Constructing single molecule restriction maps from fluorescence micrographs

3.2.1. FlatOverMerge

After acquired raw images are stored they are processed and merged together to form composite images of the original 48 microchannels of DNA deposition. This is achieved via another software program - FlatOverMerge. Although laser illumination is spatially even, further correction is necessary for accurate fluorescence intensity measurements. For this purpose, the shape and contour of the illumination field is calibrated using a photoresist-coated slide prior to sample collection (the even, spun coat of photoresist uniformly fluoresces upon illumination). ChannelCollect exports these measurements to another program, OverlapAndMerge, for additional processing. Using this calibration curve,

Fig. 5. Image processing. (A) Raw micrographs from CCD camera. (B) A raw image (left) is flattened by FlatOverMerge (right). (C) Images are overlapped into a composite microchannel view by FlatOverMerge. (D) Genomic DNA molecules are identified by PathFinder, and single molecule ordered restriction maps are generated.

Fig. 5. Image processing. (A) Raw micrographs from CCD camera. (B) A raw image (left) is flattened by FlatOverMerge (right). (C) Images are overlapped into a composite microchannel view by FlatOverMerge. (D) Genomic DNA molecules are identified by PathFinder, and single molecule ordered restriction maps are generated.

FlatOverMerge sequentially "flattens" every image, by calculating corrections for uneven illumination on a per pixel basis. Using the slight overlap between images as a guide, it correctly orients and merges contiguous images together, forming a train of overlapped images. These images record the entire length of an imaged stripe of deposited DNA molecules. This process is illustrated in Figure 5.

3.2.2. PathFinder

PathFinder is another software program written to identify and generate single DNA molecule restriction maps from these composite images. PathFinder performs four primary functions to obtain finalized data:

i. Segmentation: DNA molecules are roughly isolated from background by examining image-wide pixel intensities and setting an appropriate threshold for cutoff. The precise boundaries of each DNA restriction fragment are generated by analysis of local pixel intensity gradients and an additional thresholding cutoff. Importantly, this cutoff is automatically determined.

ii. Identification of DNA molecule backbones: A greatly modified version of Dijkstra's algorithm is used to generate a series of lines connecting the DNA molecules identified in the segmentation step. The resulting series of connected lines traverse down the length of each DNA molecule, and generally span several contiguous images.

iii. Identification of restriction fragments: Restriction enzyme cuts manifest a distinctive morphology that facilitates recognition by PathFinder. Since DNA molecules are under tension on the optical mapping surfaces, the 50-and 30-end of each restriction fragment recoils slightly, leaving a 1-2 mm visible gap. Accordingly, the ends of DNA restriction fragments show additional fluorescence due to ''DNA balls'' that result from coil relaxation at these newly formed ends. An optimized series of measurements is used by PathFinder to detect these morphological features and distinguish true restriction sites from normal fluctuations in DNA stretching and reflected by variation in fluorescence intensity.

iv. Determine the mass of each restriction fragment: The mass of each restriction fragment is determined by its integrated fluorescence intensity value. As mentioned above, DNA molecules show some fluctuations in terms of elongation on the optical mapping surfaces. Using fluorescence intensity as a measure of mass obviates any errors due to purely length-based measurements. Internal standards are added to every experiment to facilitate accurate sizing. Typically, viral DNA of known sequence and cut pattern is added to the genomic DNA preparation at a concentration of ~20pg/ml. Despite a series of errors stemming from a diverse set of issues, i.e., uneven fluorochrome incorporation, anomalous fluorescence intensity measurements, etc., PathFinder is able to determine the mass of genomic DNA fragments with a precision of ~ + 10%. By averaging multiple measurements (from multiple restriction maps) at each locus, a finalized genome-wide optical map typically determines fragment sizes with an accuracy of 2-5%.

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