Genomic mapping by anchoring random clones: a mathematical analysis.

A complete physical map of the DNA of an organism, consisting of overlapping clones spanning the genome, is an extremely useful tool for genomic analysis. Various methods for the construction of such physical maps are available. One approach is to assemble the physical map by "fingerprinting" a large number of random clones and inferring overlap between clones with sufficiently similar fingerprints. E.S. Lander and M.S. Waterman (1988, Genomics 2:231-239) have recently provided a mathematical analysis of such physical mapping schemes, useful for planning such a project. Another approach is to assemble the physical map by "anchoring" a large number of random clones--that is, by taking random short regions called anchors and identifying the clones containing each anchor. Here, we provide a mathematical analysis of such a physical mapping scheme.     

Genomic mapping by single copy landmark detection: a predictive model with a discrete mathematical approach

One of the goals of the Human Genome Project is to produce libraries of largely contiguous, ordered sets of molecular clones for use in sequencing and gene mapping projects. This is planned to be done for human and many model organisms. Theory and practice have shown that long-range contiguity and the degree to which the entire genome is covered by ordered clones can be affected by many biological variables. Many laboratories are currently experimenting with different experimental strategies and theoretical models to help plan strategies for accomplishing longrange molecular mapping of genomes. Here we describe a new mathematical model and formulas for helping to plan genome mapping projects, using various single-copy landmark (SCL) detection, or anchoring, strategies. We derive formulas that allow us to examine the effects of interactions among the following variables: average insert size of the cloning vector, average size of SCL, the number of SCL, and the redundancy in coverage of the clone library. We also examine and compare three different ways in which anchoring can be implemented: (1) anchors are selected independently of the library to be ordered (random anchoring); (2) anchors are made from end probes from both ends of clones in the library to be ordered (nonrandom anchoring); and (3) anchors are made from one end or the other, randomly, from clones in the library to be ordered (nonrandom anchoring). Our results show that, for biologically realistic conditions, nonrandom anchoring is always more effective than random anchoring for contig building, and there is little to be gained from making SCL from both ends of clones vs. only one end of clones. We compare and contrast our results with other similar mathematical models.     

Genome physical mapping with large-insert bacterial clones by fingerprint analysis: methodologies, source clone genome coverage, and contig map quality

Genome physical mapping with large-insert clones by fingerprint analysis is becoming an active area of genomics research. Here, we report two new capillary electrophoresis-based fingerprinting methods for genome physical mapping and the effects of different fingerprinting methods and source clone genome coverage on quality physical map construction revealed by computer simulations and laboratory experiments. It was shown that the manual sequencing gel-based two-enzyme fingerprinting method consistently generated larger and more accurate contigs, followed by the new capillary electrophoresis-based three-enzyme method, the new capillary electrophoresis-based five-enzyme (SNaPshot) method, the agarose gel-based one-enzyme method, and the automatic sequencing gel-based four-enzyme method, in descending order, when 1% or fewer questionable clones were allowed. Analysis of clones equivalent to 5x, 8x, 10x, and 15x genomes using the fingerprinting methods revealed that as the number of clones increased from 5x to 10x, the contig length rapidly increased for all methods. However, when the number of clones was increased from 10x to 15x coverage, the contig length at best increased at a lower rate or even decreased. The results will provide useful knowledge and strategies for effective construction of quality genome physical maps for advanced genomics research.     

Bacterial artificial chromosome-based physical map of the rice genome constructed by restriction fingerprint analysis

Genome-wide physical mapping with bacteria-based large-insert clones (e.g., BACs, PACs, and PBCs) promises to revolutionize genomics of large, complex genomes. To accelerate rice and other grass species genome research, we developed a genome-wide BAC-based map of the rice genome. The map consists of 298 BAC contigs and covers 419 Mb of the 430-Mb rice genome. Subsequent analysis indicated that the contigs constituting the map are accurate and reliable. Particularly important to proficiency were (1) a high-resolution, high-throughput DNA sequencing gel-based electrophoretic method for BAC fingerprinting, (2) the use of several complementary large-insert BAC libraries, and (3) computer-aided contig assembly. It has been demonstrated that the fingerprinting method is not significantly influenced by repeated sequences, genome size, and genome complexity. Use of several complementary libraries developed with different restriction enzymes minimized the "gaps" in the physical map. In contrast to previous estimates, a clonal coverage of 6.0-8.0 genome equivalents seems to be sufficient for development of a genome-wide physical map of approximately 95% genome coverage. This study indicates that genome-wide BAC-based physical maps can be developed quickly and economically for a variety of plant and animal species by restriction fingerprint analysis via DNA sequencing gel-based electrophoresis.     

DNA fiber mapping techniques for the assembly of high-resolution physical maps.

High-resolution physical maps are indispensable for directed sequencing projects or the finishing stages of shotgun sequencing projects. These maps are also critical for the positional cloning of disease genes and genetic elements that regulate gene expression. Typically, physical maps are based on ordered sets of large insert DNA clones from cosmid, P1/PAC/BAC, or yeast artificial chromosome (YAC) libraries. Recent technical developments provide detailed information about overlaps or gaps between clones and precisely locate the position of sequence tagged sites or expressed sequences, and thus support efforts to determine the complete sequence of the human genome and model organisms. Assembly of physical maps is greatly facilitated by hybridization of non-isotopically labeled DNA probes onto DNA molecules that were released from interphase cell nuclei or recombinant DNA clones, stretched to some extent and then immobilized on a solid support. The bound DNA, collectively called "DNA fibers," may consist of single DNA molecules in some experiments or bundles of chromatin fibers in others. Once released from the interphase nuclei, the DNA fibers become more accessible to probes and detection reagents. Hybridization efficiency is therefore increased, allowing the detection of DNA targets as small as a few hundred base pairs. This review summarizes different approaches to DNA fiber mapping and discusses the detection sensitivity and mapping accuracy as well as recent achievements in mapping expressed sequence tags and DNA replication sites.     

Randomly picked cosmid clones overlap the pyrB and oriC gap in the physical map of the E. coli chromosome.

Sets of overlapping cosmid clones generated by random sampling and fingerprinting methods complement data at pyrB (96.5') and oriC (84') in the published physical map of E. coli. A new cloning strategy using sheared DNA, and a low copy, inducible cosmid vector were used in order to reduce bias in libraries, in conjunction with micro-methods for preparing cosmid DNA from a large number of clones. Our results are relevant to the design of the best approach to the physical mapping of large genomes.     

Modeling DNA shuffling.

In vitro evolution is a new, important laboratory method to evolve molecules with desired properties. It has been used in a variety of biological studies and drug development. In this paper, we study one important mutagenesis method used in in vitro evolution experiments called DNA shuffling. We construct a mathematical model for DNA shuffling and study the properties of molecules after DNA shuffling experiments based on this model. The model for DNA shuffling consists of two parts. First we apply the Lander-Waterman model for physical mapping by fingerprinting random clones to model the distribution of regions that can be reassembled through DNA shuffling. Then we present a model for recombination between different DNA species with different mutations. We compare our theoretical results with experimental data. Finally we propose novel applications of the theoretical results to the optimal design of DNA shuffling experiments and to physical mapping using DNA shuffling.     

Genome physical mapping from large-insert clones by fingerprint analysis with capillary electrophoresis: a robust physical map of Penicillium chrysogenum

Physical mapping with large-insert clones is becoming an active area of genomics research, and capillary electrophoresis (CE) promises to revolutionize the physical mapping technology. Here, we demonstrate the utility of the CE technology for genome physical mapping with large-insert clones by constructing a robust, binary bacterial artificial chromosome (BIBAC)-based physical map of Penicillium chrysogenum. We fingerprinted 23.1× coverage BIBAC clones with five restriction enzymes and the SNaPshot kit containing four fluorescent-ddNTPs using the CE technology, and explored various strategies to construct quality physical maps. It was shown that the fingerprints labeled with one or two colors, resulting in 40–70 bands per clone, were assembled into much better quality maps than those labeled with three or four colors. The selection of fingerprinting enzymes was crucial to quality map construction. From the dataset labeled with ddTTP–dROX, we assembled a physical map for P.chrysogenum, with 2–3 contigs per chromosome and anchored the map to its chromosomes. This map represents the first physical map constructed using the CE technology, thus providing not only a platform for genomic studies of the penicillin-producing species, but also strategies for efficient use of the CE technology for genome physical mapping of plants, animals and microbes.     

Characterization of a new BAC library for rainbow trout: evidence for multi-locus duplication

A 10X rainbow trout bacterial artificial chromosome (BAC) library was constructed to aid in the physical and genetic mapping efforts of the rainbow trout genome. The library was derived from the Swanson clonal line (YY male) and consists of 184,704 clones with an average insert size of 137,500 bp (PFGE) or 118,700 bp (DNA fingerprinting). The clones were gridded onto 10 large nylon membranes to produce high-density arrays for screening the library by hybridization. The library was probed with 11 cDNAs from the NCCCWA EST project chosen because of interest in their homology to known gene sequences, seven known genes, and a Y-specific sex marker. Putative positive clones identified by hybridization were re-arrayed and gridded for secondary confirmation. FPC analysis of HindIII and EcoRV DNA fingerprinting was used to estimate the level of redundancy in the library, to construct BAC contigs and to detect duplicated loci in the semi-duplicated rainbow trout genome. A good correlation (R2 = 0.7) was found between the number of hits per probe and the number of contigs that were assembled from the positive BACs. The average number of BACs per contig was 9.6, which is in good agreement with 10X genome coverage of the library. Two-thirds of the loci screened were predicted to be duplicated as the positive BACs for those genes were assembled into two or three different contigs, which suggests that most of the rainbow trout genome is duplicated.     

A BAC-based physical map of Zhikong scallop (Chlamys farreri Jones et Preston)

Zhikong scallop (Chlamys farreri) is one of the most economically important aquaculture species in China. Physical maps are crucial tools for genome sequencing, gene mapping and cloning, genetic improvement and selective breeding. In this study, we have developed a genome-wide, BAC-based physical map for the species. A total of 81,408 clones from two BAC libraries of the scallop were fingerprinted using an ABI 3130xl Genetic Analyzer and a fingerprinting kit developed in our laboratory. After data processing, 63,641 (～5.8× genome coverage) fingerprints were validated and used in the physical map assembly. A total of 3,696 contigs were assembled for the physical map. Each contig contained an average of 10.0 clones, with an average physical size of 490 kb. The combined total physical size of all contigs was 1.81 Gb, equivalent to approximately 1.5 fold of the scallop haploid genome. A total of 10,587 BAC end sequences (BESs) and 167 markers were integrated into the physical map. We evaluated the physical map by overgo hybridization, BAC-FISH (fluorescence in situ hybridization), contig BAC pool screening and source BAC library screening. The results have provided evidence of the high reliability of the contig physical map. This is the first physical map in mollusc; therefore, it provides an important platform for advanced research of genomics and genetics, and mapping of genes and QTL of economical importance, thus facilitating the genetic improvement and selective breeding of the scallop and other marine molluscs.     

On the Consistency of a Physical Mapping Method to Reconstruct a Chromosome in Vitro

During recent years considerable effort has been invested in creating physical maps for a variety of organisms as part of the Human Genome Project and in creating various methods for physical mapping. The statistical consistency of a physical mapping method to reconstruct a chromosome, however, has not been investigated. In this paper, we first establish that a model of physical mapping by binary fingerprinting of DNA fragments is identifiable using the key assumption--for a large randomly generated recombinant DNA library, there exists a staircase of DNA fragments across the chromosomal region of interest. Then we briefly introduce epi-convergence theory of variational analysis and transform the physical mapping problem into a constrained stochastic optimization problem. By doing so, we prove epi-convergence of the physical mapping model and epi-convergence of the physical mapping method. Combining the identifiability of our physical mapping model and the epi-convergence of a physical mapping method, finally we establish strong consistency of a physical mapping method.     

Contig selection in physical mapping.

In physical mapping, one orders a set of genetic landmarks or a library of cloned fragments of DNA according to their position in the genome. Our approach to physical mapping divides the problem into smaller and easier subproblems by partitioning the probe set into independent parts (probe contigs). For this purpose we introduce a new distance function between probes, the averaged rank distance (ARD) derived from bootstrap resampling of the raw data. The ARD measures the pairwise distances of probes within a contig and smoothes the distances of probes across different contigs. It shows distinct jumps at contig borders. This makes it appropriate for contig selection by clustering. We have designed a physical mapping algorithm that makes use of these observations and seems to be particularly well suited to the delineation of reliable contigs. We evaluated our method on data sets from two physical mapping projects. On data from the recently sequenced bacterium Xylella fastidiosa, the probe contig set produced by the new method was evaluated using the probe order derived from the sequence information. Our approach yielded a basically correct contig set. On this data we also compared our method to an approach which uses the number of supporting clones to determine contigs. Our map is much more accurate. In comparison to a physical map of Pasteurella haemolytica that was computed using simulated annealing, the newly computed map is considerably cleaner. The results of our method have already proven helpful for the design of experiments aimed at further improving the quality of a map.     

Construction and partial characterization of a Corynebacterium pseudotuberculosis bacterial artificial chromosome library through genomic survey sequencing

Corynebacterium pseudotuberculosis is a gram-positive bacterium that causes caseous lymphadenitis in sheep and goats. However, despite the economic losses caused by caseous lymphadenitis, there is little information about the molecular mechanisms of pathogenesis of this bacterium. Genomic libraries constructed in bacterial artificial chromosome (BAC) vectors have become the method of choice for clone development in high-throughput genomic-sequencing projects. Large-insert DNA libraries are useful for isolation and characterization of important genomic regions and genes. In order to identify targets that might be useful for genome sequencing, we constructed a C. pseudotuberculosis BAC library in the vector pBeloBAC11. This library contains about 18,000 BAC clones, with inserts ranging in size from 25 to 120 kb, theoretically representing a 390-fold coverage of the C. pseudotuberculosis genome (estimated to be 2.5-3.1 Mb). Many genomic survey sequences (GSSs) with homology to C. diphtheriae, C. glutamicum, C. efficiens, and C. jeikeium proteins were observed within a sample of 215 sequenced clones, confirming their close phylogenetic relationship. Computer analyses of GSSs did not detect chimeric, deleted, or rearranged BAC clones, showing that this library has low redundancy. This GSSs collection is now available for further genetic and physical analysis of the C. pseudotuberculosis genome. The GSS strategy that we used to develop our library proved to be efficient for the identification of genes and will be an important tool for mapping, assembly, comparative, and functional genomic studies in a C. pseudotuberculosis genome sequencing project that will begin this year.     

Physical mapping of plasmid and cosmid clones in filamentous fungi by fiber-FISH

Fluorescence in situ hybridization to extended DNA fibers (fiber-FISH) serves as a powerful tool for direct physical mapping in plants and animals. Here, we show that fiber-FISH is useful for contig mapping as well as for estimating the physical distance between genetic markers in fungi. A five-cosmid contig from a chromosome of Nectria haematococca and four cloned genetic markers from a linkage map of Cochliobolus heterostrophus were chosen as models for the application of this technology. In N. haematococca, overlapping and non-overlapping clones were visually mapped on individual DNA fibers, confirming the results from conventional physical mapping perfectly. Fiber-FISH concomitantly indicated the gap size or the extent of overlap between two clones. In C. heterostrophus, the physical distance between the two pairs of genetic markers could be estimated from the microscopic measurements of the intervals. Chromosomal DNA isolated from a pulsed field gel was suitable for preparing the DNA fibers.     

Whole-genome validation of high-information-content fingerprinting

Fluorescent-based high-information-content fingerprinting (HICF) techniques have recently been developed for physical mapping. These techniques make use of automated capillary DNA sequencing instruments to enable both high-resolution and high-throughput fingerprinting. In this article, we report the construction of a whole-genome HICF FPC map for maize (Zea mays subsp. mays cv B73), using a variant of HICF in which a type IIS restriction enzyme is used to generate the fluorescently labeled fragments. The HICF maize map was constructed from the same three maize bacterial artificial chromosome libraries as previously used for the whole-genome agarose FPC map, providing a unique opportunity for direct comparison of the agarose and HICF methods; as a result, it was found that HICF has substantially greater sensitivity in forming contigs. An improved assembly procedure is also described that uses automatic end-merging of contigs to reduce the effects of contamination and repetitive bands. Several new features in FPC v7.2 are presented, including shared-memory multiprocessing, which allows dramatically faster assemblies, and automatic end-merging, which permits more accurate assemblies. It is further shown that sequenced clones may be digested in silico and located accurately on the HICF assembly, despite size deviations that prevent the precise prediction of experimental fingerprints. Finally, repetitive bands are isolated, and their effect on the assembly is studied.     

Theoretical analysis of library screening using a N-dimensional pooling strategy.

A solution to the problem of library screening is analysed. We examine how to retrieve those clones that are positive for a single copy landmark from a whole library while performing only a minimum number of laboratory tests: the clones are arranged on a matrix (i.e in 2 dimensions) and pooled according to the rows and columns. A fingerprint is determined for each pool and an analysis allows selection of a list containing all the positive clones, plus a few false positives. These false positives are eliminated by using another (or several other) matrix which has to be reconfigured in a way as different as possible from the previous one. We examine the use of cubes (3 dimensions) or hypercubes of any dimension instead of matrices and analyse how to reconfigure them in order to eliminate the false positives as efficiently as possible. The advantage of the method proposed is the low number of tests required and the low number of pools that require to be prepared [only 258 pools and 282 tests (258 + 24 verifications) are needed to screen the 72,000 clones of the CEPH YAC library (1) with a sequence-tagged site]. Furthermore, this method allows easy and systematic screenings and can be applied to a large physical mapping project, which will lead to an interesting map with a low, precisely known, rate of error: when fingerprinting a 150 Mb chromosome with the CEPH YAC library and 1750 sequence-tagged sites, 903,000 tests would be necessary to obtain about 20 contigs of an average length of 6.7 Mb, while only about one false positive would be expected in the resultant map. Finally, STSs can be ordered by dividing a clone library into sublibraries (corresponding to groups of microplates for example) and testing each STS on pooled clones from each sublibrary. This allows to dedicate to each STSs a fingerprint that consists in the list of the positive pools. In many cases these fingerprints will be enough to order the STSs. Indeed if large YACs (greater than 1 Mb) can be obtained, the combined screening of DNA families and YAC DNA pools would allow an integrated construction of both genetic and physical maps of the human genome, that will also reduce the optimal number of meioses needed for a 1 centimorgan linkage map.     

Construction and characterization of two rice bacterial artificial chromosome libraries from the parents of a permanent recombinant inbred mapping population

Rice is a leading grain crop and the staple food for over half of the world population. Rice is also an ideal species for genetic and biological studies of cereal crops and other monocotyledonous plants because of its small genome and well developed genetic system. To facilitate rice genome analysis leading to physical mapping, the identification of molecular markers closely linked to economic traits, and map-based cloning, we have constructed two rice bacterial artificial chromosome (BAC) libraries from the parents of a permanent mapping population (Lemont and Teqing) consisting of 400 F9 recombinant inbred lines (RILs). Lemont (japonica) and Teqing (indica) represent the two major genomes of cultivated rice, both are leading commercial varieties and widely used germplasm in rice breeding programs. The Lemont library contains 7296 clones with an average insert size of 150 kb, which represents 2.6 rice haploid genome equivalents. The Teqing library contains 14208 clones with an average insert size of 130 kb, which represents 4.4. rice haploid genome equivalents. Three single-copy DNA probes were used to screen the libraries and at least two overlapping BAC clones were isolated with each probe from each library, ranging from 45 to 260 kb in insert size. Hybridization of BAC clones with chloroplast DNA probes and fluorescent in situ hybridization using BAC DNA as probes demonstrated that both libraries contain very few clones of chloroplast DNA origin and are likely free of chimeric clones. These data indicate that both BAC libraries should be suitable for map-based cloning of rice genes and physical mapping of the rice genome.     

Chromosomal assignment of the canine melanophilin gene (MLPH): a candidate gene for coat color dilution in Pinschers

Pinschers affected by coat color dilution show a specific pigmentation phenotype. The dilute pigmentation phenotype leads to a silver-blue appearance of the eumelanin-containing fur and a pale sandy color of pheomelanin-containing fur. In Pinscher breeding, dilute black-and-tan dogs are called "blue," and dilute red or brown animals are termed "fawn" or "Isabella fawn." Coat color dilution in Pinschers is sometimes accompanied by hair loss and a recurrent infection of the hair follicles. In human and mice, several well-characterized genes are responsible for similar pigment variations. To investigate the genetic cause of the coat color dilution in Pinschers, we isolated BAC clones containing the canine ortholog of the known murine color dilution gene Mlph. RH mapping of the canine MLPH gene was performed using an STS marker derived from BAC sequences. Additionally, one MLPH BAC clone was used as probe for FISH mapping, and the canine MLPH gene was assigned to CFA25q24.     

Application of restriction fragment fingerprinting with a rice microsatellite sequence to assembling rice YAC clones.

To refine the current physical map of rice, we have established a restriction fragment fingerprinting method for identifying overlap between pairs of rice yeast artificial chromosome (YAC) clones and defining the physical arrangement of YACs within contiguous fragments (contigs). In this method, Southern blots of rice YAC DNAs digested with a restriction endonuclease are probed with a rice microsatellite probe, (GGC)5. The probe produces a unique fingerprint profile characteristic of each YAC clone. The profile is then digitized, processed in a computer, and a statistic that represents the degree of overlap between two YACs is calculated. The statistics have been used to detect overlaps among YAC clones, thereby filling a gap between two neighbouring contigs and organizing overlapping rice YAC clones into contiguous fragments. We applied this method to rearranging YACs that had previously been assigned to rice chromosome 6 by anchoring with RFLP markers.