Periodic Reporting for period 3 - BeyondSeq (Genomic diagnostics beyond the sequence) Reporting period: 2018-05-01 to 2019-04-30 Summary of the context and overall objectives of the project Accurate diagnosis is a prerequisite for efficient health care. Ideally, if signs of pathogenesis could be detected before or at early stages of disease onset, then effective intervention and treatment may be administered. Many disease states are associate with genomic aberrations such as specific genetic mutations, epigenetic modifications or DNA damage lesions. Characterization of such aberrations at early stage relies on ultra-sensitive genomic analysis that is only partially addressed by the current available technologies. Diagnostics based on cytogenetic approaches provides information on the single-chromosome level but suffers from low resolution and low throughput. In contrast, next generation sequencing (NGS) based diagnostics provides single base resolution and high throughput but suffers from short reads that prevents the analysis of large genomic aberrations, as well as being prone to bias due to PCR-amplification and erasure of epigenetic information.The ground-breaking goal of this research project is to establish a robust set of diagnostic assays via single-molecule, genetic/epigenetic profiling of native chromosomal DNA. A successful accomplishment of this goal will allow low cost and rapid characterization of genomic aberrations and infectious organisms. Dissemination and commercialization of the developed technologies will open new horizons in biomedical diagnostics and personalized medicine. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far The beyondSeq project has fostered exciting new collaborations between the member teams. these collaborations yielded over 30 high publications, many in leading journals in the respective fields, and several others are currently in the pipeline. This high publication yield testifies to the quality of research and extent of scientific progress achieved by the partner teams. Upon completion of the project, several goals have materialized into exploitable outcomes that are now available to the research and biomedical community.• We have developed a DNA labeling toolbox that can be used for genetic mapping as well as for quantifying epigenetic modifications and DNA damage levels. Kits have been distributed to the research community and early steps towards commercialization of these tools have been taken. • Microfluidic devices for Cell and DNA sorting and purification have been developed and tested. These serve as components for an integrated Lab On Chip device which is yet to be developed. Nevertheless, the proof of concept experiments attracted commercial interest and funds were secured for continued development.• A screen for bacterial infections and antibiotic resistance has been introduced and tested on real life infection breakouts in hospitals. Funds were secured for larger scale clinical testing and assay automation.• A diagnostic screen for various cancers based on quantifying the epigenetic modification 5-hydroxymethylcytosine (5hmC) has been developed and tested successfully on 30 colon cancer patients and healthy individuals. Funds were secured for larger scale clinical testing.• An assay for diagnosis of colorectal and lung cancer via detection of KRAS mutations was developed and validated on medical samples. The assay is under continuous development towards clinical utility and commercialization.The strategy of diversifying various medical applications utilizing a set of single molecule tools has proven successful. Overall, many of the goals set front at the beginning of the project have been significantly approached, with several outcomes reaching readiness for commercialization. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) This research project aims to establish a robust experimental toolbox for genetic and epigenetic profiling of unamplified genomic DNA molecules. We focus on emerging DNA mapping technologies and their transition from lab to clinic with the potential to dramatically expand diagnostic capabilities based on genomic information.The accomplishments achieved within this project are expected to significantly promote the state-of-the-art within several key areas of both basic life science research and medical sciences. The developed toolbox and resulting assays will open new diagnosis and prognosis avenues and may lead to exciting new discoveries that will broadly impact the genetics and epigenetics research communities by providing unprecedented potential for studying the genomic aspects of pathogenesis.We specifically addressed three types of challenges to current genomic-based diagnostics: (i) Loss of relevant information such as DNA damage, rare mutations or epigenetic marks following PCR amplification.(ii) Limitations in resolving long-range variations in genomic layout, preventing large scale screens (including DNA repeats, structural variations and copy number variations). (iii) Limitations imposed by the sample such as low sample amounts (micro biopsy) or inhomogeneous/ highly variable samples (bacterial cultures)Within the scope of this project we have delivered a set of diagnostic tools in the form of functional assays that demonstrate the utility of our single-molecule approach and provide concrete solutions for pressing biomedical diagnostic challenges. The general concepts and methods developed in this project will open new avenues for the future development of additional innovative diagnostic tools. In addition, instrumentation and devices for deposition, sensing and imaging of DNA molecules have been developed and tested by several partner groups. The new devices have shown performance beyond the state of the art in terms of sensitivity and utility for single molecule characterization. Bridging the gap between Cytogenetics and next generation sequencing by single-molecule genomics
