Shiguo Zhou, Jill Herschleb and David C. Schwartz

Laboratory for Molecular and Computational Genomics, UW Biotechnology Center, Laboratory of Genetics and Department of Chemistry, University of Wisconsin, 425 Henry

Mall, Madison, 53706, USA


Abstract 266

1. Introduction 266

1.1. Presentation of long, restriction enzyme-digested DNA molecules 267

1.2. Image acquisition, processing, and machine vision: moving from images to data files 269

1.3. Data management, system network, map construction and analysis tools 269 1.3.1. Summary 271

1.4. The history of optical mapping 271

2. The optical mapping system 273

2.1. DNA preparation methods for optical mapping 273

2.1.1. Limitations and constraints of dealing with large DNA molecules: shearing and PFGE sample preparation 274

2.1.2. Extraction of DNA from PFGE inserts 274

2.1.3. Direct DNA extraction via heat lysis 274

2.2. Optical mapping surface preparation 275

2.2.1. Surface cleaning 275

2.2.2. Silane derivitization 276

2.3. Microfluidic device fabrication 276

2.4. DNA mounting, overlay, digestion, and staining 277

2.4.1. Mounting/overlay 278

2.4.2. Digestion and staining 279

3. The optical mapping system: image acquisition, processing, and analysis 280

3.1. A single molecule scanning system - ''Genome Zephyr'' 280

3.2. Constructing single molecule restriction maps from fluorescence micrographs 282

3.2.1. FlatOverMerge 282

3.2.2. PathFinder 283

3.3. Data storage, file management and visualization 284

3.4. Optical map assembly and alignment 285 3.4.1. De novo map assembly 285

PERSPECTIVES IN BIOANALYSIS, VOLUME 2 ISSN 1871-0069 DOI: 10.1016/S1871-0069(06)02009-X

© 2007 Elsevier B.V. All rights reserved

3.4.2. Map Aligner: pairwise alignment of single DNA molecule optical maps against a reference map 286

3.4.3. Cluster computing 286

4. Applications of optical mapping 287

4.1. Use of optical maps to dissect complex genome structures and facilitate sequence assembly 287

4.2. Use of optical maps for microbial comparative genomics 287

4.3. Use of optical maps for microbial identification and infectious disease diagnosis 288

4.4. Discovering structural alterations in mammalian genomes 291

5. Comparison of optical mapping and alternate methods for genome analysis 292

5.1. Microarray-based methods 292

5.2. Pulsed-field gel electrophoresis 294

5.3. Cytogenetics 294

5.4. Paired-end sequencing 294

6. Optical sequencing 294 References 298


Optical mapping is a fully automated single molecule system for creating ordered restriction maps directly from genomic DNA molecules. The system has integrated components drawn from chemistry, physics, genetics, computer science, statistics, and engineering allowing whole genome analysis through analysis of large collections of individual DNA molecules. Such large-scale analysis is potentiated by analytes consisting of high molecular weight genomic DNA molecules that are arrayed using microfluidic devices; this step fosters high-throughput acquisition of image data. DNA "barcodes" are created using the action of restriction endonucleases on arrayed molecules from the analysis of images acquired by automated microscopy embedded within an integrated system. This system links together machine vision, barcode assembly and comparative analysis software with the massive computational power of cluster computing. After assembly of these barcodes into genome-wide physical maps, complex genome structure is revealed, characterizing genomic alterations, in addition to providing scaffolds for genome sequence assembly, or validation. Further sequence information is obtainable from barcoded DNA molecules using optical sequencing technology, delivering strings of nucleotide data from barcoded loci.

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