Physical Mapping Projects Research Articles

“A Dot on a Map”: Cartographies of Erasure in Garifuna Territory

AbstractThis article explores the complexities of territorial dispossession in a post‐Washington‐Consensus global development policy context. In particular, it explores a contemporary development paradox in Honduras: the transnational recognition of the rights of indigenous people alongside massive land dispossession of the Afro‐Indigenous Garifuna in the name of development. Cartography is considered both in terms of physical mapping projects and ideological boundary‐making through rhetorical dispossession. In state‐sponsored communal mapping projects from the late 1990s into the early 2000s, the Garifuna were denied both currently inhabited land and that which they historically accessed. Anything that sat beyond mapped borders became “open” to foreign purchase. Legislation passed after the 2009 coup d’état further erased the Garifuna's historical occupation of coastal lands by embracing "model city” development and megatourism. Despite post–Washington Consensus development discourses of equality and official rhetoric of inclusion and celebration of indigenous rights, this case study demonstrates cartographic processes continue to erase Garifuna historical rights to territory.

Assembling global maps of cellular function through integrative analysis of physical and genetic networks

To take full advantage of high-throughput genetic and physical interaction mapping projects, the raw interactions must first be assembled into models of cell structure and function. PanGIA (for physical and genetic interaction alignment) is a plug-in for the bioinformatics platform Cytoscape, designed to integrate physical and genetic interactions into hierarchical module maps. PanGIA identifies 'modules' as sets of proteins whose physical and genetic interaction data matches that of known protein complexes. Higher-order functional cooperativity and redundancy is identified by enrichment for genetic interactions across modules. This protocol begins with importing interaction networks into Cytoscape, followed by filtering and basic network visualization. Next, PanGIA is used to infer a set of modules and their functional inter-relationships. This module map is visualized in a number of intuitive ways, and modules are tested for functional enrichment and overlap with known complexes. The full protocol can be completed between 10 and 30 min, depending on the size of the data set being analyzed.

A simple, high throughput method to locate single copy sequences from Bacterial Artificial Chromosome (BAC) libraries using High Resolution Melt analysis

BackgroundThe high-throughput anchoring of genetic markers into contigs is required for many ongoing physical mapping projects. Multidimentional BAC pooling strategies for PCR-based screening of large insert libraries is a widely used alternative to high density filter hybridisation of bacterial colonies. To date, concerns over reliability have led most if not all groups engaged in high throughput physical mapping projects to favour BAC DNA isolation prior to amplification by conventional PCR.ResultsHere, we report the first combined use of Multiplex Tandem PCR (MT-PCR) and High Resolution Melt (HRM) analysis on bacterial stocks of BAC library superpools as a means of rapidly anchoring markers to BAC colonies and thereby to integrate genetic and physical maps. We exemplify the approach using a BAC library of the model plant Arabidopsis thaliana. Super pools of twenty five 384-well plates and two-dimension matrix pools of the BAC library were prepared for marker screening. The entire procedure only requires around 3 h to anchor one marker.ConclusionsA pre-amplification step during MT-PCR allows high multiplexing and increases the sensitivity and reliability of subsequent HRM discrimination. This simple gel-free protocol is more reliable, faster and far less costly than conventional PCR screening. The option to screen in parallel 3 genetic markers in one MT-PCR-HRM reaction using templates from directly pooled bacterial stocks of BAC-containing bacteria further reduces time for anchoring markers in physical maps of species with large genomes.

Use of Commercially Available Radiation Hybrid Panels

Several panels are available for puchase, and this unit provides update information on the use of the three commercially available panels and on the interpretation of mapping results using the Internet. Radiation hybrid panels continue to serve as integral biological reagents in physical mapping projects and positional cloning.

Integrated Mapping Package—A Physical Mapping Software Tool Kit

We have developed an integrated physical mapping computer software package (IMP), originally designed to support the physical mapping of human chromosome 13 and expanded to support several gene-identification projects based on the positional candidate approach. IMP displays map data in a form that provides useful guidelines to the end users. An integrated map with high resolution and confidence is constructed from different types of mapping data, including hybridization experiments, STS-based PCR assays, genetic linkage mapping, cDNA localization, and FISH data. The map is also designed to provide suggestions for specific experiments that are required to obtain maps with even higher resolution and confidence. To this end, the optimization employs multiple constraints that take into account already established STS “scaffold” maps. This software thus serves as an important general tool kit for physical mapping, sequencing, and gene-hunting projects.

Modification of bacterial artificial chromosome clones using Cre recombinase: introduction of selectable markers for expression in eukaryotic cells.

Bacterial artificial chromosome clones (BACs) are widely used at present in human genome physical mapping projects. To extend the utility of these clones for functional genomic studies, we have devised a method to modify BACs using Cre recombinase to introduce a gene cassette into the loxP sequence, which is present in the vector portion of the BAC clone. Cre-mediated integration is site specific and thus maintains the integrity of the genomic insert sequences, while eliminating the steps that are involved in restriction digest-based DNA cloning strategies. The success of this method depends on the use of a DNA construct, RETRObac, which contains the reporter marker green fluorescent protein (GFP) and the selectable marker neomycin phosphotransferase (neo), but does not contain a bacterial origin of replication. BAC clones have been modified successfully using this method and the genomic insert shows no signs of deletions or rearrangements. Transfection efficiencies of the modified BACs into human or murine cell lines ranged from 1% to 6%. After culture in media containing G418 for 3 weeks, approximately 0. 1% of cells previously sorted for GFP expression acquired stable antibiotic resistance. Introduction of a human BAC clone that contains genomic p53 sequences into murine NIH3T3 cells led to expression of human p53 mRNA as determined by RT-PCR, demonstrating that sequences contained on the BAC are expressed. We believe that GFP-neo modified BAC clones will be a valuable resource in efforts to study biological effects of known genes as well as in efforts to clone and analyze new genes and regulatory regions.

HOSEpipe--a WWW-hosted data management and analysis system for STS content mapping projects.

We have developed a data management system, 'HOSEpipe' (High Output STS Evaluation pipeline) to aid sample tracking and data analysis in STS content mapping projects. The system is based around a World Wide Web (WWW) server that provides a number of pages including forms for sample processing and data entry accessible via a standard WWW browser application. The system is split into two main modules: firstly, a sequence evaluation and annotation module that takes de novo sequence for a potential STS, screens it against existing STSs and DNA sequence databases, followed by appropriate primer sequence design; secondly, a module that handles YAC library STS screening and includes facilities for both sample tracking and experimental data analysis. We present the design and rationale of the HOSEpipe system and its development to support a whole chromosomal physical mapping project. This software and design approach is potentially applicable to physical mapping projects of varying sizes and resolution and to similar projects, such as sample sequencing and the construction of sequence-ready maps.

Coverage processes in physical mapping by anchoring random clones.

The aim of this paper is to provide general results for predicting progress in a physical mapping project by anchoring random clones, when clones and anchors are not homogeneously distributed along the genome. A complete physical map of the DNA of an organism consists of overlapping clones spanning the entire genome. Several schemes can be used to construct such a map, depending on the way that clones overlap. We focus here on the approach consisting of assembling clones sharing a common random short sequence called an anchor. Some mathematical analyses providing statistical properties of anchored clones have been developed in the stationary case. Modeling the clone and anchor processes as nonhomogeneous Poisson processes provides such an analysis in a general nonstationary framework. We apply our results to two natural nonhomogeneous models to illustrate the effect of inhomogeneity. This study reveals that using homogeneous processes for clones and anchors provides an overly optimistic assessment of the progress of the mapping project.

Algorithms for computing and integrating physical maps using unique probes.

Current physical mapping projects based on STS-probes involve additional clues such as the fact that some probes are anchored to a known map and that others come from the ends of clones. Because of the disparate combinatorial contributions of these varied data items, it is difficult to design a "tailored" algorithm that incorporates them all. Moreover, it is inevitable that new experiments will provide new kinds of data, making obsolete any such algorithm. We show how to convert the physical mapping problem into a 0/1 linear programming (LP) problem. We further show how one can incorporate additional clues as additional constraints in the LP formulation. We give a simple relaxation of the 0/1 LP problem, which solves problems of the same scale as previously reported tailored algorithms, to equal or greater optimization levels. We also present a theorem proving that when the data is 100% accurate, then the relaxed and integer solutions coincide. The LP algorithm suffices to solve problems on the order of 80-100 probes--the typical size of the 2- or 3-connected contigs of Arratia et al. (1991). We give a heuristic algorithm which attempts to order and link the set of LP-solved contigs. Unlike previous work, this algorithm only links and orders contigs when the join is 90% or more likely to be correct. It is our view that there is no value in computing an optimal solution with respect to some criteria over very noisy data as this optimal solution rarely corresponds to the true solution. The paper involves extensive empirical trials over real and simulated data.

Cloning of the human NADH: ubiquinone oxidoreductase subunit B13: localization to chromosome 7q32 and identification of a pseudogene on 11p15.

Cloning of the human NADH: ubiquinone oxidoreductase subunit B13: localization to chromosome 7q32 and identification of a pseudogene on 11p15.

Use of ACEDB as a database for YAC library data management.

AbstractIncreasingly, the libraries that are the basic genomic DNA resources of physical mapping projects are stored in ordered arrays in the wells of 96- or 384-well microtiter plates (see Chapters 2–4). Localization of individual genomic clones to single wells of 96-well microtiter plates rather than the traditional random plating has meant that screening of these libraries is no longer done “in isolation.” The results of all library screens may be accumulated over time and positive signals ascribed to the individual clones. In this way potential links between markers are more quickly identified and any worker who has access to the library can obtain a clone that has been previously identified positive with a particular marker. Thus the resources available to the mapping community have become less parochial and more highly organized. The distribution of yeast artificial chromosome (YAC) and other libraries by their constructors to many centers around the world, ensures that, at least in principle, an investigator has access to the sum of all data generated in that library, and the power of the ordered genomic resource strategy is further enhanced. However, this change in the way genomic DNA libraries are used has brought with it the need to store efficiently and display the screening data for libraries consisting of hundreds of thousands of clones. The very fact that a YAC library carries with it a burgeoning history of screening that is communicable presents the problem of how to store, display, and communicate these data.KeywordsMicrotiter PlateHybridization PatternYeast Artificial ChromosomeVirtual GridYeast Artificial Chromosome CloneThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

LabBase: managing lab data in a large-scale genome-mapping project

The Whitehead/MIT Center for Genome Research is engaged in several high-throughput genome-mapping projects, including physical mapping of the human genome and genetic-linkage mapping of the mouse genome. The scale and complexity of the laboratory workflows for these projects make a laboratory information system a necessity. For example, the human physical mapping project has performed over 1.2 million experimental steps to date, many of which involve numerous biochemical assays. The result is a physical map containing over 13 thousand sequence-tagged-site markers, with a near-term goal of 15 thousand markers. These markers will likely be used as a starting point for producing a higher resolution map, which in turn will provide the raw material for sequencing the human genome. LabBase is a database management system (DBMS) tailored to the needs of laboratory information systems that support such projects. It is designed to make it easy to keep track of laboratory samples, the experimental steps performed on them, and the results of these experiments. LabBase is functionally specialised because it provides special support for the requirements of managing laboratory data over and above what would be provided by a generic, off-the-shelf DBMS. >

Efficient high-resolution genetic mapping of mouse interspersed repetitive sequence PCR products, toward integrated genetic and physical mapping of the mouse genome.

The ability to carry out high-resolution genetic mapping at high throughput in the mouse is a critical rate-limiting step in the generation of genetically anchored contigs in physical mapping projects and the mapping of genetic loci for complex traits. To address this need, we have developed an efficient, high-resolution, large-scale genome mapping system. This system is based on the identification of polymorphic DNA sites between mouse strains by using interspersed repetitive sequence (IRS) PCR. Individual cloned IRS PCR products are hybridized to a DNA array of IRS PCR products derived from the DNA of individual mice segregating DNA sequences from the two parent strains. Since gel electrophoresis is not required, large numbers of samples can be genotyped in parallel. By using this approach, we have mapped > 450 polymorphic probes with filters containing the DNA of up to 517 backcross mice, potentially allowing resolution of 0.14 centimorgan. This approach also carries the potential for a high degree of efficiency in the integration of physical and genetic maps, since pooled DNAs representing libraries of yeast artificial chromosomes or other physical representations of the mouse genome can be addressed by hybridization of filter representations of the IRS PCR products of such libraries.

Predicting Progress in Directed Mapping Projects

Several recent mapping efforts have used so-called "directed" approaches to construct their maps. However, most, but not all, published methods for modeling the progress in physical mapping projects have been focused on random approaches, such as bottom-up fingerprinting and STS-content mapping. In addition, those few efforts that did model directed approaches used methods that required assuming that all insert lengths were the same. This assumption is unnecessary. Using properties of stationary processes, one can derive simple asymptotic formulas that apply equally to constant and variable clone lengths. Also, in the case of constant clone lengths, these results are equivalent to, and extend, those published results for directed mapping derived by other methods. Simulations show that these methods provide estimates well within the limits of uncertainty inherent in any mapping project.

Engineering automated systems for physical mapping

The automated physical mapping system described here is currently being used to complete physical maps of both the human and the mouse genomes. The originally targeted 5,000 screenings per day has been surpassed with about 18,000 daily screenings on a routine basis. Thus far, 3,500 STSs have been mapped and the remaining 6,500 required to make the first low-density STS physical map are likely to be completed very early in 1995. Many of the biochemical and automation techniques used for this physical mapping project may also have applications in other areas such as DNA genotyping, and DNA diagnostics. For example, it may be possible to screen large populations for particular genetic diseases (such as cystic fibrosis and a propensity for certain cancers) before they exhibit any symptoms, thereby allowing potential treatments to start as early as possible.< <ETX xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">&gt;</ETX>

Automatable screening of yeast artificial-chromosome libraries based on the oligonucleotide-ligation assay

The systematic screening of yeast artificial-chromosome (YAC) libraries is the limiting step in many physical mapping projects. To improve the screening throughout for a human YAC library, we designed an automatable strategy to identify YAC clones containing a specific segment of DNA. Our approach combines amplification of the target sequence from pooled YAC DNA by the polymerase chain reaction (PCR) with detection of the sequence by an ELISA-based oligonucleotide-ligation assay (OLA). The PCR-OLA approach eliminates the use of radioactive isotopes and gel electrophoresis, two of the major obstacles to automated YAC screening. Furthermore, the use of the OLA to test for the presence of sequences internal to PCR primers provides an additional level of sensitivity and specificity in comparison to methods that rely solely on the PCR.

Molecular cloning of lin-29, a heterochronic gene required for the differentiation of hypodermal cells and the cessation of molting in C.elegans.

The lin-29 gene product of C.elegans activates a temporal developmental switch for hypodermal cells. Loss-of-function lin-29 mutations result in worms that fail to execute a stage-specific pattern of hypodermal differentiation that includes exist from the cell cycle, repression of larval cuticle genes, activation of adult cuticle genes, and the cessation of molting. Combined genetic and physical mapping of restriction fragment length polymorphisms (RFLPs) was used to identify the lin-29 locus. A probe from the insertion site of a Tc1 (maP1), closely linked and to the left of lin-29 on the genetic map, was used to identify a large set of overlapping cosmid, lambda and yeast artificial chromosome (YAC) clones assembled as part of the C.elegans physical mapping project. Radiolabeled DNA from one YAC clone identified two distinct allele-specific alterations that cosegregated with the lin-29 mutant phenotype in lin-29 intragenic recombinants. lin-29 sequences were severely under-represented in all cosmid and lambda libraries tested, but were readily cloned in a YAC vector, suggesting that the lin-29 region contains sequences incompatible with standard prokaryotic cloning techniques.

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.

Genomic mapping by fingerprinting random clones: A mathematical analysis

Results from physical mapping projects have recently been reported for the genomes of Escherichia coli, Saccharomyces cerevisiae, and Caenorhabditis elegans, and similar projects are currently being planned for other organisms. In such projects, the physical map is assembled by first “fingerprinting” a large number of clones chosen at random from a recombinant library and then inferring overlaps between clones with sufficiently similar fingerprints. Although the basic approach is the same, there are many possible choices for the fingerprint used to characterize the clones and the rules for declaring overlap. In this paper, we derive simple formulas showing how the progress of a physical mapping project is affected by the nature of the fingerprinting scheme. Using these formulas, we discuss the analytic considerations involved in selecting an appropriate fingerprinting scheme for a particular project.