR-2.0 5 modifies the network structure on the basis of NetTCR-1.0 and uses paired CDR3 α / β data as input instead of CDR3 β information only. Here, we exclusively employed the CDR3 β single-input model from NetTCR-2.0 for a fair comparison, omitting the utilization of the paired CDR3 α / β dual-input model. ERGO 21 applies two encoding methods, Long Short Term Memory (LSTM) acceptor encoding, and Autoencoder-based encoding, for predicting TCR-peptide binding. DLpTCR 22 uses a variety of mixed encoding methods, including one-hot, PCP, and PCA encoding, and integrates FCN, LeNet-5 and ResNet-20 to predict the interaction between peptide and TCR.

The codes for the aforementioned models is sourced from their respective GitHub repositories. The models were retrained and evaluated on our dataset. We trained the models on the IEDB dataset 30 , which comprises of 76,348 peptide-TCR pairs, using 5-fold cross-validation. The dataset was randomly divided into five equal parts, and the models were evaluated in each part in a rotating manner (Methods). For independent testing, we used data collected from the VDJdb 31 , which contains peptide-TCR pairs with confidence scores ranging from 0 to 3, where higher scores indicate more reliable records. Peptide-TCR pairs present in the training data were excluded from the testing data. Throughout our analysis, the area under the curve (AUC) of the receiver operating characteristic (ROC) was used as the metric to estimate model performance. All experiments were performed with default parameter settings.

As shown in Fig.  2 , the AUC scores of all models generally increased with improved dataset quality. However, due to a limited number of data pairs in VDJdb with a confidence score greater than or equal to 3 (only 32 pairs), yielding statistically significant results became impractical, leading us to exclude them. Additionally, the AUCs of HeteroTCR reached 0.6658, 0.6712, and 0.7039 in VDJdb with a confidence score greater than or equal to 0, 1, and 2 respectively, surpassing those of other published models (paired t-test P-value between HeteroTCR’s AUC and that of any other model was <0.00001) (details in Supplementary Tables  1 , 2 ).

figure 2

All boxplots: The center line of each boxplot marks the sample median, and the box extends from the lower to the upper quartile. Each colored point represents one data point. Each boxplot represents the results of n = 5 independent experiments. Our results show that HeteroTCR outperforms tested methods, as indicated by a paired t-test P-value of <0.00001 for comparisons with any other model.

Four types of data splitting methods to evaluate the generalization ability of the model

To further demonstrate the generalizability of HeteroTCR, we evaluated models based on four different types of data splitting methods (Methods), namely pair-based, TCR-based, antigen-based, and strict-based data sets. For the pair-based data sets, the peptide-TCR pairs present in the training dataset were removed from the testing dataset. For TCR-based data sets, peptide-TCR pairs involving TCRs present in the training dataset are excluded from the testing dataset. Similarly, for antigen-based data sets, peptide-TCR pairs involving peptides appearing in the training dataset are excluded from the testing dataset. In the case of strict-based data sets, peptide-TCR pairs involving either TCRs or peptides present in the training dataset are excluded from the testing dataset.

In this analysis, we trained models on IEDB data using four types of data splitting methods, and took McPAS-TCR 32 as the validation dataset for parameter selection. The evaluation was conducted on VDJdb with varying confidence scores (Methods). Importantly, the validation dataset was completely independent from the training set, and likewise, the testing dataset was also independent from both training and validation sets. This approach confirmed the model’s grasp of essential peptide-TCR binding properties, rather than confounding factors within the database.

As shown in Fig.  3 (performance comparison with other VDJdb confidence scores are detailed in Supplementary Figs.  1 – 3 ), we observed a general decrease in AUC scores across all models as data splitting methods became stricter. This decline in performance could stem from potential violations of the assumption of independent and identically distributed (i.i.d.) data across training, validation, and testing sets 7 . Additionally, stricter data splitting methods might lead to weaker data leakage and consequent performance degradation. Given the sparsity of current datasets, it is challenging for models to generalize to unseen epitopes. Notably, all models exhibited substantial performance drops on antigen-based and strict-based data sets as anticipated.

figure 3

For pair-based data sets, the validation dataset McPAS-TCR consists of 1574 pairs, and the testing dataset consists of 9206 pairs. For TCR-based data sets, antigen-based data sets, and strict-based data sets, the number of pairs in McPAS-TCR is 1436, 836, and 784, respectively, while VDJdb contains 7374, 1600, and 1208 pairs, respectively. All boxplots: The center line of each boxplot marks the sample median, the colored points scattered along each boxplot represent all the actual data points, and the box extends from the lower to upper quartile. Each boxplot represents the results of n  = 5 independent experiments. The paired t-test P-value between the AUC of HeteroTCR and that of any other model is < 0.00001, indicating that HeteroTCR performs significantly better than other methods.

HeteroTCR significantly outperformed other models across all dataset splitting with varying VDJdb confidence scores (paired t-test P-value between the AUC of HeteroTCR and that of any other model was <0.00001) (see Supplementary Tables  3 , 4 for more details). Although generalizability on strict-based data sets remained limited, HeteroTCR’s performance (AUC = 0.6535) surpassed random guessing (AUC = 0.5). These findings indicated HeteroTCR’s ability to learn from existing datasets and extrapolate predictions to unseen epitopes and TCRs.

Additionally, the lower variance in AUC for HeteroTCR compared to other methods was likely attributed to the robustness of the GNN model, which consistently performs well across diverse datasets and demonstrates a more stable predictive performance. This stability is a noteworthy characteristic that enhances the reliability and consistency of HeteroTCR’s predictive capabilities.

Comparisons to the state-of-the-art models

ImRex 23 and TITAN 7 are two state-of-the-art (SOTA) models for predicting unseen epitopes and TCRs on independent datasets. ImRex relies on an interaction map, which combines the physicochemical properties of both interactors on the amino-acid level, to predict peptide-TCR recognition under different dataset settings. TITAN exploits convolutions with an interpretable attention mechanism to aggregate local information and integrates the modalities, from which binding probabilities are predicted. To compare HeteroTCR directly, we trained and tested it under their respective training and testing data.

In ImRex (Table  1 ), the VDJdb dataset was used for training with 5-fold cross-validation, and the McPAS-TCR dataset served as an independent testing set, which was split into two subsets: one containing peptides already present in the training set (shared-epitope data), and the other containing peptides not seen during ImRex training (unseen-epitope data, which was referred as the unique-epitope data by the authors of ImRex 23 ). Negative interaction pairs were generated using a shuffling approach for each subset. We observed that HeteroTCR consistently outperformed ImRex on both independent testing sets.

For comparison with TITAN (Table  2 ), two different data splitting methods were utilized to evaluate model generalizability using 10-fold cross-validation. The TCR split ensured each TCR appeared in only one fold, avoiding overlap between validation and training datasets. The strict split extended this approach to both TCRs and peptides, ensuring that validation data were unseen during training. To ensure the separation of TCRs and peptides in their folds, negative data were generated by shuffling within each fold.

We found that HeteroTCR, utilizing only CDR3 sequences of TCRs and AA sequences of peptides, outperformed TITAN’s best model, which was pretrained on BindingDB 33 and fine-tuned on training data with full TCR sequences and SMILES encodings 34 of peptides, featuring a frozen epitope input channel with augmentations to enrich the data. HeteroTCR exhibited improved performance on unseen epitopes and TCRs with the strict data-splitting method.

The Heterogeneous GNN module is essential for the improved performance of HeteroTCR

To emphasize the advantages of HeteroTCR, we conducted ablation studies by establishing a baseline model. In computational terms, ablation studies refer to the systematic removal of components from the computational model to assess its impact on overall performance, rather than modifying predicted residues of peptides or TCRs. In our baseline model, we excluded the Heterogeneous GNN module, enabling us to assess the module’s contribution to enhancing both between-type (peptide-TCR) interaction and within-type (TCR-TCR or peptide-peptide) similarity. Models were trained on IEDB data using four types of data splitting methods, with McPAS-TCR as the validation dataset. Further evaluation took place independently on VDJdb with a confidence score greater than or equal to 0.

The AUC of HeteroTCR markly surpassed that of the baseline model (Fig.  4 ), indicating that the information on between-type interaction and within-type similarity learned by Heterogeneous GNN greatly enhanced the model’s performance (further details can be found in Supplementary Tables  5 and 6 ).

figure 4

All boxplots: The center line of each boxplot marks the sample median, the colored points scattered along each boxplot represent all the actual data points, and the box extends from the lower to upper quartile. Each boxplot represents the results of n  = 5 independent experiments. T-test P-value between HeteroTCR and the baseline model is <0.001.

Furthermore, we investigated the relationship between model performance and the parameter K (Supplementary Table  7 ). We considered K values ranging from 1 to 6. Our findings indicate a non-linear relationship between K and model performance, characterized by an initial improvement followed by a subsequent decline. Notably, the model achieves the highest AUC at K  = 4. However, considering the associated increase in training depth and computational cost at higher K values, we recommend adopting K  = 3 as it provides a balanced trade-off between time complexity and performance.

An inherent challenge in generic peptide-TCR models is their potential to memorize TCR motifs regardless of the peptide partner, leading to an inability to capture the true underlying molecular forces governing the binding process and merely managing to memorize spurious motifs present within the TCR training data 23 . To assess whether HeteroTCR captures essential binding features or memorizes spurious TCR motifs, we visualized model-extracted representations of peptide-TCR pairs in a 2-dimensional space using t-distributed stochastic neighbor embedding (t-SNE) 35 (Fig.  5a, b ). Due to the large number of peptide-TCR pairs in VDJdb with a confidence score greater than or equal to 0, we selected representative visuals from VDJdb with a confidence score greater than or equal to 1. Notably, using numeric representations extracted by the full model, cognate TCRs for a specific antigen clustered more tightly compared to a model without the Heterogeneous GNN module. Additionally, with the Heterogeneous GNN module, we observed a better separation between the orange dots (representing positive samples, indicating peptide-TCR interactions) and the purple dots (indicating negative samples, denoting no peptide-TCR interactions).

figure 5

a , b The t-SNE plot for the peptide-TCR pairs of the given peptide with and without a Heterogeneous GNN module. Each point represents a pair of peptide-TCR and is colored purple or orange, where purple represents no interaction and orange represents an interaction. Without Heterogeneous GNN module, the colored dots are scattered, and no clear boundary can be observed between purple and orange dots. In contrast, with Heterogeneous GNN, the colored dots are more clearly clustered and classified. c The degree of aggregation of the colored points (peptides with ≥15 cognate TCRs) is assessed by computing the center of the point set and the average distance of each point to the center. All boxplots: The center line of each boxplot marks the sample median, the colored points scattered along each boxplot represent all the actual data points, and the box extends from the lower to upper quartile. Each boxplot represents the results of n  = 5 independent experiments. d The per-peptide (with ≥15 cognate TCRs) AUC for the models with and without Heterogeneous GNN module, which are trained five times on IEDB and tested on VDJdb with confidence score greater than or equal to 1, based on the pair-based setting. All boxplots: The center line of each boxplot marks the sample median, the colored points scattered along each boxplot represent all the actual data points, and the box extends from the lower to upper quartile. Each boxplot represents the results of n  = 5 independent experiments.

To measure the degree of aggregation of positive and negative samples for the same antigen, we calculated the center of the point set and the average distance of each point to the center (Fig.  5c ). We found that the degree of aggregation of the samples with the Heterogeneous GNN module, is smaller than that without the Heterogeneous GNN module, indicating that cognate TCRs for a specific antigen cluster more tightly (paired t-test P-value between the models with and without Heterogeneous GNN module is \(3.9\times {10}^{-10}\) ) (additional details in Supplementary Table  8 ). Figure  5d shows the AUC in per-peptide classification of positive and negative samples (≥15 cognate TCRs), demonstrating the Heterogeneous GNN module’s reasonable performance (paired t-test P-value between the models with and without Heterogeneous GNN module is \(1.2\times {10}^{-7}\) ) (additional details in Supplementary Table  9 ). This observation reveals that the information on between-type (peptide-TCR) interactions and within-type (TCR-TCR or peptide-peptide) similarity learned by HeteroTCR prevents reliance on TCR motif patterns for interaction prediction.

TCRs recognizing the same antigen can exhibit diverse amino acid sequences. To investigate the impact of TCR sequence diversity on HeteroTCR, we evaluated model performance using TCRs with decreasing similarity within the training data (Fig.  6a ), and trained with the full data but tested using TCRs with decreasing similarity to the training data (Fig.  6b ). TCR similarity was measured using the mean Levenshtein ratio for each TCR to all TCRs in the training data (Methods). A higher mean Levenshtein ratio suggests a TCR sequence is closer to the TCRs in the training data. AUC analysis was conducted for TCRs below specified Levenshtein ratio cut-offs (Methods). AUC scores are shown for model trained (Fig.  6a ) or tested (Fig.  6b ) with TCRs above each cut-off. The outcomes reveal HeteroTCR’s relative robust as TCR similarity levels decrease. With diminishing TCR similarity within the training set, HeteroTCR’s performance slightly drops (Fig.  6a ), indicating a subtle influence of TCR motifs on the model. As the similarity between testing and the training TCRs (Fig.  6b ) wanes, AUC scores remain steady, indicating HeteroTCR’s capability to generalize across diverse TCR motifs.

figure 6

a Performance of HeteroTCR with decreasing similarity of TCRs within the training data. The abscissa represents the TCR Levenshtein ratio, with higher values indicating greater similarity. The blue bars represent AUC scores (left ordinate), and the red line indicates the number of training data points (right ordinate) for each cut-off. The cut-offs used are 0.30, 0.29, 0.28, 0.27, 0.26, 0.25, and 0.24. b Performance of HeteroTCR with decreasing similarity of TCRs between the training and testing data. The abscissa represents the TCR Levenshtein ratio, where higher values indicate that testing TCRs are more similar to the training TCRs. The blue bars represent AUC scores (left ordinate), and the red line indicates the number of testing data points (right ordinate) for each cut-off. The cut-offs used are 0.30, 0.29, 0.28, 0.27, 0.26, 0.25, and 0.24, corresponding to thresholds where similar TCR data points are progressively removed from the testing dataset.

Additionally, we investigated the impact of the number of peptides on model performance (Fig.  7 ). In this analysis, we trained models on the IEDB dataset using strict-based datasets and utilized McPAS-TCR as the validation dataset for parameter selection. The evaluation was performed on VDJdb with a confidence score greater than or equal to 0. In the experiments, the initial training dataset consisted of 559 unique peptides. We randomly subsampled them to reduce the quantity to 100, 200, 300, 400, and 500. Our observations revealed that the model performance continued to improve with increased input data, suggesting that HeteroTCR benefited from the inclusion of more peptides (additional details in Supplementary Table  10 ). In summary, our experiments demonstrate that increasing the number of peptides in the training dataset consistently improves the performance of the model, underscoring the importance of diverse antigenic exposures in enhancing predictive accuracy.

figure 7

The shaded area depicts the mean ± the standard deviation over five repeated experiments. The blue points represent the mean data points, while the grey points ( n  = 5) scattered around each mean point represent all the actual data points. In the validation dataset and testing dataset, the number of pairs for different numbers of subsampled peptides is 784 and 1208, respectively. For different numbers of subsampled peptides (ranging from 100 to 559), the corresponding numbers of pairs in the training dataset are 7180, 22440, 40932, 52636, 69816, 76348, respectively.

HeteroTCR binding predictions correlate with experimentally derived pMHC-T cell binding fractions

To assess the performance of the model from an alternative angle, we investigated whether the predictions of the HeteroTCR model correlate with experimentally derived pMHC-T cell binding fractions (Methods). The data utilized in our study was generated from the 10x Genomics Chromium Single Cell Immune Profiling platform 36 (Methods). We analyzed single-cell datasets containing profiles of CD8 + T cells specific to 44 different pMHC multimers, sourced from four healthy donors. The binding specificity between each T-cell and tested pMHC was quantified by counting the number of unique molecular identifier (UMI) sequences associated with each specific pMHC in the T-cell. After data curation and the computation of binding fractions (Methods), we proceeded to calculate the Spearman correlation coefficient between HeteroTCR-predicted binding probabilities and binding fractions, i.e. the fraction of a T cell clone bound to a specific TCR.

Figure  8a–d illustrates the correlation between the predicted binding probabilities of HeteroTCR and binding fractions. Notably, the dataset includes cases where the binding fraction is 0, illustrating an absence of binding specificity. A positive correlation observed in the figures indicates the association between the model’s predicted probabilities and the binding events. In Fig.  8e–h , the correlation between the two variables in data excluding instances where the binding fraction equals 0. Consequently, the figures exclusively comprise data with binding specificity, and a positive correlation depicts that the predicted binding probabilities of HeteroTCR are associated with binding strength in instances where binding occurs.

figure 8

a – d Binding predictions for donors 1–4 correlate with binding fractions, where binding fractions include cases equal to 0. The number of data for each donor is 29495, 62956, 41496, and 13430, respectively. e – h Binding predictions for donors 1–4 correlate with binding fractions, where binding fractions exclude cases equal to 0. The number of data for each donor is 9909, 24156, 13114, and 4792, respectively.

In this study, we introduce HeteroTCR, a Heterogeneous Graph Neural Network method leveraging the sequence information form the peptide and CDR3 β region of TCR for predicting peptide-TCR binding probability. HeteroTCR is designed to extract both information on between-type (peptide-TCR) interaction and within-type (TCR-TCR or peptide-peptide) similarity from input sequence. As far as we know, HeteroTCR is the first model to apply graph neural networks to peptide-TCR interaction prediction tasks. Our evaluation demonstrated that HeteroTCR outperforms existing methods in predicting peptide-TCR binding probabilities, particularly across various independent testing datasets and dataset settings. Although the generalization capability of HeteroTCR on strict-based data sets is limited, its overall performance remains robust. These results highlight HeteroTCR’s enhanced reliability and robustness in predicting immunogenic peptides recognized by TCR. The HeteroTCR program, trained model parameters, machine learning platform and hardware (Methods), and datasets are available on GitHub ( https://github.com/yuzilan/HeteroTCR ).

To further analyze the superiority of HeteroTCR design, we establish a baseline model for ablation studies. Our results show that the information on between-type interaction and within-type similarity learned by the Heterogeneous GNN module enhances model performance considerably. Utilizing t-SNE visualization on numeric vectors processed with and without the Heterogeneous GNN module, we uncover HeteroTCR’s ability to capture fundamental molecular binding forces rather than memorizing TCR pseudo motifs. Additionally, we investigate the impact of TCR pattern similarity on HeteroTCR by varying Levenshtein ratio cut-offs on training and testing sets, illustrating that HeteroTCR’s strong performance is partly contributed by within-type similarity.

Moreover, we investigated the correlation between HeteroTCR’s binding predictions and experimentally derived pMHC-T cell binding fractions using publicly available 10x Genomics data from single cell droplet sequences in the presence of DNA barcoded pMHC dextramers. While the model is promising in aiding the identification of immunogenic epitopes, a wide range of binding strengths were observed even among TCR with the highest HeteroTCR GNN-inferred interaction scores. This underscores that HeteroTCR may have the utility to prioritize candidate TCRs of clinical relevance, but binding prediction will require experimental validation.

Heterogeneous GNN, integrating diverse node and edge information 37 , opens promising avenues for future research to expand on HeteroTCR. Additional information, including the alpha chains of TCRs, V / J genes, other CDR loops, and the MHC contexts, can be incorporated into the graph as distinct node types with different vector dimensions and embedding methods. Edges can also have weights attached to represent different degrees of importance and the proportion of message transmission. Notably, HeteroTCR has important implications for designing TCR-T or neoantigen vaccine therapies, and offers a modeling framework for various molecular interactions like protein-protein or antibody-antigen interactions.

Here, we collected data from three well-known databases: IEDB 30 , VDJdb 31 , and McPAS-TCR 32 . The Immune Epitope Database (IEDB) is a freely available resource that contains a comprehensive collection of antibody and T cell epitopes in humans, non-human primates, and other animal species in the context of infectious disease, allergy, autoimmunity, and transplantation. The IEDB dataset was downloaded from http://www.iedb.org on November 17, 2021. The VDJdb is a curated database of TCR sequences with known antigen specificities and confidence scores to highlight the most reliable records. The McPAS-TCR is a manually curated catalog that links TCR sequences to their antigen targets or to the pathology and organ with which they are associated. The VDJdb and McPAS-TCR were collected from https://vdjdb.cdr3.net and http://friedmanlab.weizmann.ac.il/McPAS-TCR/ , both on June 12, 2021. To construct reliable datasets, we took five preprocessing steps on the raw data.

Step 1. filtering data: (i) The data used in this study were restricted to TCR β chain sequences due to the scarcity of paired chain data. (ii) Peptide-CDR3 β pairs were adopted because the complementarity-determining region 3 (CDR3) is the key determinant of specificity in antigen recognition. (iii) Given that peptide with lengths ranging from 8 to 15 amino acids (AAs) and CDR3 β sequences containing 10–20 AAs account for the majority of datasets 22 , we focused on the CDR3 β sequences with 10–20 AAs and peptides with 8–15 AAs presented by human MHC class I molecules, as they are likely to represent the most common and biologically relevant cases.

Step 2. removing invalid sequences: Since the majority of CDR3 β chains begin with the amino acid ‘C’ and end with ‘F’ 38 , we excluded peptide-CDR3 β pairs whose CDR3 β sequences do not adhere to this pattern. Furthermore, we eliminated peptide-CDR3 β sequences containing lowercase letters or letters that are not amino acids as these likely result from errors during database collection and collation.

Step 3. removing redundant sequence pairs: Duplicate sequence pairs may exist in the databases due to the omission of information such as the CDR1 and CDR2 regions and HLA molecules. To address this, we removed redundant data within each database by retaining only unique peptide-CDR3 β sequence pairs.

Step 4. screening antigen-specific TCR CDR3s: iSMART 14 is a computational method that performs a specially parameterized pairwise local alignment on TCR CDR3 sequences to group them into antigen-specific clusters with high efficiency. Since TCRs usually share conserved sequence features when recognizing the same pMHC epitope 22 , we employed iSMART as a filter to identify and cluster antigen-specific TCR CDR3s, removing the CDR3s that are not selected in the cluster.

Step 5. constructing datasets: The peptide-CDR3 pairs processed in Steps 1–4 can be added to the set of interacting pairs of CDR3s and peptides. However, to train supervised models, both positive and negative examples, which are pairs of CDR3s and peptides that do not recognize each other, are required. To generate negative data, each CDR3 sequence in the positive dataset was paired with a peptide that has not been shown to interact with the corresponding CDR3 by shuffling or mismatching the sequences. The enormous diversity of potential TCR and peptide sequences makes it unlikely for a randomly selected TCR to bind to a specific peptide, thereby making the shuffling method feasible 39 . In this study, the shuffling method was used instead of sampling uniformly from healthy CDR3 reference repertoire sets, as using an external negative reference dataset may introduce an inherent bias that may result in over-optimistic performance 23 .

In the Results 2.2 section, we used IEDB dataset as the training dataset with 5-fold cross-validation to generate five optimal parameter models, and evaluated these five models on an independent testing dataset (VDJdb) to get five AUC metrics.

In the Results 2.3 section, we trained the model on the data from IEDB dataset, took McPAS-TCR as the validation dataset to select the optimal parameter model, and evaluated the model on VDJdb. We repeated this process five times, so we got five optimal parameter models each time and five AUC metrics on the independent testing dataset.

In the Results 2.4 section, to avoid being accused of manipulating data using complex filtering steps, we directly used the data of SOTA models to train and evaluate HeteroTCR and compared the results in their papers.

Four types of data splitting methods

In order to further refine our experiment and show the performance of HeteroTCR, we redefined the data splitting methods into four types:

Pair-based data sets/seen epitopes and seen TCRs.

TCR-based data sets/unseen TCRs.

Antigen-based data sets/unseen epitopes.

Strict-based data sets/unseen epitopes and unseen TCRs.

For the pair-based data sets, it simply guarantees that the interacting peptide-TCR pairs in the testing dataset are not contained in the training dataset. It is worth noting that the peptide or TCR alone in the training dataset may exist in the testing dataset. This division method has been widely adopted by most models.

For the TCR-based data sets, if a TCR appears in the training dataset, it cannot be present in the testing dataset.

For the antigen-based data sets, the peptide that appears in the training dataset will no longer appear in the testing dataset.

For the strict-based data sets, it is necessary to ensure that both the TCR and the peptide in the training dataset do not appear in the testing dataset. This approach differs from the pair-based data sets, where the model’s generalization ability is evaluated by predicting whether TCRs or peptides that are not in the database can stably bind.

In addition to the above four types of data splitting methods, our model utilized cross-validation across different datasets to demonstrate that our model learns essential information about TCRs and peptides, rather than simply memorizing the specifics of a single database. More details regarding the datasets can be found in the results section.

Embedding of TCRs and peptides

Firstly, the TCR sequences were zero-padded to a maximum length of 20, while the peptide sequences to a maximum length of 15. We then encoded AA sequences of TCRs and peptides using the BLOSUM50 matrix, in which each AA is represented as the score for substituting the AA with all the 20 amino AAs. Hence, the BLOSUM encoding scheme maps a sequence with a length of l to an array with a size of l × 20. Secondly, the TCR sequences and the peptide sequences were separately deconvoluted by different convolution kernels with kernel size {1, 3, 5, 7, 9}, in which different features were integrated through different convolution kernels to filter the whole sequence. For each kernel size, the convolutional output was max-pooled and the resulting feature vectors were concatenated in a single vector with 160 entries (80 for each input sequence). Thirdly, the 160-dimensional vector was fed into a dense layer of 32 hidden neurons and the output consists of one single neuron, giving the probability of a peptide-TCR pair to bind (Fig.  1 ). The activation function used through the network is the sigmoid function. The model was trained for 1000 epochs with early stopping and patience of 50 epochs. The weights were updated using the Adam optimizer with a learning rate of 0.001. The batch size is 512 and the loss function is binary cross-entropy. Finally, the 160-dimensional max-pooling layers of the pre-trained CNN module were extracted as numeric embeddings of TCRs and peptides to input the next step.

Heterogeneous GNN module

Firstly, we regard the entire dataset as a global graph. A circle node represents a TCR, while a triangle node represents a peptide (Fig.  1 ). The edge of the solid line (label 1) represents an interaction between a TCR and a peptide, while the dotted line (label 0) represents no interaction, and no connection represents unknown. The difference between solid and dotted lines is only used for the calculation of loss function and backpropagation of the MLP in the training dataset, as well as the evaluating the model in the testing dataset, and it does not participate in any other process to cause data leakage. In other words, when initializing the graph, the nature of the relationship between peptides and TCRs is unknown, and we can only rely on the input training pairs to identify the existence of a relationship. We get the weight matrix by iterating 1000 epochs of training to learn and adjust the nature of the relationship between the given TCR and peptide, whether it is a solid line or a dotted line.

Secondly, the idea behind Heterogeneous GNN can be broadly divided into two steps. We use a set of aggregator functions to learn aggregate feature information from K th -order neighborhood of a node, with a default K value of 3. The activation function used through Heterogeneous GNN is the Leaky ReLUs function. The input of Heterogeneous GNN is an 80-dimensional numeric embedding of TCR and an 80-dimensional numeric embedding of peptide, and the output is a 1024-dimensional TCR vector and a 1024-dimensional peptide vector.

Let \(T=\{{t}_{1},{t}_{2},\ldots ,{t}_{m}\}(\left|T\right|=m)\) and \(P=\{{p}_{1},{p}_{2},\ldots ,{p}_{n}\}(\left|P\right|=n)\) denote the set of TCRs and peptides, respectively. Let \({I}^{+}=\{{y}_{t,p}{|t}\in T,{p}\in P\}\) denote the positive interactions, where \({y}_{t,p}\) indicates that TCR t has interacted with peptide p . Let \({I}^{-}=\{{y}_{t,p}{|t}\in T,{p}\in P\}\) denote the negative interactions, where \({y}_{t,p}\) indicates that TCR t has no interacted with peptide p . Moreover, a peptide-TCR interaction graph is constructed, denoted as \({{{{{\mathscr{G}}}}}}({{{{{\mathscr{V}}}}}}{{{{{\mathscr{,}}}}}}{{{{{\mathscr{E}}}}}})\) , where \({{{{{\mathscr{V}}}}}}{{{{{\mathscr{=}}}}}}T\cup P\) is the set of nodes and \({{{{{\mathscr{E}}}}}}=\{(t,p)|{y}_{t,p}\in I,t\in T,p\in P,I={I}^{+}\cup {I}^{-}\}\) is the edge set.

More formally, for the Graph \({{{{{\mathscr{G}}}}}}({{{{{\mathscr{V}}}}}}{{{{{\mathscr{,}}}}}}{{{{{\mathscr{E}}}}}})\) , the input features of TCRs or peptides are \(\left\{{x}_{v},\,\forall v\in {{{{{\mathscr{V}}}}}}\right\}\) , and the edges \(\left\{\forall e{{{{{\mathscr{\in }}}}}}{{{{{\mathscr{E}}}}}}\right\}\) represent the relationships between TCRs and peptides, whether positive or negative. The TCR or peptide embedding \({z}_{v}\) generation algorithm is described as follows:

where \({h}_{v}^{k}\) is the representation of node v at k-th order. \({h}_{v}^{0}\) is initialized by the input features of TCRs or peptides. \({{{{{\mathscr{N}}}}}}(v)\) denotes the neighbors of node v . \({z}_{v}\) is final representations of node v . \({W}_{k}\) and \({B}_{k}\) are trainable weight matrices, the depth K is 3 by default and the non-linearity σ is Leaky ReLU. The differentiable aggregator functions AGGREGATE is the average of neighbor’s previous layer embeddings. \({h}_{{{{{{\mathscr{N}}}}}}(v)}^{k}\) is trivially computed by \({h}_{{{{{{\mathscr{N}}}}}}(v)}^{k}={\sum}_{u\in {{{{{\mathscr{N}}}}}}(v)}\frac{{h}_{u}^{k-1}}{\left|{{{{{\mathscr{N}}}}}}(v)\right|}\) with |·| donating the number of the node v neighbors. To generate the representation of node v , it first aggregates the representations of \({{{{{\mathscr{N}}}}}}(v)\) and then updates the representation of v by concatenating its representation at (k-1)-th order and the aggregated representations.

MLP classifier

A linear layer transforms one vector into another vector, which refers to a single-layer neural network without hidden layers. The MLP is a stack of linear layers with hidden layers. For classification tasks, HeteroTCR uses an MLP with two hidden layers (Fig.  1 ). The two numerical vectors (2048 dimension) from the Heterogeneous GNN module are concatenated into a single layer with 512 neurons activated by ReLU, followed by a dense layer with 256 neurons also activated by ReLU, and the last layer with a single neuron with sigmoid activation. Mathematically, the output is a continuous variable between 0 and 1, representing the predicted binding strength between TCR and peptide. HeteroTCR was trained for 1000 epochs with 512 batch sizes using the Adam optimizer with a learning rate of 0.0001 and a weight decay of 0 by default. The loss function is binary cross-entropy.

Levenshtein similarity ratio

Here, the Levenshtein ratio was used as a measure of the similarity between TCR sequences. The Levenshtein ratio is based on the ldist , which is not Levenshtein distance, but the sum of the costs. Given two strings, the ldist describes the count of modifications needed to transform one word into another. The possible changes are deletion, insertion, and replacement. The count of deletion and insertion is 1, while the count of replacement is 2. The Levenshtein ratio is given by the formula

where u and v represent two TCR sequences, and |∙| defines their length.

We defined the Levenshtein similarity ratio of a TCR as the average of the Levenshtein ratio from the TCR to each TCR in the training set, and then removed the data exceeding the specified ratio cut-offs. In Fig.  6a , we calculated the average of each TCR’s Levenshtein ratio in the training set to all of the training TCRs and thus reduced TCR similarity within the training set. In Fig.  6b we calculated the average of each TCR’s Levenshtein ratio in the testing set to all of the training TCRs and thus reduced the TCR similarity between the testing set and the training set.

Data curation of the 10× Genomics platform and calculation of binding fractions

The data utilized in our study was generated from the 10× Genomics Chromium Single Cell Immune Profiling platform ( https://www.10xgenomics.com/resources/datasets ). The raw data is accessible for download at https://zenodo.org/records/6952657 . Closely examining four single-cell datasets, we analyzed profiles of CD8 + T cells specific to a highly multiplexed panel consisting of 44 different pMHC multimers, alongside 6 negative control pMHC multimers, sourced from four healthy donors. The binding specificity between T cells and each pMHC complex was quantified using the UMI counts as a binding indicator.

In general, a T cell typically expresses only one pair of functional TCRs, and thus, we selectively retain clones of T cells expressing a single pair of TCR α and TCR β chains. We focused only on expanded clones, as the UMI counts of each cell were inherently noisy due to dropouts and high variances in single-cell experiments. Consequently, we opted to utilize the binding fraction of each T cell clone with the same TCR as a measure of antigen affinity to a TCR. The binding fraction of a clone is determined by the following formula:

where m represents the number of T cells within the clone exhibiting a higher UMI count for a given antigen compared to the maximum UMI count among the 6 negative control antigens, and n denotes the clone size, i.e., the number of T cells with identical TCRs. It is noteworthy that instances where m equals 0 are only considered in the following two scenarios: when the UMI count of T cells within the clone for a given antigen is less than the maximum UMI count among the 6 negative control antigens, or when the UMI count of T cells within the clone for a given antigen equals the maximum UMI count among the 6 negative control antigens, and both are non-zero. Specifically, we do not consider cases where m equals 0 when the UMI count of T cells within the clone for a given antigen is 0, and the maximum UMI count among the 6 negative control antigens is also 0. This omission is due to the sparse nature of the original data, characterized by numerous zero values, leading to excessive noise and inclusion of many meaningless data points. Finally, we calculated the Spearman correlation coefficient between predicted binding probabilities and binding fractions.

Machine learning platform and hardware

The pre-trained CNN module was implemented with Keras 2.6.0 ( https://keras.io ) using the Tensorflow backend and Python 3.7.0. The Heterogeneous GNN module and the MLP classifier were implemented with PyTorch 1.9.1 ( https://pytorch.org ) using Python 3.7.11. PyG (PyTorch Geometric) 37 , a library built upon PyTorch to easily write and train GNNs, was employed for modeling and processing graph-structured data. The metric evaluation was implemented with TorchMetrics package 40 . All deep learning models were trained on a single NVIDIA A40 graphics card.

Reporting summary

Further information on research design is available in the  Nature Portfolio Reporting Summary linked to this article.

Data availability

All additional data that support the conclusions from this manuscript are available in the Supplementary Information. Source data and codes for the main figures, as well as all data utilized for HeteroTCR training and evaluation, are shared on the github repository ( https://github.com/yuzilan/HeteroTCR ) and its Zenodo ( https://doi.org/10.5281/zenodo.11120879 ). The dataset used for training and testing were collected from IEDB , VDJdb ( https://vdjdb.cdr3.net ) and McPAS-TCR ( http://friedmanlab.weizmann.ac.il/McPAS-TCR/ ). The detailed information of the 10x Genomics cohort is available at https://www.10xgenomics.com/resources/datasets . The raw data for single-cell datasets is available at https://zenodo.org/records/6952657 .

Code availability

HeteroTCR is available on github ( https://github.com/yuzilan/HeteroTCR ) and its Zenodo ( https://doi.org/10.5281/zenodo.11120879 ), together with a usage documentation and several example testing datasets.

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Acknowledgements

This work was funded by grants from the Tsinghua University Independent Research Project (ID: 52302102423), the Tsinghua University Spring Breeze Fund, and the Beijing Institute of Technology’s Proof of Concept Project for Tumor Neoantigen Personalized TCR-T Therapy.

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These authors contributed equally: Zilan Yu, Mengnan Jiang.

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School of Medicine, Tsinghua University, 100084, Beijing, China

Zilan Yu, Mengnan Jiang & Xun Lan

Centre for Life Sciences, Tsinghua University, 100084, Beijing, China

Zilan Yu & Xun Lan

Tsinghua-Peking Center for Life Sciences, MOE Key Laboratory of Tsinghua University, Beijing, China

MOE Key Laboratory of Bioinformatics, Tsinghua University, 100084, Beijing, China

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Zilan Yu, Mengnan Jiang, and Xun Lan designed the framework of this study. Zilan Yu devised the model architecture and implemented the code. Mengnan Jiang contributed biological insights, created figures, and acquired and preprocessed data for analysis. Zilan Yu wrote the paper with the help of other authors. Xun Lan supervised the study. All authors read and approved the final paper.

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Correspondence to Xun Lan .

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Yu, Z., Jiang, M. & Lan, X. HeteroTCR: A heterogeneous graph neural network-based method for predicting peptide-TCR interaction. Commun Biol 7 , 684 (2024). https://doi.org/10.1038/s42003-024-06380-6

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What Happens to Your Body When You Take NAD

Getty Images / PhotoAlto/Laurence Mouton

Understanding NAD

  • Potential Effects

Current Research

Dosage and delivery methods, natural ways to boost nad.

It's a typical weekday morning. Before the hustle and bustle of the day begins, you sip your collagen coffee while scrolling your social feeds. One after the other, almost every swipe is a post about the next best anti-aging elixir, compound, exercise, or food. Then, NAD pops up—a supplement touted by the likes of Hailey Bieber and Kendall Jenner as an anti-aging goldmine worthy of using for a lifetime. But is it really, though?

NAD—short for nicotinamide adenine dinucleotide—is a cellular coenzyme responsible for turning the food you eat into usable energy. It also plays a major role in DNA repair, cellular processes, and immune system function. Even though the body produces NAD naturally, its production of NAD declines as you age.

That means the older you get, the harder it is for your body to keep you healthy and process nutrients into energy. That's why you feel more sluggish, and your risk of illness or injury increases. It's also why people became interested in NAD supplementation.

The idea is that supplementing with NAD could equate to more energy, better health, and maybe even a longer, healthier life. But before you hit the submit button on your virtual shopping cart, let's uncover what happens to your body when you take NAD supplements.

NAD acts as a coenzyme within the body to help several metabolic processes happen. Essentially, coenzymes assist other molecules in whatever job they're designed to do. Without this metabolic pathway, you would not function.

Then there's DNA, which is easily damaged via life stressors, UV radiation, and alkylating and oxidative molecules. Fortunately, your body is quite good at maintaining DNA integrity by sending in NAD to get the job done. Without getting into the complex nature of DNA repair pathways, NAD calls in molecules to quickly nourish and rejuvenate the damaged DNA cells.

As mentioned earlier, NAD acts as an assistant or middleman, so to speak, making things happen and maximizing communication between cells throughout the body. Without enough NAD, simple processes in the body may take longer, partly because of poor communication. This might look like a compromised immune system (AKA you get sick more often), poor stress response, and an increased risk for obesity, type 2 diabetes , metabolic syndrome, cancer, and other degenerative diseases.

Potential Effects of NAD Supplementation

With the inevitable decline in NAD as you age, your energy production and cellular repair also decline. This reduction leads to energy loss, mental fatigue, wrinkles, and frequent sickness. Though there's no way to stop this from happening, you can fill in the gaps with NAD supplementation.

Boosted Cellular Energy

When you eat food , it's broken down into molecules to produce energy in the form of ATP. During this process, NAD acts as an enzyme facilitating the conversion of NADH to ATP. Without enough NAD, the body has a difficult time producing ATP in a timely manner, which can lead to feeling sluggish and tired. Supplementing with NAD could increase cellular energy production by boosting ATP levels, potentially improving metabolism and reducing fatigue.

Research on people with chronic fatigue syndrome evaluated the safety and effectiveness of NAD supplementation on fatigue, quality of life, and sleep quality. The overall consensus is that supplementing with NAD has a positive effect on quality of life and improves health parameters like anxiety, energy, and immune system response.

Enhanced Cellular Repair

Because NAD is involved in mechanisms of DNA repair that help protect cells from damage caused by stress, oxidative damage, and aging, higher levels (as a result of supplementation) may promote a more efficient process.

For instance, one study investigated the role of NAD supplementation on DNA repair. Researchers found that supplementation decreased the accumulation of endogenous DNA damage and improved DNA repair capacity. Ultimately, supplementing with NAD may positively impact DNA repair in the context of aging and in neurodegenerative diseases like Alzheimer's disease.

Improved Cellular Communication

Cellular communication is essential for overall health, well-being, and longevity. If cells cannot get messages to each other promptly, aging will advance.

Because NAD acts like an assistant, its job is to ensure that things happen quickly and efficiently. Restoring NAD to youthful levels means your body's checking things off its daily to-do list fast. In doing so, it restores cell-signaling function and reduces stress.

Potential Negative Side Effects

If all of this sounds too good to be true, it might be because some studies report mild side effects of using NAD supplements, especially in high doses. Excess of 500 milligrams of nicotinamide isn't recommended. Research shows that excess supplementation increases the risk of vascular disease, neurodegenerative disease, and chronic kidney disease. Other symptoms, including diarrhea, easy bruising, and increased wound bleeding, may be observed. Excess of 3,000 milligrams may cause nausea, vomiting, and liver damage.

It's important to consult with a healthcare provider before taking NAD, especially if you have a preexisting condition or are taking medication.

While there is existing research on NAD supplementation in humans, it is not complete or conclusive. There is always room for ongoing studies and findings.

That said, there are some promising benefits to using a NAD supplement for conditions like age-related cognitive decline, neurodegenerative diseases (Alzheimer's, Parkinson's), and cardiovascular health. Here's what research has shown so far.

  • Age-related cognitive decline: Research on using NAD supplements for cognitive decline is promising. However, the majority of the findings were in animal studies. Despite this, a recent review found that NAD supplementation may preserve cognitive health across a variety of age-related diseases, including Alzheimer's disease and dementia.
  • Neurodegenerative diseases: Healthy brain aging is extremely important especially in the prevention of neurodegenerative diseases like Alzheimer's, Parkinson's, and Huntington diseases. During normal brain aging, NAD levels decline. Over time this leads to cellular damage, improper cellular waste disposal, impaired stress response, DNA repair delay, stem cell exhaustion, and inflammation. Supplementing with NAD may return NAD to healthy levels and prevent further damage to brain cells. Though promising, more research in humans is needed.
  • Cardiovascular health: Cardiovascular diseases often involve metabolic disorders (abnormal lipid and glucose metabolism), oxidative stress (cell damage), and inflammation (poor cell signaling). These stress markers make it difficult for the heart to function properly and contribute to cardiovascular disease. Even within the heart, NAD homeostasis is important. One review found that boosting levels of NAD with supplements has favorable effects on heart health and may have a profound influence on cardiovascular disease.

The two most common delivery methods for NAD are oral tablets and IV injections. NAD tablets are available online, usually as an NAD precursor nicotinamide riboside (NR) and/or niacin (NADH). These precursor supplements strongly influence positive NAD levels.

NAD+ IV injections are available at local clinics and are generally not covered by insurance. These intravenous injections use a more immediate source of NAD. However, it must be dripped over the course of at least two hours to prevent adverse events like rapid heartbeat, nausea, and anxiousness.

The best dosage and delivery method of NAD is still under investigation. However, most reported side effects occur only in doses higher than those used clinically or for those used for daily supplementation. Therefore, take the dosage recommended by a healthcare provider and avoid exceeding this to prevent side effects and potential health risks.

If you're not ready to take the NAD supplement leap, there are ways to boost NAD naturally with simple lifestyle changes. Here's what you need to know.

  • Exercise : Regular aerobic and resistance training exercise has a regenerative effect on NAD levels and can even return them to young adult levels. A 2019 study found that aerobic and resistance training increase NAMPT, the precursor to NAD.
  • Circadian Rhythm & Sleep : Your circadian rhythm (AKA internal 24-hour clock) and sleep patterns directly correlate with NAD+ production. Getting enough sleep is key for maintaining adequate levels—sleep deprivation and disrupted sleep patterns are associated with decreased NAD+ synthesis.
  • Environmental Stress (Heat/Cold) : Cold plunge pools and saunas may help boost NAD. Exposure to extreme hot and cold temperatures may place extra stress on the body, triggering the production of NAD+.
  • Certain foods : A balanced diet can provide all the NAD you need, especially if you include foods rich in vitamin B3 (niacin) and leafy green vegetables. Vitamin B3 (aka niacin) can easily be found in meat, fish, dairy products, avocado, and whole grains and is the precursor to NAD. Niacin converts to NAD for use in cellular metabolism. Tryptophan, a precursor for NAD, is found in turkey, chicken, fish, nuts, and soy. Leafy green vegetables, including broccoli, colored cabbages, and green onions, also contain compounds that positively influence NAD levels.

Bottom Line

While NAD is promising as an anti-aging powerhouse, there are still many unknowns regarding NAD supplementation. Before making any changes to your healthcare routine and supplement lineup, talk to a healthcare provider, such as a registered dietitian to ensure you're making the right choice for you. In the meantime, fueling your body with nourishing foods and living a healthy lifestyle free from tobacco and other stressors is the cornerstone for promoting cellular health and overall well-being.

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Cantó C, Menzies KJ, Auwerx J. NAD(+) Metabolism and the Control of Energy Homeostasis: A Balancing Act between Mitochondria and the Nucleus. Cell Metab . 2015 Jul 7;22(1):31-53. doi:10.1016/j.cmet.2015.05.023

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Poljšak B, Kovač V, Špalj S, Milisav I. The central role of the NAD+ molecule in the development of aging and the prevention of chronic age-related diseases: Strategies for NAD+ modulation. Int J Mol Sci . 2023 Feb 3;24(3):2959. doi:10.3390/ijms24032959

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Wilk A, Hayat F, Cunningham R, Li J, Garavaglia S, Zamani L, Ferraris DM, Sykora P, Andrews J, Clark J, Davis A, Chaloin L, Rizzi M, Migaud M, Sobol RW. Extracellular NAD+ enhances PARP-dependent DNA repair capacity independently of CD73 activity. Sci Rep. 2020 Jan 20;10(1):651. doi:10.1038/s41598-020-57506-9

Poljšak B, Kovač V, Špalj S, Milisav I. The central role of the nad+ molecule in the development of aging and the prevention of chronic age-related diseases: strategies for nad+ modulation .  IJMS . 2023;24(3):2959. doi:10.3390/ijms24032959

National Institutes of Health: Office of Dietary Supplements. Consumer Fact Sheets: Niacin .

Campbell JM. Supplementation with NAD+ and its precursors to prevent cognitive decline across disease contexts. Nutrients. 2022 Aug 7;14(15):3231. doi:10.3390/nu14153231

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Okabe K, Yaku K, Uchida Y, Fukamizu Y, Sato T, Sakurai T, Tobe K, Nakagawa T. Oral Administration of Nicotinamide Mononucleotide Is Safe and Efficiently Increases Blood Nicotinamide Adenine Dinucleotide Levels in Healthy Subjects. Front Nutr. 2022 Apr 11;9:868640. doi:10.3389/fnut.2022.868640

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By Shoshana Pritzker RD, CDN, CSSD, CISSN Shoshana Pritzker RD, CDN is a sports and pediatric dietitian, the owner of Nutrition by Shoshana, and is the author of "Carb Cycling for Weight Loss." Shoshana received her B.S in dietetics and nutrition from Florida International University. She's been writing and creating content in the health, nutrition, and fitness space for over 15 years and is regularly featured in Oxygen Magazine, JennyCraig.com, and more.

  • Open access
  • Published: 27 May 2024

Current status of community resources and priorities for weed genomics research

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  • Richard P. Dale 20 ,
  • Barend Juan Vorster   ORCID: orcid.org/0000-0003-3518-3508 21 ,
  • Bodo Peters 11 ,
  • Jens Lerchl   ORCID: orcid.org/0000-0002-9633-2653 22 ,
  • Patrick J. Tranel   ORCID: orcid.org/0000-0003-0666-4564 23 ,
  • Roland Beffa   ORCID: orcid.org/0000-0003-3109-388X 24 ,
  • Alexandre Fournier-Level   ORCID: orcid.org/0000-0002-6047-7164 25 ,
  • Mithila Jugulam   ORCID: orcid.org/0000-0003-2065-9067 15 ,
  • Kevin Fengler 18 ,
  • Victor Llaca   ORCID: orcid.org/0000-0003-4822-2924 18 ,
  • Eric L. Patterson   ORCID: orcid.org/0000-0001-7111-6287 14 &
  • Todd A. Gaines   ORCID: orcid.org/0000-0003-1485-7665 1  

Genome Biology volume  25 , Article number:  139 ( 2024 ) Cite this article

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Weeds are attractive models for basic and applied research due to their impacts on agricultural systems and capacity to swiftly adapt in response to anthropogenic selection pressures. Currently, a lack of genomic information precludes research to elucidate the genetic basis of rapid adaptation for important traits like herbicide resistance and stress tolerance and the effect of evolutionary mechanisms on wild populations. The International Weed Genomics Consortium is a collaborative group of scientists focused on developing genomic resources to impact research into sustainable, effective weed control methods and to provide insights about stress tolerance and adaptation to assist crop breeding.

Each year globally, agricultural producers and landscape managers spend billions of US dollars [ 1 , 2 ] and countless hours attempting to control weedy plants and reduce their adverse effects. These management methods range from low-tech (e.g., pulling plants from the soil by hand) to extremely high-tech (e.g., computer vision-controlled spraying of herbicides). Regardless of technology level, effective control methods serve as strong selection pressures on weedy plants and often result in rapid evolution of weed populations resistant to such methods [ 3 , 4 , 5 , 6 , 7 ]. Thus, humans and weeds have been locked in an arms race, where humans develop new or improved control methods and weeds adapt and evolve to circumvent such methods.

Applying genomics to weed science offers a unique opportunity to study rapid adaptation, epigenetic responses, and examples of evolutionary rescue of diverse weedy species in the face of widespread and powerful selective pressures. Furthermore, lessons learned from these studies may also help to develop more sustainable control methods and to improve crop breeding efforts in the face of our ever-changing climate. While other research fields have used genetics and genomics to uncover the basis of many biological traits [ 8 , 9 , 10 , 11 ] and to understand how ecological factors affect evolution [ 12 , 13 ], the field of weed science has lagged behind in the development of genomic tools essential for such studies [ 14 ]. As research in human and crop genetics pushes into the era of pangenomics (i.e., multiple chromosome scale genome assemblies for a single species [ 15 , 16 ]), publicly available genomic information is still lacking or severely limited for the majority of weed species. Recent reviews of current weed genomes identified 26 [ 17 ] and 32 weed species with sequenced genomes [ 18 ]—many assembled to a sub-chromosome level.

Here, we summarize the current state of weed genomics, highlighting cases where genomics approaches have successfully provided insights on topics such as population genetic dynamics, genome evolution, and the genetic basis of herbicide resistance, rapid adaptation, and crop dedomestication. These highlighted investigations all relied upon genomic resources that are relatively rare for weedy species. Throughout, we identify additional resources that would advance the field of weed science and enable further progress in weed genomics. We then introduce the International Weed Genomics Consortium (IWGC), an open collaboration among researchers, and describe current efforts to generate these additional resources.

Evolution of weediness: potential research utilizing weed genomics tools

Weeds can evolve from non-weed progenitors through wild colonization, crop de-domestication, or crop-wild hybridization [ 19 ]. Because the time span in which weeds have evolved is necessarily limited by the origins of agriculture, these non-weed relatives often still exist and can be leveraged through population genomic and comparative genomic approaches to identify the adaptive changes that have driven the evolution of weediness. The ability to rapidly adapt, persist, and spread in agroecosystems are defining features of weedy plants, leading many to advocate agricultural weeds as ideal candidates for studying rapid plant adaptation [ 20 , 21 , 22 , 23 ]. The insights gained from applying plant ecological approaches to the study of rapid weed adaptation will move us towards the ultimate goals of mitigating such adaptation and increasing the efficacy of crop breeding and biotechnology [ 14 ].

Biology and ecological genomics of weeds

The impressive community effort to create and maintain resources for Arabidopsis thaliana ecological genomics provides a motivating example for the emerging study of weed genomics [ 24 , 25 , 26 , 27 ]. Arabidopsis thaliana was the first flowering plant species to have its genome fully sequenced [ 28 ] and rapidly became a model organism for plant molecular biology. As weedy genomes become available, collection, maintenance, and resequencing of globally distributed accessions of these species will help to replicate the success found in ecological studies of A. thaliana [ 29 , 30 , 31 , 32 , 33 , 34 , 35 ]. Evaluation of these accessions for traits of interest to produce large phenomics data sets (as in [ 36 , 37 , 38 , 39 , 40 ]) enables genome-wide association studies and population genomics analyses aimed at dissecting the genetic basis of variation in such traits [ 41 ]. Increasingly, these resources (e.g. the 1001 genomes project [ 29 ]) have enabled A. thaliana to be utilized as a model species to explore the eco-evolutionary basis of plant adaptation in a more realistic ecological context. Weedy species should supplement lessons in eco-evolutionary genomics learned from these experiments in A. thaliana .

Untargeted genomic approaches for understanding the evolutionary trajectories of populations and the genetic basis of traits as described above rely on the collection of genotypic information from across the genome of many individuals. While whole-genome resequencing accomplishes this requirement and requires no custom methodology, this approach provides more information than is necessary and is prohibitively expensive in species with large genomes. Development and optimization of genotype-by-sequencing methods for capturing reduced representations of newly sequence genomes like those described by [ 42 , 43 , 44 ] will reduce the cost and computational requirements of genetic mapping and population genetic experiments. Most major weed species do not currently have protocols for stable transformation, a key development in the popularity of A. thaliana as a model organism and a requirement for many functional genomic approaches. Functional validation of genes/variants believed to be responsible for traits of interest in weeds has thus far relied on transiently manipulating endogenous gene expression [ 45 , 46 ] or ectopic expression of a transgene in a model system [ 47 , 48 , 49 ]. While these methods have been successful, few weed species have well-studied viral vectors to adapt for use in virus induced gene silencing. Spray induced gene silencing is another potential option for functional investigation of candidate genes in weeds, but more research is needed to establish reliable delivery and gene knockdown [ 50 ]. Furthermore, traits with complex genetic architecture divergent between the researched and model species may not be amenable to functional genomic approaches using transgenesis techniques in model systems. Developing protocols for reduced representation sequencing, stable transformation, and gene editing/silencing in weeds will allow for more thorough characterization of candidate genetic variants underlying traits of interest.

Beyond rapid adaptation, some weedy species offer an opportunity to better understand co-evolution, like that between plants and pollinators and how their interaction leads to the spread of weedy alleles (Additional File 1 : Table S1). A suite of plant–insect traits has co-evolved to maximize the attraction of the insect pollinator community and the efficiency of pollen deposition between flowers ensuring fruit and seed production in many weeds [ 51 , 52 ]. Genetic mapping experiments have identified genes and genetic variants responsible for many floral traits affecting pollinator interaction including petal color [ 53 , 54 , 55 , 56 ], flower symmetry and size [ 57 , 58 , 59 ], and production of volatile organic compounds [ 60 , 61 , 62 ] and nectar [ 63 , 64 , 65 ]. While these studies reveal candidate genes for selection under co-evolution, herbicide resistance alleles may also have pleiotropic effects on the ecology of weeds [ 66 ], altering plant-pollinator interactions [ 67 ]. Discovery of genes and genetic variants involved in weed-pollinator interaction and their molecular and environmental control may create opportunities for better management of weeds with insect-mediated pollination. For example, if management can disrupt pollinator attraction/interaction with these weeds, the efficiency of reproduction may be reduced.

A more complete understanding of weed ecological genomics will undoubtedly elucidate many unresolved questions regarding the genetic basis of various aspects of weediness. For instance, when comparing populations of a species from agricultural and non-agricultural environments, is there evidence for contemporary evolution of weedy traits selected by agricultural management or were “natural” populations pre-adapted to agroecosystems? Where there is differentiation between weedy and natural populations, which traits are under selection and what is the genetic basis of variation in those traits? When comparing between weedy populations, is there evidence for parallel versus non-parallel evolution of weediness at the phenotypic and genotypic levels? Such studies may uncover fundamental truths about weediness. For example, is there a common phenotypic and/or genotypic basis for aspects of weediness among diverse weed species? The availability of characterized accessions and reference genomes for species of interest are required for such studies but only a few weedy species have these resources developed.

Population genomics

Weed species are certainly fierce competitors, able to outcompete crops and endemic species in their native environment, but they are also remarkable colonizers of perturbed habitats. Weeds achieve this through high fecundity, often producing tens of thousands of seeds per individual plant [ 68 , 69 , 70 ]. These large numbers in terms of demographic population size often combine with outcrossing reproduction to generate high levels of diversity with local effective population sizes in the hundreds of thousands [ 71 , 72 ]. This has two important consequences: weed populations retain standing genetic variation and generate many new mutations, supporting weed success in the face of harsh control. The generation of genomic tools to monitor weed populations at the molecular level is a game-changer to understanding weed dynamics and precisely testing the effect of artificial selection (i.e., management) and other evolutionary mechanisms on the genetic make-up of populations.

Population genomic data, without any environmental or phenotypic information, can be used to scan the genomes of weed and non-weed relatives to identify selective sweeps, pointing at loci supporting weed adaptation on micro- or macro-evolutionary scales. Two recent within-species examples include weedy rice, where population differentiation between weedy and domesticated populations was used to identify the genetic basis of weedy de-domestication [ 73 ], and common waterhemp, where consistent allelic differences among natural and agricultural collections resolved a complex set of agriculturally adaptive alleles [ 74 , 75 ]. A recent comparative population genomic study of weedy barnyardgrass and crop millet species has demonstrated how inter-specific investigations can resolve the signatures of crop and weed evolution [ 76 ] (also see [ 77 ] for a non-weed climate adaptation example). Multiple sequence alignments across numerous species provide complementary insight into adaptive convergence over deeper timescales, even with just one genomic sample per species (e.g., [ 78 , 79 ]). Thus, newly sequenced weed genomes combined with genomes available for closely related crops (outlined by [ 14 , 80 ]) and an effort to identify other non-weed wild relatives will be invaluable in characterizing the genetic architecture of weed adaptation and evolution across diverse species.

Weeds experience high levels of genetic selection, both artificial in response to agricultural practices and particularly herbicides, and natural in response to the environmental conditions they encounter [ 81 , 82 ]. Using genomic analysis to identify loci that are the targets of selection, whether natural or artificial, would point at vulnerabilities that could be leveraged against weeds to develop new and more sustainable management strategies [ 83 ]. This is a key motivation to develop genotype-by-environment association (GEA) and selective sweep scan approaches, which allow researchers to resolve the molecular basis of multi-dimensional adaptation [ 84 , 85 ]. GEA approaches, in particular, have been widely used on landscape-wide resequencing collections to determine the genetic basis of climate adaptation (e.g., [ 27 , 86 , 87 ]), but have yet to be fully exploited to diagnose the genetic basis of the various aspects of weediness [ 88 ]. Armed with data on environmental dimensions of agricultural settings, such as focal crop, soil quality, herbicide use, and climate, GEA approaches can help disentangle how discrete farming practices have influenced the evolution of weediness and resolve broader patterns of local adaptation across a weed’s range. Although non-weedy relatives are not technically required for GEA analyses, inclusion of environmental and genomic data from weed progenitors can further distinguish genetic variants underpinning weed origins from those involved in local adaptation.

New weeds emerge frequently [ 89 ], either through hybridization between species as documented for sea beet ( Beta vulgaris ssp. maritima) hybridizing with crop beet to produce progeny that are well adapted to agricultural conditions [ 90 , 91 , 92 ], or through the invasion of alien species that find a new range to colonize. Biosecurity measures are often in place to stop the introduction of new weeds; however, the vast scale of global agricultural commodity trade precludes the possibility of total control. Population genomic analysis is now able to measure gene flow between populations [ 74 , 93 , 94 , 95 ] and identify populations of origin for invasive species including weeds [ 96 , 97 , 98 ]. For example, the invasion route of the pest fruitfly Drosophila suzukii from Eastern Asia to North America and Europe through Hawaii was deciphered using Approximate Bayesian Computation on high-throughput sequencing data from a global sample of multiple populations [ 99 ]. Genomics can also be leveraged to predict invasion rather than explain it. The resequencing of a global sample of common ragweed ( Ambrosia artemisiifolia L.) elucidated a complex invasion route whereby Europe was invaded by multiple introductions of American ragweed that hybridized in Europe prior to a subsequent introduction to Australia [ 100 , 101 ]. In this context, the use of genomically informed species distribution models helps assess the risk associated with different source populations, which in the case of common ragweed, suggests that a source population from Florida would allow ragweed to invade most of northern Australia [ 102 ]. Globally coordinated research efforts to understand potential distribution models could support the transformation of biosecurity from perspective analysis towards predictive risk assessment.

Herbicide resistance and weed management

Herbicide resistance is among the numerous weedy traits that can evolve in plant populations exposed to agricultural selection pressures. Over-reliance on herbicides to control weeds, along with low diversity and lack of redundancy in weed management strategies, has resulted in globally widespread herbicide resistance [ 103 ]. To date, 272 herbicide-resistant weed species have been reported worldwide, and at least one resistance case exists for 21 of the 31 existing herbicide sites of action [ 104 ]—significantly limiting chemical weed control options available to agriculturalists. This limitation of control options is exacerbated by the recent lack of discovery of herbicides with new sites of action [ 105 ].

Herbicide resistance may result from several different physiological mechanisms. Such mechanisms have been classified into two main groups, target-site resistance (TSR) [ 4 , 106 ] and non-target-site resistance (NTSR) [ 4 , 107 ]. The first group encompasses changes that reduce binding affinity between a herbicide and its target [ 108 ]. These changes may provide resistance to multiple herbicides that have a common biochemical target [ 109 ] and can be effectively managed through mixture and/or rotation of herbicides targeting different sites of action [ 110 ]. The second group (NTSR), includes alterations in herbicide absorption, translocation, sequestration, and/or metabolism that may lead to unpredictable pleotropic cross-resistance profiles where structurally and functionally diverse herbicides are rendered ineffective by one or more genetic variant(s) [ 47 ]. This mechanism of resistance threatens not only the efficacy of existing herbicidal chemistries, but also ones yet to be discovered. While TSR is well understood because of the ease of identification and molecular characterization of target site variants, NTSR mechanisms are significantly more challenging to research because they are often polygenic, and the resistance causing element(s) are not well understood [ 111 ].

Improving the current understanding of metabolic NTSR mechanisms is not an easy task, since genes of diverse biochemical functions are involved, many of which exist as extensive gene families [ 109 , 112 ]. Expression changes of NTSR genes have been implicated in several resistance cases where the protein products of the genes are functionally equivalent across sensitive and resistant plants, but their relative abundance leads to resistance. Thus, regulatory elements of NTSR genes have been scrutinized to understand their role in NTSR mechanisms [ 113 ]. Similarly, epigenetic modifications have been hypothesized to play a role in NTSR, with much remaining to be explored [ 114 , 115 , 116 ]. Untargeted approaches such as genome-wide association, selective sweep scans, linkage mapping, RNA-sequencing, and metabolomic profiling have proven helpful to complement more specific biochemical- and chemo-characterization studies towards the elucidation of NTSR mechanisms as well as their regulation and evolution [ 47 , 117 , 118 , 119 , 120 , 121 , 122 , 123 , 124 ]. Even in cases where resistance has been attributed to TSR, genetic mapping approaches can detect other NTSR loci contributing to resistance (as shown by [ 123 ]) and provide further evidence for the role of TSR mutations across populations. Knowledge of the genetic basis of NTSR will aid the rational design of herbicides by screening new compounds for interaction with newly discovered NTSR proteins during early research phases and by identifying conserved chemical structures that interact with these proteins that should be avoided in small molecule design.

Genomic resources can also be used to predict the protein structure for novel herbicide target site and metabolism genes. This will allow for prediction of efficacy and selectivity for new candidate herbicides in silico to increase herbicide discovery throughput as well as aid in the design and development of next-generation technologies for sustainable weed management. Proteolysis targeting chimeras (PROTACs) have the potential to bind desired targets with great selectivity and degrade proteins by utilizing natural protein ubiquitination and degradation pathways within plants [ 125 ]. Spray-induced gene silencing in weeds using oligonucleotides has potential as a new, innovative, and sustainable method for weed management, but improved methods for design and delivery of oligonucleotides are needed to make this technique a viable management option [ 50 ]. Additionally, success in the field of pharmaceutical drug discovery in the development of molecules modulating protein–protein interactions offers another potential avenue towards the development of herbicides with novel targets [ 126 , 127 ]. High-quality reference genomes allow for the design of new weed management technologies like the ones listed here that are specific to—and effective across—weed species but have a null effect on non-target organisms.

Comparative genomics and genome biology

The genomes of weed species are as diverse as weed species themselves. Weeds are found across highly diverged plant families and often have no phylogenetically close model or crop species relatives for comparison. On all measurable metrics, weed genomes run the gamut. Some have smaller genomes like Cyperus spp. (~ 0.26 Gb) while others are larger, such as Avena fatua (~ 11.1 Gb) (Table  1 ). Some have high heterozygosity in terms of single-nucleotide polymorphisms, such as the Amaranthus spp., while others are primarily self-pollinated and quite homozygous, such as Poa annua [ 128 , 129 ]. Some are diploid such as Conyza canadensis and Echinochloa haploclada while others are polyploid such as C. sumetrensis , E. crus-galli , and E. colona [ 76 ]. The availability of genomic resources in these diverse, unexplored branches of the tree of life allows us to identify consistencies and anomalies in the field of genome biology.

The weed genomes published so far have focused mainly on weeds of agronomic crops, and studies have revolved around their ability to resist key herbicides. For example, genomic resources were vital in the elucidation of herbicide resistance cases involving target site gene copy number variants (CNVs). Gene CNVs of 5-enolpyruvylshikimate-3-phosphate synthase ( EPSPS ) have been found to confer resistance to the herbicide glyphosate in diverse weed species. To date, nine species have independently evolved EPSPS CNVs, and species achieve increased EPSPS copy number via different mechanisms [ 153 ]. For instance, the EPSPS CNV in Bassia scoparia is caused by tandem duplication, which is accredited to transposable element insertions flanking EPSPS and subsequent unequal crossing over events [ 154 , 155 ]. In Eleusine indica , a EPSPS CNV was caused by translocation of the EPSPS locus into the subtelomere followed by telomeric sequence exchange [ 156 ]. One of the most fascinating genome biology discoveries in weed science has been that of extra-chromosomal circular DNAs (eccDNAs) that harbor the EPSPS gene in the weed species Amaranthus palmeri [ 157 , 158 ]. In this case, the eccDNAs autonomously replicate separately from the nuclear genome and do not reintegrate into chromosomes, which has implications for inheritance, fitness, and genome structure [ 159 ]. These discoveries would not have been possible without reference assemblies of weed genomes, next-generation sequencing, and collaboration with experts in plant genomics and bioinformatics.

Another question that is often explored with weedy genomes is the nature and composition of gene families that are associated with NTSR. Gene families under consideration often include cytochrome P450s (CYPs), glutathione- S -transferases (GSTs), ABC transporters, etc. Some questions commonly considered with new weed genomes include how many genes are in each of these gene families, where are they located, and which weed accessions and species have an over-abundance of them that might explain their ability to evolve resistance so rapidly [ 76 , 146 , 160 , 161 ]? Weed genome resources are necessary to answer questions about gene family expansion or contraction during the evolution of weediness, including the role of polyploidy in NTSR gene family expansion as explored by [ 162 ].

Translational research and communication with weed management stakeholders

Whereas genomics of model plants is typically aimed at addressing fundamental questions in plant biology, and genomics of crop species has the obvious goal of crop improvement, goals of genomics of weedy plants also include the development of more effective and sustainable strategies for their management. Weed genomic resources assist with these objectives by providing novel molecular ecological and evolutionary insights from the context of intensive anthropogenic management (which is lacking in model plants), and offer knowledge and resources for trait discovery for crop improvement, especially given that many wild crop relatives are also important agronomic weeds (e.g., [ 163 ]). For instance, crop-wild relatives are valuable for improving crop breeding for marginal environments [ 164 ]. Thus, weed genomics presents unique opportunities and challenges relative to plant genomics more broadly. It should also be noted that although weed science at its core is an applied discipline, it draws broadly from many scientific disciplines such as, plant physiology, chemistry, ecology, and evolutionary biology, to name a few. The successful integration of weed-management strategies, therefore, requires extensive collaboration among individuals collectively possessing the necessary expertise [ 165 ].

With the growing complexity of herbicide resistance management, practitioners are beginning to recognize the importance of understanding resistance mechanisms to inform appropriate management tactics [ 14 ]. Although weed science practitioners do not need to understand the technical details of weed genomics, their appreciation of the power of weed genomics—together with their unique insights from field observations—will yield novel opportunities for applications of weed genomics to weed management. In particular, combining field management history with information on weed resistance mechanisms is expected to provide novel insights into evolutionary trajectories (e.g. [ 6 , 166 ]), which can be utilized for disrupting evolutionary adaptation. It can be difficult to obtain field history information from practitioners, but developing an understanding among them of the importance of such information can be invaluable.

Development of weed genomics resources by the IWGC

Weed genomics is a fast-growing field of research with many recent breakthroughs and many unexplored areas of study. The International Weed Genomics Consortium (IWGC) started in 2021 to address the roadblocks listed above and to promote the study of weedy plants. The IWGC is an open collaboration among academic, government, and industry researchers focused on producing genomic tools for weedy species from around the world. Through this collaboration, our initial aim is to provide chromosome-level reference genome assemblies for at least 50 important weedy species from across the globe that are chosen based on member input, economic impact, and global prevalence (Fig.  1 ). Each genome will include annotation of gene models and repetitive elements and will be freely available through public databases with no intellectual property restrictions. Additionally, future funding of the IWGC will focus on improving gene annotations and supplementing these reference genomes with tools that increase their utility.

figure 1

The International Weed Genomics Consortium (IWGC) collected input from the weed genomics community to develop plans for weed genome sequencing, annotation, user-friendly genome analysis tools, and community engagement

Reference genomes and data analysis tools

The first objective of the IWGC is to provide high-quality genomic resources for agriculturally important weeds. The IWGC therefore created two main resources for information about, access to, or analysis of weed genomic data (Fig.  1 ). The IWGC website (available at [ 167 ]) communicates the status and results of genome sequencing projects, information on training and funding opportunities, upcoming events, and news in weed genomics. It also contains details of all sequenced species including genome size, ploidy, chromosome number, herbicide resistance status, and reference genome assembly statistics. The IWGC either compiles existing data on genome size, ploidy, and chromosome number, or obtains the data using flow cytometry and cytogenetics (Fig.  1 ; Additional File 2 : Fig S1-S4). Through this website, users can request an account to access our second main resource, an online genome database called WeedPedia (accessible at [ 168 ]), with an account that is created within 3–5 working days of an account request submission. WeedPedia hosts IWGC-generated and other relevant publicly accessible genomic data as well as a suite of bioinformatic tools. Unlike what is available for other fields, weed science did not have a centralized hub for genomics information, data, and analysis prior to the IWGC. Our intention in creating WeedPedia is to encourage collaboration and equity of access to information across the research community. Importantly, all genome assemblies and annotations from the IWGC (Table  1 ), along with the raw data used to produce them, will be made available through NCBI GenBank. Upon completion of a 1-year sponsoring member data confidentiality period for each species (dates listed in Table  1 ), scientific teams within the IWGC produce the first genome-wide investigation to submit for publication including whole genome level analyses on genes, gene families, and repetitive sequences as well as comparative analysis with other species. Genome assemblies and data will be publicly available through NCBI as part of these initial publications for each species.

WeedPedia is a cloud-based omics database management platform built from the software “CropPedia” and licensed from KeyGene (Wageningen, The Netherlands). The interface allows users to access, visualize, and download genome assemblies along with structural and functional annotation. The platform includes a genome browser, comparative map viewer, pangenome tools, RNA-sequencing data visualization tools, genetic mapping and marker analysis tools, and alignment capabilities that allow searches by keyword or sequence. Additionally, genes encoding known target sites of herbicides have been specially annotated, allowing users to quickly identify and compare these genes of interest. The platform is flexible, making it compatible with future integration of other data types such as epigenetic or proteomic information. As an online platform with a graphical user interface, WeedPedia provides user-friendly, intuitive tools that encourage users to integrate genomics into their research while also allowing more advanced users to download genomic data to be used in custom analysis pipelines. We aspire for WeedPedia to mimic the success of other public genomic databases such as NCBI, CoGe, Phytozome, InsectBase, and Mycocosm to name a few. WeedPedia currently hosts reference genomes for 40 species (some of which are currently in their 1-year confidentiality period) with additional genomes in the pipeline to reach a currently planned total of 55 species (Table  1 ). These genomes include both de novo reference genomes generated or in progress by the IWGC (31 species; Table  1 ), and publicly available genome assemblies of 24 weedy or related species that were generated by independent research groups (Table  2 ). As of May 2024, WeedPedia has over 370 registered users from more than 27 countries spread across 6 continents.

The IWGC reference genomes are generated in partnership with the Corteva Agriscience Genome Center of Excellence (Johnston, Iowa) using a combination of single-molecule long-read sequencing, optical genome maps, and chromosome conformation mapping. This strategy has already yielded highly contiguous, phased, chromosome-level assemblies for 26 weed species, with additional assemblies currently in progress (Table  1 ). The IWGC assemblies have been completed as single or haplotype-resolved double-haplotype pseudomolecules in inbreeding and outbreeding species, respectively, with multiple genomes being near gapless. For example, the de novo assemblies of the allohexaploids Conyza sumatrensis and Chenopodium album have all chromosomes captured in single scaffolds and most chromosomes being gapless from telomere to telomere. Complementary full-length isoform (IsoSeq) sequencing of RNA collected from diverse tissue types and developmental stages assists in the development of gene models during annotation.

As with accessibility of data, a core objective of the IWGC is to facilitate open access to sequenced germplasm when possible for featured species. Historically, the weed science community has rarely shared or adopted standard germplasm (e.g., specific weed accessions). The IWGC has selected a specific accession of each species for reference genome assembly (typically susceptible to herbicides). In collaboration with a parallel effort by the Herbicide Resistant Plants committee of the Weed Science Society of America, seeds of the sequenced weed accessions will be deposited in the United States Department of Agriculture Germplasm Resources Information Network [ 186 ] for broad access by the scientific community and their accession numbers will be listed on the IWGC website. In some cases, it is not possible to generate enough seed to deposit into a public repository (e.g., plants that typically reproduce vegetatively, that are self-incompatible, or that produce very few seeds from a single individual). In these cases, the location of collection for sequenced accessions will at least inform the community where the sequenced individual came from and where they may expect to collect individuals with similar genotypes. The IWGC ensures that sequenced accessions are collected and documented to comply with the Nagoya Protocol on access to genetic resources and the fair and equitable sharing of benefits arising from their utilization under the Convention on Biological Diversity and related Access and Benefit Sharing Legislation [ 187 ]. As additional accessions of weed species are sequenced (e.g., pangenomes are obtained), the IWGC will facilitate germplasm sharing protocols to support collaboration. Further, to simplify the investigation of herbicide resistance, the IWGC will link WeedPedia with the International Herbicide-Resistant Weed Database [ 104 ], an already widely known and utilized database for weed scientists.

Training and collaboration in weed genomics

Beyond producing genomic tools and resources, a priority of the IWGC is to enable the utilization of these resources across a wide range of stakeholders. A holistic approach to training is required for weed science generally [ 188 ], and we would argue even more so for weed genomics. To accomplish our training goals, the IWGC is developing and delivering programs aimed at the full range of IWGC stakeholders and covering a breadth of relevant topics. We have taken care to ensure our approaches are diverse as to provide training to researchers with all levels of existing experience and differing reasons for engaging with these tools. Throughout, the focus is on ensuring that our training and outreach result in impacts that benefit a wide range of stakeholders.

Although recently developed tools are incredibly enabling and have great potential to replace antiquated methodology [ 189 ] and to solve pressing weed science problems [ 14 ], specialized computational skills are required to fully explore and unlock meaning from these highly complex datasets. Collaboration with, or training of, computational biologists equipped with these skills and resources developed by the IWGC will enable weed scientists to expand research programs and better understand the genetic underpinnings of weed evolution and herbicide resistance. To fill existing skill gaps, the IWGC is developing summer bootcamps and online modules directed specifically at weed scientists that will provide training on computational skills (Fig.  1 ). Because successful utilization of the IWGC resources requires more than general computational skills, we have created three targeted workshops that teach practical skills related to genomics databases, molecular biology, and population genomics (available at [ 190 ]). The IWGC has also hosted two official conference meetings, one in September of 2021 and one in January of 2023, with more conferences planned. These conferences have included invited speakers to present successful implementations of weed genomics, educational workshops to build computational skills, and networking opportunities for research to connect and collaborate.

Engagement opportunities during undergraduate degrees have been shown to improve academic outcomes [ 191 , 192 ]. As one activity to help achieve this goal, the IWGC has sponsored opportunities for US undergraduates to undertake a 10-week research experience, which includes an introduction to bioinformatics, a plant genomics research project that results in a presentation, and access to career building opportunities in diverse workplace environments. To increase equitable access to conferences and professional communities, we supported early career researchers to attend the first two IWGC conferences in the USA as well as workshops and bootcamps in Europe, South America, and Australia. These hybrid or in-person travel grants are intentionally designed to remove barriers and increase participation of individuals from backgrounds and experiences currently underrepresented within weed/plant science or genomics [ 193 ]. Recipients of these travel awards gave presentations and gained the measurable benefits that come from either virtual or in-person participation in conferences [ 194 ]. Moving forward, weed scientists must amass skills associated with genomic analyses and collaborate with other area experts to fully leverage resources developed by the IWGC.

The tools generated through the IWGC will enable many new research projects with diverse objectives like those listed above. In summary, contiguous genome assemblies and complete annotation information will allow weed scientists to join plant breeders in the use of genetic mapping for many traits including stress tolerance, plant architecture, and herbicide resistance (especially important for cases of NTSR). These assemblies will also allow for investigations of population structure, gene flow, and responses to evolutionary mechanisms like genetic bottlenecking and artificial selection. Understanding gene sequences across diverse weed species will be vital in modeling new herbicide target site proteins and designing novel effective herbicides with minimal off-target effects. The IWGC website will improve accessibility to weed genomics data by providing a single hub for reference genomes as well as phenotypic and genotypic information for accessions shared with the IWGC. Deposition of sequenced germplasm into public repositories will ensure that researchers are able to access and utilize these accessions in their own research to make the field more standardized and equitable. WeedPedia allows users of all backgrounds to quickly access information of interest such as herbicide target site gene sequence or subcellular localization of protein products for different genes. Users can also utilize server-based tools such as BLAST and genome browsing similar to other public genomic databases. Finally, the IWGC is committed to training and connecting weed genomicists through hosting trainings, workshops, and conferences.

Conclusions

Weeds are unique and fascinating plants, having significant impacts on agriculture and ecosystems; and yet, aspects of their biology, ecology, and genetics remain poorly understood. Weeds represent a unique area within plant biology, given their repeated rapid adaptation to sudden and severe shifts in the selective landscape of anthropogenic management practices. The production of a public genomics database with reference genomes and annotations for over 50 weed species represents a substantial step forward towards research goals that improve our understanding of the biology and evolution of weeds. Future work is needed to improve annotations, particularly for complex gene families involved in herbicide detoxification, structural variants, and mobile genetic elements. As reference genome assemblies become available; standard, affordable methods for gathering genotype information will allow for the identification of genetic variants underlying traits of interest. Further, methods for functional validation and hypothesis testing are needed in weeds to validate the effect of genetic variants detected through such experiments, including systems for transformation, gene editing, and transient gene silencing and expression. Future research should focus on utilizing weed genomes to investigate questions about evolutionary biology, ecology, genetics of weedy traits, and weed population dynamics. The IWGC plans to continue the public–private partnership model to host the WeedPedia database over time, integrate new datasets such as genome resequencing and transcriptomes, conduct trainings, and serve as a research coordination network to ensure that advances in weed science from around the world are shared across the research community (Fig.  1 ). Bridging basic plant genomics with translational applications in weeds is needed to deliver on the potential of weed genomics to improve weed management and crop breeding.

Availability of data and materials

All genome assemblies and related sequencing data produced by the IWGC will be available through NCBI as part of publications reporting the first genome-wide analysis for each species.

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Peer review information

Wenjing She was the primary editor of this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

The International Weed Genomics Consortium is supported by BASF SE, Bayer AG, Syngenta Ltd, Corteva Agriscience, CropLife International (Global Herbicide Resistance Action Committee), the Foundation for Food and Agriculture Research (Award DSnew-0000000024), and two conference grants from USDA-NIFA (Award numbers 2021–67013-33570 and 2023-67013-38785).

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Department of Agricultural Biology, Colorado State University, 1177 Campus Delivery, Fort Collins, CO, 80523, USA

Jacob Montgomery, Sarah Morran & Todd A. Gaines

Protecting Crops and the Environment, Rothamsted Research, Harpenden, Hertfordshire, UK

Dana R. MacGregor

Department of Crop, Soil, and Environmental Sciences, Auburn University, Auburn, AL, USA

J. Scott McElroy

Department of Plant and Environmental Sciences, University of Copenhagen, Taastrup, Denmark

Paul Neve & Célia Neto

IFEVA-Conicet-Department of Ecology, University of Buenos Aires, Buenos Aires, Argentina

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Department of Ecology, Faculty of Agronomy, University of Buenos Aires, Buenos Aires, Argentina

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Department of Botany, The University of British Columbia, Vancouver, BC, Canada

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Institute of Crop Sciences, Zhejiang University, Hangzhou, China

Longjiang Fan

Department of Biology, University of Massachusetts Amherst, Amherst, MA, USA

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Department of Plant and Wildlife Sciences, Brigham Young University, Provo, UT, USA

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Bayer AG, Weed Control Research, Frankfurt, Germany

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Contributions

JMo and TG conceived and outlined the article. TG, DM, EP, RB, JSM, PJT, MJ wrote grants to obtain funding. MMI, BSG, and MJ performed mitotic chromosome visualization. VL performed sequencing. VL and KF assembled the genomes. LC and ELP annotated the genomes. JMo, SM, DRM, JSM, PN, CN, MV, MVS, AIM, JMK, LF, ALC, PJM, BABM, JMi, AC, MVB, LC, AFL, and ELP wrote the first draft of the article. All authors edited the article and improved the final version.

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Correspondence to Todd A. Gaines .

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Some authors work for commercial agricultural companies (BASF, Bayer, Corteva Agriscience, or Syngenta) that develop and sell weed control products.

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Supplementary Information

13059_2024_3274_moesm1_esm.docx.

Additional file 1. List of completed and in-progress genome assemblies of weed species pollinated by insects (Table S1).

13059_2024_3274_MOESM2_ESM.docx

Additional file 2. Methods and results for visualizing and counting the metaphase chromosomes of hexaploid Avena fatua (Fig S1); diploid Lolium rigidum  (Fig S2); tetraploid Phalaris minor (Fig S3); and tetraploid Salsola tragus (Fig S4).

Additional file 3. Review history.

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Montgomery, J., Morran, S., MacGregor, D.R. et al. Current status of community resources and priorities for weed genomics research. Genome Biol 25 , 139 (2024). https://doi.org/10.1186/s13059-024-03274-y

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    Background: Autism is a normal part of cognitive diversity, resulting in communication and sensory processing differences, which can become disabling in a neurotypical world. Autistic people have an increased likelihood of physical and mental co-occurring conditions and die earlier than neurotypical peers. Inaccessible healthcare may contribute to this. Autism Health Passports (AHPs) are paper ...

  28. NAD Supplement: Benefits, Side Effects, and Efficacy

    Learn more about potential research-supported NAD benefits, including increased energy and cellular repair, to discover whether NAD is right for you. ... making things happen and maximizing communication between cells throughout the body. Without enough NAD, simple processes in the body may take longer, partly because of poor communication ...

  29. Current status of community resources and priorities for weed genomics

    Weeds are attractive models for basic and applied research due to their impacts on agricultural systems and capacity to swiftly adapt in response to anthropogenic selection pressures. Currently, a lack of genomic information precludes research to elucidate the genetic basis of rapid adaptation for important traits like herbicide resistance and stress tolerance and the effect of evolutionary ...

  30. Adaptive attack recognition method based on probability model for

    Traditional attack detection methods, relying on static anomaly thresholds from data distributions, falter in complex driving scenarios. Recent studies have sought more nuanced detection techniques, including deep learning and inter-vehicle communication, but these approaches face limitations related to data security and environmental ...