More than a decade ago, as a PhD student at Uppsala University, Sweden, the first-year syllabus introduced the textbook What Is This Thing Called Science? by Alan Chalmers, which explores the nature of scientific inquiry. In Chapter 2, "Observation as Practical Intervention," Chalmers discusses how Galileo Galilei challenged the geocentric model and supported heliocentrism through systematic observation and experimentation—key principles of the scientific method. Using his telescope, Galileo provided empirical evidence that contradicted Aristotelian cosmology. He discovered the moons of Jupiter, proving that not all celestial bodies orbit Earth, observed the phases of Venus, confirming its orbit around the Sun, and noted the Moon’s uneven surface, challenging the idea of perfect celestial spheres. Galileo also observed that Mars and Venus appeared to change in size through a telescope, consistent with the predictions of the Copernican model. Chalmers uses Galileo’s work to illustrate how science progresses through observation, hypothesis testing, and theory revision, emphasizing that knowledge should be grounded in empirical evidence rather than authority.

As scientists, we are all committed to “letting experimental data determine the truth”. Meticulously collected data from well-designed experiments are generally considered objective. However, the truth is that data does not speak for itself. Any dataset must be interpreted within a web of concepts, theories, and methodologies which continue to be refined over time. Naturally, this background knowledge is rarely free from bias. We may overlook how our perspectives and assumptions have shaped findings when we assert that certain data provides evidence for a specific conclusion. Understanding this is crucial: not to undermine the data, but to recognise the complex process of interpretation that underlies every scientific insight. Often, we look at contradictions in science with suspicion and fear. Nevertheless, philosophers and scientists have advocated for the value of contradictions in driving new and exciting discoveries¹.

This challenge is exemplified in Heinrich Hertz’s experiments on electromagnetic waves, as discussed in Chapter 3, “Experiment” of What Is This Thing Called Science?. Hertz successfully demonstrated the existence of these waves, confirming Maxwell’s theoretical predictions. However, in his own interpretation of the results, Hertz adhered to the mechanical ether model, believing that electromagnetic phenomena could be explained within the prevailing framework of mechanical physics. His commitment to this theory led him to describe his findings in ways that did not fully anticipate the later abandonment of the ether concept. Despite this, Hertz’s experiments laid the foundation for the understanding of electromagnetism, and his results were later reinterpreted in light of the new conceptual framework developed by Einstein and others. This example illustrates how the theoretical lens through which data is viewed can shape, and sometimes constrain, scientific understanding.

Several Springer Nature journals have introduced the “Matters Arising” article type, recognising the value of fostering contradictory viewpoints^2,3. Designed for post-publication discussion, Matters Arising encourages discourse that can bring more profound insights into published research. This format goes beyond traditional forms of scholarly correspondence to create a platform where critiques of published work are not only possible but formally integrated into the scientific record. Matters Arising enables direct engagement with the original authors and encourages rigorous discussion by inviting constructive criticism, offering a method to probe findings, identify limitations, and highlight alternative interpretations, thereby helping to ensure scientific rigour. Constructive criticism and rigorous debate are integral to scientific progress. Through Matters Arising, Springer Nature journals embrace this ethos, helping refine the scientific record and advancing the field. Matters Arising serves as a venue for embracing contradictions and propelling the pursuit of new insights.

Our encounter with contradiction: the story of black-bone chicken

Uncovering accurate genomic structures is crucial for understanding genetic traits in the complex and ever-evolving field of genomics. The Fm locus in chickens is specifically associated with the unique “fibromelanosis” (Fm) phenotype found in Silkie and other black-bone breeds. Bateson and Punnet described the autosomal dominant nature of the Fm locus in the 1900s⁴. However, scientists first unravelled the genomic organisation of the Fm locus in Silkie chickens only a decade ago. Since then, research into Silkie and other black-bone chicken breeds has delved into the rich genetic history, copy number variations, and diversity patterns at this locus. The hyperpigmentation that makes Silkie chickens stand out visually also impacts traits like immune system development, showcasing how one genetic feature can influence multiple biological processes. This detailed examination has elevated the Fm locus to a textbook example of a remarkable phenotype linked to copy number variation, highlighting its significance in genetic studies. Our recent Matters Arising article sought to address and clarify an important aspect of this region—building upon the foundational research presented by Zhu et al. in Communications Biology⁵.

The Foundation: Zhu et al.’s Contributions to Avian Genomics

Zhu et al. have laid a critical foundation, demonstrating the power of modern genome assemblies to resolve long-standing questions. The CAU_Silkie genome does not just fill gaps in knowledge—it reshapes the landscape of avian genetics and sets a new benchmark for excellence in genome assembly research. Their meticulous attention to detail, coupled with innovative sequencing and assembly strategies, has provided an unprecedented resolution of the Fm locus, offering an invaluable resource for geneticists worldwide.

The extraordinary skill and rigorous methodology behind their study have delivered a profound leap forward in our understanding of the Fm locus and beyond. By integrating multi-platform sequencing approaches, Zhu et al. have gone beyond conventional genome assembly practices, achieving a level of accuracy and completeness rarely seen in avian genomics. Their work not only refines our knowledge of genetic structures but also lays the groundwork for further studies into gene regulation, evolutionary biology, and structural variation across species.

Zhu et al.’s approach represents a landmark contribution to avian genetics, opening the door for further exploration into the intricate structure of the Fm locus. Future research will undoubtedly build upon this groundbreaking effort, leveraging their insights to unlock new discoveries in comparative genomics, genetic trait mapping, and evolutionary adaptation. The CAU_Silkie genome stands as a testament to the transformative power of cutting-edge genomic science and the lasting impact of meticulous, high-quality research.

Why Reassessing the Fm Locus Structure Matters

The Fm locus on chromosome 20 includes two key duplicated regions, Dup1 (~127 Kb) and Dup2 (~170 Kb), separated by an intermediate region (~412 Kb). In black-bone breeds, this locus undergoes a complex chromosomal rearrangement, leading to fibromelanosis. While Zhu et al. proposed the *Fm_1 arrangement based on their assembly, other studies—using genetic crosses and short-read data—have supported an alternative structure called the *Fm_2 scenario^6–8.

The discrepancy between these structural hypotheses (*Fm_1 vs *Fm_2) holds significance for ongoing research. Establishing an accurate model for the Fm locus is essential to correctly understanding gene regulation and expression in this region. Since structural variations, like duplications and inversions, may impact regulatory networks and trait expression, indisputably establishing the correct structure of the Fm locus is essential. Thus, our work aimed to reexamine the structure of the Fm locus using an alternative approach, adding further clarity to this question.

Our Approach: Leveraging Read-Backed Phasing for Accurate Structural Insights

For our analysis, we adopted a haplotype phasing method to reassess the Fm locus structure in Silkie chickens. We could map reads to specific haplotypes using long-read sequencing data, allowing us to determine which structural scenario matched the observed genomic sequences precisely.

This read-backed phasing approach provided two distinct advantages:

1. Enhanced Resolution of Structural Boundaries: We used long reads to identify haplotype-defining positions (HDPs) that spanned the duplicated regions, enabling us to reconstruct the structure of the Fm locus with a high degree of confidence.
2. Independence from Assembly Artifacts: By mapping the long reads directly to the genome, we could bypass potential artefacts that sometimes arise in de novo assemblies, where mosaic haplotypes may misrepresent true genomic organisation.

Through this approach, our analysis consistently pointed to the *Fm_2 scenario as the accurate structure of the Fm locus, confirming the results of several prior studies.

Results: Clarifying the Fm Locus Structure

Our findings indicate that the original assembly by Zhu et al. inadvertently favoured the *Fm_1 scenario due to the presence of mosaic haplotypes in the assembly process. This mosaic haplotype structure likely emerged from technical challenges during assembly and insufficient post-assembly validation. Our read-backed phasing approach identified specific haplotype-consistent tiling paths that extended across the duplicated regions, aligning with the *Fm_2 scenario.

In Chapter 5 of What Is This Thing Called Science?, titled "Introducing Falsificationism," Alan Chalmers discusses Karl Popper's philosophy that scientific theories cannot be conclusively proven but can be rigorously tested and potentially falsified. Chalmers emphasizes that for a theory to be considered scientific, it must be falsifiable; that is, it should make precise predictions that can be tested and potentially proven wrong. He also highlights that the more falsifiable a theory is, the more informative and valuable it becomes to scientific progress.

Applying Chalmers' insights to our research, we recognize the importance of subjecting our proposed *Fm_2 structure of the Fm locus to stringent tests that could potentially falsify it. By analyzing data from various sequencing platforms (ONT and PacBio) and independent datasets from different black-bone chicken populations, we have sought opportunities to challenge our hypothesis. The consistency of our findings across these diverse datasets, along with the support from haplotype-specific analysis of Hi-C data from Zhu et al., indicates that our hypothesis has withstood rigorous testing. This resilience in the face of potential falsification enhances our confidence in the *Fm_2 structure as the accurate representation of the Fm locus. Therefore, our approach aligns with the falsificationist philosophy discussed by Chalmers, where scientific knowledge advances through bold conjectures that are subjected to rigorous attempts at refutation, and only those that withstand such scrutiny are tentatively accepted.

Implications for Future Research

Clarifying the structure of the Fm locus has wide-reaching implications for genetic research on the Fm trait and similar loci. Structural accuracy in genome assemblies is essential, as misinterpretations can lead to errors in downstream analyses and functional studies. For complex loci like the Fm locus, which contain repetitive sequences and rearrangements, thorough validation steps can ensure that the observed structures truly reflect the biological reality.

The discovery of Neptune serves as a powerful example of how scientific progress is driven by the refinement of theories through empirical testing and falsification. In Chapter 6 (Sophisticated falsificationism, novel predictions and the growth of science) of What Is This Thing Called Science?, Chalmers discusses how Newton’s theory of gravitation was initially challenged by discrepancies in the orbit of Uranus. Rather than rejecting the theory outright, scientists like Urbain Le Verrier hypothesized that an unseen planet could explain these irregularities. This predictive power of scientific theories led to the eventual observation of Neptune in 1846, reinforcing the strength of Newtonian mechanics rather than falsifying it.

Similarly, our study underscores the importance of rigorous validation in genome assembly. Just as astronomers refined Newton’s model by incorporating new observations, our approach integrates long-read sequencing and haplotype phasing to ensure the structural accuracy of the Fm locus. By identifying potential assembly artefacts and resolving complex regions, we strengthen genomic research in the same way that the discovery of Neptune strengthened celestial mechanics.

Moving forward, as sequencing technologies advance, collaborative research efforts will refine our understanding of complex loci. Just as scientific inquiry does not end with a single discovery, genome assembly projects should be iterative—identifying inconsistencies, making predictions, and testing them through additional sequencing and computational analysis. By embracing this dynamic approach, we can uncover deeper insights into the genetic basis of traits and the evolutionary forces shaping genomes.

In Chapter 13 (The New Experimentalism) of What Is This Thing Called Science?, Chalmers discusses Ian Hacking's stories concerning the use of microscopes. A miniature grid with labelled squares is photographically reduced until it becomes invisible to the naked eye. When viewed through a microscope, however, the grid reappears, demonstrating that the microscope magnifies and does so reliably—without requiring a theoretical explanation of how it works. Another example is that of a biologist, using an electron microscope, to observe dense bodies within red blood platelets and suspecting they might be artifacts of the instrument. To test this, the biologist notes their positions on a reference grid and then examines the sample with a fluorescence microscope, which operates on entirely different physical principles. When the same dense bodies appear in the same positions, it becomes highly improbable that both instruments would produce identical artifacts. This example illustrates how scientific confidence is built through independent validation rather than mere theoretical justification. We use independent data from the PacBio sequencer, Nanopore sequencer, the Hi-C library sequenced using a Illumina sequencer to ensure the haplotypes consistently recover the *Fm_2 scenario.

Moving Forward: A Call for Collaboration in Genomic Research

The complexity of the Fm locus and the challenges in resolving its structure underscore the value of collaborative, iterative research in genomics. Zhu et al.’s work has made a substantial impact, advancing the capabilities of genome assemblies in chickens and providing a foundation for further refinement. Our Matters Arising article is intended as a constructive continuation of this effort, contributing additional analysis to clarify the Fm locus structure. In a related study⁹, we identified a chromosomal rearrangement on the Z chromosome located within the Z amplicon repeat units associated with the Id locus. Together, these studies exemplify the dynamic nature of scientific discovery.

Just as the elucidation of the DNA double helix by Watson and Crick¹⁰ was only possible because it built upon a foundation laid by others—such as Rosalind Franklin's X-ray diffraction images and Chargaff’s rules—our current understanding of the Fm locus is similarly cumulative¹¹. Each study, from early genetic descriptions to the latest long-read assemblies, adds a layer of insight, challenges assumptions, and refines prevailing models. Like Watson and Crick, who integrated disparate findings into a transformative model of DNA structure, researchers studying fibromelanosis synthesize historical and contemporary data to piece together complex genomic rearrangements, emphasizing the collaborative and iterative nature of scientific progress.

As sequencing technologies continue to improve, combining long-read sequencing, read-backed phasing, and computational approaches will enable more precise genomic analyses. By embracing contradictions and refining methodologies, the scientific community can better understand genetic traits and the evolutionary forces shaping genomes. A very interesting paper that got published recently¹² uses population genomics tools to analyse a large dataset of over 500 BBC to establish *Fm_2 as the correct scenario. Notably, the newer version of the Silkie genome (CAU_Silkie 2.0) has also been published¹³. Which of the two scenarios, *Fm_1^5,14 or *Fm_2^15,16 do you think makes sense? How did you decide which scenario is correct?

References

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