Periodic Reporting for period 4 - MultiplexGenomics (Exploring the Epigenome by Multiplexed Physical Mapping of Individual Chromosomes)
Reporting period: 2024-04-01 to 2025-03-31
Modern genetic testing is typically limited to analyzing a single type of genomic information, such as specific mutations, which constrains the clinical insights that can be drawn from patient samples. The MultiplexGenomics project addresses this limitation by developing technologies that enable the simultaneous measurement of multiple genomic features, including genetic structure, epigenetic modifications, and DNA damage, on the same DNA molecule. The overall objectives are to create a comprehensive toolbox of detection methods, validate them on clinically relevant samples, and facilitate their adoption by the broader scientific and medical communities. Ultimately, these advances aim to improve disease detection, diagnosis, and patient care, while also providing powerful new research tools for understanding complex disease mechanisms.
From the outset, the MultiplexGenomics project focused on developing and integrating advanced technologies for comprehensive genomic analysis at the single-molecule level. The team first concentrated on optical single-molecule genomics, successfully developing and implementing the Continuously Controlled Spectral Resolution (CoCoS) microscopy platform. This innovative approach enabled high-throughput, multi-color imaging of DNA molecules, allowing for the simultaneous detection of multiple genomic features. Building on this, the DeepQR method was introduced, offering rapid and accurate multiplexed color registration, which further improved the efficiency and reliability of single-molecule analyses. These technologies were adapted for clinical applications, including the dual labeling of DNA methylation marks, and were applied to the genetic and epigenetic profiling of cancer samples.
In parallel, significant progress was made in the area of tag-enhanced nanopore sequencing. The project advanced a chemo-enzymatic DNA labeling technique that produces distinct electrical signatures, enabling the direct detection of chemically modified nucleotides during nanopore sequencing. Additionally, the team explored the use of DNA damage as a contrast-enhancing feature for sequencing and published a comprehensive review of the Repair Assisted Damage Detection (RADD) approach, highlighting its potential for mapping DNA damage at the single-molecule level.
Work on nanochannel devices for electro-optical mapping also yielded important results. Using nanopore-based platforms, the team was able to map structural variants and large chromosomal rearrangements with high resolution. Comparative analyses with existing genomic mapping and sequencing methods demonstrated the advantages and complementarity of these new approaches.
Throughout the project, these technological breakthroughs were validated on both clinical and biological samples, demonstrating their utility for detecting structural variants, epigenetic modifications, and DNA damage. The project’s multidisciplinary nature, combining expertise from chemistry, physics, biology, and computational sciences, was instrumental in achieving these results.
In terms of exploitation and dissemination, a patent application covering key technologies is currently under review and at different stages of PCT. The team organized a workshop to train researchers from other laboratories, facilitating broader adoption of the methods developed. Results from the project have been widely disseminated through publications in high-impact journals and preprints, with additional manuscripts currently under review.
The research findings have also been extensively presented at international and national conferences, seminars, and academic meetings, stakeholders' events (patient forums, clinical assembly,etc. ), ensuring broad reach within the scientific and nonscientific community. Additionally, public talks were delivered to elementary and high school students.
Overall, the MultiplexGenomics project has delivered a suite of innovative tools that significantly advance the field of genomic analysis. These achievements are already having an impact on both clinical diagnostics and biomedical research, and ongoing efforts are focused on ensuring their continued adoption and further development within the scientific and medical communities.
In parallel, significant progress was made in the area of tag-enhanced nanopore sequencing. The project advanced a chemo-enzymatic DNA labeling technique that produces distinct electrical signatures, enabling the direct detection of chemically modified nucleotides during nanopore sequencing. Additionally, the team explored the use of DNA damage as a contrast-enhancing feature for sequencing and published a comprehensive review of the Repair Assisted Damage Detection (RADD) approach, highlighting its potential for mapping DNA damage at the single-molecule level.
Work on nanochannel devices for electro-optical mapping also yielded important results. Using nanopore-based platforms, the team was able to map structural variants and large chromosomal rearrangements with high resolution. Comparative analyses with existing genomic mapping and sequencing methods demonstrated the advantages and complementarity of these new approaches.
Throughout the project, these technological breakthroughs were validated on both clinical and biological samples, demonstrating their utility for detecting structural variants, epigenetic modifications, and DNA damage. The project’s multidisciplinary nature, combining expertise from chemistry, physics, biology, and computational sciences, was instrumental in achieving these results.
In terms of exploitation and dissemination, a patent application covering key technologies is currently under review and at different stages of PCT. The team organized a workshop to train researchers from other laboratories, facilitating broader adoption of the methods developed. Results from the project have been widely disseminated through publications in high-impact journals and preprints, with additional manuscripts currently under review.
The research findings have also been extensively presented at international and national conferences, seminars, and academic meetings, stakeholders' events (patient forums, clinical assembly,etc. ), ensuring broad reach within the scientific and nonscientific community. Additionally, public talks were delivered to elementary and high school students.
Overall, the MultiplexGenomics project has delivered a suite of innovative tools that significantly advance the field of genomic analysis. These achievements are already having an impact on both clinical diagnostics and biomedical research, and ongoing efforts are focused on ensuring their continued adoption and further development within the scientific and medical communities.
Our work has pushed beyond the state of the art by enabling the simultaneous detection of genetic, epigenetic, and structural features on the same DNA molecule at single-molecule resolution. The combination of innovative optical and electrical detection technologies with novel DNA labeling chemistries has delivered unprecedented sensitivity, resolution, and throughput. These breakthroughs have opened new avenues for exploring complex genomic architectures, particularly in heterogeneous clinical samples such as tumors.
In the final phase, we applied our integrated toolbox in clinically relevant proof-of-concept experiments using patient-derived samples. These studies demonstrated the practical utility of our technologies for early disease detection, molecular diagnosis, and patient stratification. The comprehensive genomic signatures we obtained correlated strongly with clinical outcomes, underscoring the translational potential of our approach.
By the conclusion of the project, we validated our technologies in real-world clinical contexts, established standardized protocols, and disseminated our findings through high-impact publications, patent applications, and training workshops. Collectively, these outcomes confirm the feasibility and impact of multidimensional single-molecule genomic analysis and position these technologies for adoption in both research and clinical settings, setting a new benchmark in the field.
The impact and versatility of our technological platform is further evidenced by the successful securing of two additional Proof of Concept grants that build upon and extend the ERC project foundations: the Multiplex microRNA detection platform (Miracle) and Targeted Microarrays for 5-hydroxymethylcytosine-based Diagnosis of Hematological Malignancies (Base 6). These projects demonstrate the broader applicability of our core technologies beyond the original ERC scope.
In the final phase, we applied our integrated toolbox in clinically relevant proof-of-concept experiments using patient-derived samples. These studies demonstrated the practical utility of our technologies for early disease detection, molecular diagnosis, and patient stratification. The comprehensive genomic signatures we obtained correlated strongly with clinical outcomes, underscoring the translational potential of our approach.
By the conclusion of the project, we validated our technologies in real-world clinical contexts, established standardized protocols, and disseminated our findings through high-impact publications, patent applications, and training workshops. Collectively, these outcomes confirm the feasibility and impact of multidimensional single-molecule genomic analysis and position these technologies for adoption in both research and clinical settings, setting a new benchmark in the field.
The impact and versatility of our technological platform is further evidenced by the successful securing of two additional Proof of Concept grants that build upon and extend the ERC project foundations: the Multiplex microRNA detection platform (Miracle) and Targeted Microarrays for 5-hydroxymethylcytosine-based Diagnosis of Hematological Malignancies (Base 6). These projects demonstrate the broader applicability of our core technologies beyond the original ERC scope.