1 Super-Resolution Genome Mapping in Silicon Nanochannels Jonathan Jeffet † , Asaf Kobo † , Tianxiang Su ⊥ ○, Assaf Grunwald † , Ori Green † , Adam N. Nilsson § , Eli Eisenberg † , Tobias Ambjörnsson § , Fredrik Westerlund ∥ , Elmar Weinhold ∇ , Doron Shabat † , Prashant K. Purohit ‡ , and Yuval Ebenstein *† † Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Tel Aviv 6997801, Israel ‡ Department of Mechanical Engineering and Applied Mechanics, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States ⊥ School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts 02138, United States § Department of Astronomy and Theoretical Physics, Lund University, SE-221 00 Lund, Sweden ∥ Department of Biology and Biological Engineering, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden ∇ Institute of Organic Chemistry, RWTH Aachen University, Aachen D-52056, Germany ABSTRACT: Optical genome mapping in nanochannels is a powerful genetic analysis method, complementary to deoxyribonucleic acid (DNA) sequencing. The method is based on detecting a pattern of fluorescent labels attached along individual DNA molecules. When such molecules are extended in nanochannels, the labels create a fluorescent genetic barcode that is used for mapping the DNA molecule to its genomic locus and identifying large-scale variation from the genome reference. Mapping resolution is currently limited by two main factors: the optical diffraction limit and the thermal fluctuations of DNA molecules suspended in the nanochannels. Here, we utilize single-molecule tracking and super-resolution localization in order to improve the mapping accuracy and resolving power of this genome mapping technique and achieve a 15-fold increase in resolving power compared to currently practiced methods. We took advantage of a naturally occurring genetic repeat array and labeled each repeat with custom-designed Trolox conjugated fluorophores for enhanced photostability. This model system allowed us to acquire extremely long image sequences of the equally spaced fluorescent markers along DNA molecules, enabling detailed characterization of nanoconfined DNA dynamics and quantitative comparison to the Odijk theory for confined polymer chains. We present a simple method to overcome the thermal fluctuations in the nanochannels and exploit single-step photobleaching to resolve subdiffraction spaced fluorescent markers along fluctuating DNA molecules with ∼100 bp resolution. In addition, we show how time-averaging over just ∼50 frames of 40 ms enhances mapping accuracy, improves mapping P-value cores by 3 orders of magnitude compared to nonaveraged alignment, and provides a significant advantage for analyzing structural variations between DNA molecules with similar sequence composition. KEYWORDS: nanochannels, super-resolution, single-molecule, optical genome mapping, DNA labeling, confined polymers With recent advancements in nanofabrication and single-molecule microscopy, optical genome mapping has reemerged as a valuable complement to deoxyribonucleic acid (DNA) sequencing. 1,2 The method is based on labeling long DNA fragments with fluorescent molecules indicating specific sequence motifs to create a specific pattern along the DNA that serves as a barcode for genetic identification 3–5 (Figure 1A). The DNA is then extended in nanofluidic channels and visualized by fluorescence microscopy, revealing the DNA contour decorated with a pattern of fluorescent spots