Imaging the Genome
Figure 7: ORCA workflow and implementation in SRX. (A) An overview of ORCA workflow. A set of oligonucleotide probes is designed to label a genomic region of interest. Each segment carries a unique barcode and represents a single step of the ORCA walkthrough. Segments are imaged sequentially to build the 3D recon- struction of chromatin architecture. The 3D positioning of each segment is then used to build a distance map that is comparable to a Hi-C map. (B) A representative 3D reconstruction of chromatin architecture image visualized in SRX software. (C) A contact frequency map generated in SRX software by averaging the distance maps associated with ORCA walkthroughs of many cells. Panel A was adapted with permission from its original version [35].
When comparing STORM, OligoSTORM, and ORCA,
decreasing the step size increases the sequence resolution. STORM gives an overview of the shape of the structure without sequence information, OligoSTORM adds in the sequence context to the structure, and ORCA results in a finer sequence resolution but does not provide the space-filling component that is obtained with OligoSTORM. Te techniques are complementary, and their use depends on experimental capabilities, as well as the features being studied. ORCA is a good choice for a high-throughput study of a small genomic region, whereas OligoSTORM is preferred for tak- ing larger steps to image a larger genomic region. Te ultimate goal is to have seamless integration within
SRX of imaging data with standard genomic soſtware plat- forms, and analysis for a variety of genomic imaging work- flows, including ORCA and OligoSTORM. Tis will enable
2020 November •
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easier genomic modeling and Hi-C mapping of genomic data, helping to bridge the gap between single-cell imaging data and larger-scale data from ensemble methods.
References [1] J Dekker et al., Science 295 (2002) 1306–11. [2] S Sati and G Cavalli, Chromosoma 126 (2017) 33–44. [3] KS Matthews, Microbiol Rev 56 (1992) 123–36. [4] E Abbe, Arch Mikroskop Anat 9 (1873) 413–18. [5] SW Hell and J Wichmann, Optics Lett 19 (1994) 780–82. [6] MJ Rust et al., Nat Methods 3 (2006) 793–95. [7] E Betzig, Optics Lett 20 (1995) 237–39. [8] E Betzig et al., Science 313 (2006) 1642–45. [9] ST Hess et al., Biophys J 91 (2006) 4258–72. [10] MGL Gustafsson, J Microsc 198 (2000) 82–87.
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