DNA Physical Mapping Research Articles

DDmap: a MATLAB package for the double digest problem using multiple genetic operators

BackgroundIn computational biology, the physical mapping of DNA is a key problem. We know that the double digest problem (DDP) is NP-complete. Many algorithms have been proposed for solving the DDP, although it is still far from being resolved.ResultsWe present DDmap, an open-source MATLAB package for solving the DDP, based on a newly designed genetic algorithm that combines six genetic operators in searching for optimal solutions. We test the performance of DDmap by using a typical DDP dataset, and we depict exact solutions to these DDP instances in an explicit manner. In addition, we propose an approximate method for solving some hard DDP scenarios via a scaling-rounding-adjusting process.ConclusionsFor typical DDP test instances, DDmap finds exact solutions within approximately 1 s. Based on our simulations on 1000 random DDP instances by using DDmap, we find that the maximum length of the combining fragments has observable effects towards genetic algorithms for solving the DDP problem. In addition, a Maple source code for illustrating DDP solutions as nested pie charts is also included.

Single-molecule DNA visualization using AT-specific red and non-specific green DNA-binding fluorescent proteins.

The recent advances in the single cell genome analysis are generating a considerable amount of novel insights into complex biological systems. However, there are still technical challenges because each cell has a single copy of DNA to be amplified in most single cell genome analytical methods. In this paper, we present a novel approach to directly visualize a genomic map on a large DNA molecule instantly stained with red and green DNA-binding fluorescent proteins without DNA amplification. For this visualization, we constructed a few types of fluorescent protein-fused DNA-binding proteins: H-NS (histone-like nucleoid-structuring protein), DNA-binding domain of BRCA1 (breast cancer 1), high mobility group-1 (HMG), and lysine tryptophan (KW) repeat motif. Because H-NS and HMG preferentially bind A/T-rich regions, we combined A/T specific binder (H-NS-mCherry and HMG-mCherry as red color) and a non-specific complementary DNA binder (BRCA1-eGFP and 2(KW)2-eGFP repeat as green color) to produce a sequence-specific two-color DNA physical map for efficient optical identification of single DNA molecules.

DNA nanomapping using CRISPR-Cas9 as a programmable nanoparticle

Progress in whole-genome sequencing using short-read (e.g., <150 bp), next-generation sequencing technologies has reinvigorated interest in high-resolution physical mapping to fill technical gaps that are not well addressed by sequencing. Here, we report two technical advances in DNA nanotechnology and single-molecule genomics: (1) we describe a labeling technique (CRISPR-Cas9 nanoparticles) for high-speed AFM-based physical mapping of DNA and (2) the first successful demonstration of using DVD optics to image DNA molecules with high-speed AFM. As a proof of principle, we used this new “nanomapping” method to detect and map precisely BCL2–IGH translocations present in lymph node biopsies of follicular lymphoma patents. This HS-AFM “nanomapping” technique can be complementary to both sequencing and other physical mapping approaches.

Isolation, Characterization, and Bioinformatic Analyses of Lytic Salmonella Enteritidis Phages and Tests of Their Antibacterial Activity in Food.

Salmonella Enteritidis remains a major threat for food safety. To take efforts to develop phage-based biocontrol for S. Enteritidis contamination in food, in this study, the phages against S. Enteritidis were isolated from sewage samples, characterized by host range assays, DNA restriction enzyme pattern analyses, and transmission electron microscope observations, and tested for antibacterial activity in food; some potent phages were further characterized by bioinformatic analyses. Results showed that based on the plaque quality and host range, seven lytic phages targeting S. Enteritidis were selected, considered as seven distinct phages through DNA physical maps, and classified as Myoviridae or Siphoviridae family by morphologic observations; the combined use of such seven strain phages as a "food additive" could succeed in controlling the artificial S. Enteritidis contamination in the different physical forms of food at a range of temperatures; by bioinformatic analyses, both selected phage BPS11Q3 and BPS15Q2 seemed to be newfound obligate lytic phage strains with no indications for any potentially harmful genes in their genomes. In conclusion, our results showed a potential of isolated phages as food additives for controlling S. Enteritidis contamination in some salmonellosis outbreak-associated food vehicles, and there could be minimized potential risk associated with using BPS11Q3 and BPS15Q2 in food.

Characterization of eleven monosomic alien addition lines added from Gossypium anomalum to Gossypium hirsutum using improved GISH and SSR markers.

Background Gossypium anomalum (BB genome) possesses the desirable characteristics of drought tolerance, resistance to diseases and insect pests, and the potential for high quality fibers. However, it is difficult to transfer the genes associated with these desirable traits into cultivated cotton (G. hirsutum, AADD genome). Monosomic alien addition lines (MAALs) can be used as a bridge to transfer desired genes from wild species into G. hirsutum. In cotton, however, the high number and smaller size of the chromosomes has resulted in difficulties in discriminating chromosomes from wild species in cultivated cotton background, the development of cotton MAALs has lagged far behind many other crops. To date, no set of G. hirsutum-G. anomalum MAALs was reported. Here the amphiploid (AADDBB genome) derived from G. hirsutum × G. anomalum was used to generate a set of G. hirsutum-G. anomalum MAALs through a combination of consecutive backcrossing, genomic in situ hybridization (GISH), morphological survey and microsatellite marker identification.ResultsWe improved the GISH technique used in our previous research by using a mixture of two probes from G. anomalum and G. herbaceum (AA genome). The results indicate that a ratio of 4:3 (G. anomalum : G. herbaceum) is the most suitable for discrimination of chromosomes from G. anomalum and the At-subgenome of G. hirsutum. Using this improved GISH technique, 108 MAAL individuals were isolated. Next, 170 G. hirsutum- and G. anomalum-specific codominant markers were obtained and employed for characterization of these MAAL individuals. Finally, eleven out of 13 MAALs were identified. Unfortunately, we were unable to isolate Chrs. 1Ba and 5Ba due to their very low incidences in backcrossing generation, as these remained in a condition of multiple additions.ConclusionsThe characterized lines can be employed as bridges for the transfer of desired genes from G. anomalum into G. hirsutum, as well as for gene assignment, isolation of chromosome-specific probes, development of chromosome-specific “paints” for fluorochrome-labeled DNA fragments, physical mapping, and selective isolation and mapping of cDNAs/genes for a particular G. anomalum chromosome.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0913-2) contains supplementary material, which is available to authorized users.

OMWare: a tool for efficient assembly of genome-wide physical maps.

BackgroundPhysical mapping of DNA with restriction enzymes allows for the characterization and assembly of much longer molecules than is feasible with sequencing. However, assemblies of physical map data are sensitive to input parameters, which describe noise inherent in the data collection process. One possible way to determine the parameter values that best describe a dataset is by trial and error.ResultsHere we present OMWare, a tool that efficiently generated 405 de novo map assemblies of a single datasets collected from the cotton species Gossypium raimondii. The assemblies were generated using various input parameter values, and were completed more efficiently by re-using compatible intermediate results. These assemblies were assayed for contiguity, internal consistency, and accuracy.ConclusionsResulting assemblies had variable qualities. Although highly accurate assemblies were found, contiguity and internal consistency metrics were poor predictors of accuracy.

Study of Hybrid DNA Physical Mapping Based on Approximation Algorithm with Errors

A human chromosome is a DNA molecule with approximately 10 8 base pairs. The techniques developed to date for sequencing are restricted to pieces of DNA with up to tens of thousands of base pairs. This means that when a piece is sequenced, only an extremely small part of a chromosome can be seen. Molecular biologists use special techniques to deal with DNA molecules comparable in size to a chromosome. These techniques enable them to create maps of an entire chromosome or of significant fractions of chromosomes. Computational techniques were studied that could potentially aid biologists in the map-generation process. An algorithm that solves the consecutive 1s problem was studied. Such a problem is a good model of hybridization mapping when there are no errors and when probes are unique. If errors are present, another approach is needed, and the approximation algorithm is a prospective problem solver for hybridization physical mapping of DNA with errors.

Construction of a complete set of alien chromosome addition lines from Gossypium australe in Gossypium hirsutum: morphological, cytological, and genotypic characterization

Key message We report the first complete set of alien addition lines of G. hirsutum . The characterized lines can be used to introduce valuable traits from G. australe into cultivated cotton. Gossypium australe is a diploid wild cotton species (2n = 26, GG) native to Australia that possesses valuable characteristics unavailable in the cultivated cotton gene pool, such as delayed pigment gland morphogenesis in the seed and resistances to pests and diseases. However, it is very difficult to directly transfer favorable traits into cultivated cotton through conventional gene recombination due to the absence of pairing and crossover between chromosomes of G. australe and Gossypium hirsutum (2n = 52, AADD). To enhance the transfer of favorable genes from wild species into cultivated cotton, we developed a set of hirsutum–australe monosomic alien chromosome addition lines (MAAL) using a combination of morphological survey, microsatellite marker-assisted selection, and molecular cytogenetic analysis. The amphidiploid (2n = 78, AADDGG) of G. australe and G. hirsutum was consecutively backcrossed with upland cotton to develop alien addition lines of individual G. australe chromosomes in G. hirsutum. From these backcross progeny, we generated the first complete set of chromosome addition lines in cotton; 11 of 13 lines are monosomic additions, and chromosomes 7Ga and 13Ga are multiple additions. MAALs of 1Ga and 11Ga were the first to be isolated. The chromosome addition lines can be employed as bridges for the transfer of desired genes from G. australe into G. hirsutum, as well as for gene assignment, isolation of chromosome-specific probes, flow sorting and microdissection of chromosome, development of chromosome-specific ‘‘paints’’ for fluorochrome-labeled DNA fragments, physical mapping, and selective isolation and mapping of cDNAs for a particular G. australe chromosome.Electronic supplementary materialThe online version of this article (doi:10.1007/s00122-014-2283-1) contains supplementary material, which is available to authorized users.

Probe interval graphs and probe unit interval graphs on superclasses of cographs

Graph Theory A graph is probe (unit) interval if its vertices can be partitioned into two sets: a set of probe vertices and a set of nonprobe vertices, so that the set of nonprobe vertices is a stable set and it is possible to obtain a (unit) interval graph by adding edges with both endpoints in the set of nonprobe vertices. Probe (unit) interval graphs form a superclass of (unit) interval graphs. Probe interval graphs were introduced by Zhang for an application concerning the physical mapping of DNA in the human genome project. The main results of this article are minimal forbidden induced subgraphs characterizations of probe interval and probe unit interval graphs within two superclasses of cographs: P4-tidy graphs and tree-cographs. Furthermore, we introduce the concept of graphs class with a companion which allows to describe all the minimally non-(probe G) graphs with disconnected complement for every graph class G with a companion.

Bioinformatics Tools Allow Targeted Selection of Chromosome Enumeration Probes and Aneuploidy Detection

Accurate determination of cellular chromosome complements is a highly relevant issue beyond prenatal/pre-implantation genetic analyses or stem cell research, because aneusomy may be an important mechanism by which organisms control the rate of fetal cellular proliferation and the fate of regenerating tissues. Typically, small amounts of individual cells or nuclei are assayed by in situ hybridization using chromosome-specific DNA probes. Careful probe selection is fundamental to successful hybridization experiments. Numerous DNA probes for chromosome enumeration studies are commercially available, but their use in multiplexed hybridization assays is hampered due to differing probe-specific hybridization conditions or a lack of a sufficiently large number of different reporter molecules. Progress in the International Human Genome Project has equipped the scientific community with a wealth of unique resources, among them recombinant DNA libraries, physical maps, and data-mining tools. Here, we demonstrate how bioinformatics tools can become an integral part of simple, yet powerful approaches to devise diagnostic strategies for detection of aneuploidy in interphase cells. Our strategy involving initial in silico optimization steps offers remarkable savings in time and costs during probe generation, while at the same time significantly increasing the assay’s specificity, sensitivity, and reproducibility.

Identical Functional Organization of Nonpolytene and Polytene Chromosomes in Drosophila melanogaster

Salivary gland polytene chromosomes demonstrate banding pattern, genetic meaning of which is an enigma for decades. Till now it is not known how to mark the band/interband borders on physical map of DNA and structures of polytene chromosomes are not characterized in molecular and genetic terms. It is not known either similar banding pattern exists in chromosomes of regular diploid mitotically dividing nonpolytene cells. Using the newly developed approach permitting to identify the interband material and localization data of interband-specific proteins from modENCODE and other genome-wide projects, we identify physical limits of bands and interbands in small cytological region 9F13-10B3 of the X chromosome in D. melanogaster, as well as characterize their general molecular features. Our results suggests that the polytene and interphase cell line chromosomes have practically the same patterns of bands and interbands reflecting, probably, the basic principle of interphase chromosome organization. Two types of bands have been described in chromosomes, early and late-replicating, which differ in many aspects of their protein and genetic content. As appeared, origin recognition complexes are located almost totally in the interbands of chromosomes.

A NOTE ON COMPLEXITY OF GENETIC MUTATIONS

Genes, the functional segments of DNA, can be modeled akin to DNA by quaternary strings. Pevzner and Waterman posed various problems pertaining to DNA, e.g. DNA physical mapping, DNA sequencing, and DNA sequence comparison. DNA sequence comparison methods aim to compute the genetic mutation distance. Genetic mutation, a change that happens within a gene, can be modeled by an operation like a transposition or a reversal over a string where a string models a gene. The complexity of transforming quaternary strings under a given operation is equivalent to the complexity of computing the corresponding genetic mutation distance. We show that the string transformation distance under signed prefix reversals, translocations and transreversals is NP-complete.

Probe interval and probe unit interval graphs on superclasses of cographs

Abstract Probe (unit) interval graphs form a superclass of (unit) interval graphs. A graph is probe (unit) interval if its vertices can be partitioned into two sets: a set of probe vertices and a set of nonprobe vertices, so that the set of nonprobe vertices is a stable set and it is possible to obtain a (unit) interval graph by adding edges with both endpoints in the set of nonprobe vertices. Probe interval graphs were introduced by Zhang for an application concerning with the physical mapping of DNA in the human genome project. In this work, we present characterizations by minimal forbidden induced subgraphs of probe interval and probe unit interval graphs within two superclasses of cographs: P4-tidy graphs and tree-cographs.

Analysis of DNA replication parameters in isolated organs of drosophila melanogaster by molecular combing

Methods of DNA physical mapping and the direct visualization of the replication and transcription in specific genome regions play a crucial role in the study of structural and functional organization of eukaryotic genomes. Since DNA strands in cells are organized into high-fold structure and structurally present as highly compacted chromosomes, the majority of methods at the chromosome level are of low-resolution. An approach to increasing the resolution and accuracy is molecular combing. The method is based on the process of stretching and alignment of DNA molecules that are covalently attached with one of the ends to the coverglass surface. In this article, we describe the major methodological steps of molecular combing and their adaptation to study the parameters of DNA replication in polyploid and diploid tissues of drosophila larvae.

Using Bacterial Artificial Chromosomes in Leukemia Research: The Experience at the University Cytogenetics Laboratory in Brest,France

The development of the bacterial artificial chromosome (BAC) system was driven in part by the human genome project in order to construct genomic DNA libraries and physical maps for genomic sequencing. The availability of BAC clones has become a valuable tool for identifying cancer genes. We report here our experience in identifying genes located at breakpoints of chromosomal rearrangements and in defining the size and boundaries of deletions in hematological diseases. The methodology used in our laboratory consists of a three-step approach using conventional cytogenetics followed by FISH with commercial probes, then BAC clones. One limitation to the BAC system is that it can only accommodate inserts of up to 300 kb. As a consequence, analyzing the extent of deletions requires a large amount of material. Array comparative genomic hybridization (array-CGH) using a BAC/PAC system can be an alternative. However, this technique has limitations also, and it cannot be used to identify candidate genes at breakpoints of chromosomal rearrangements such as translocations, insertions, and inversions.

On the Maximal Interval Subgraph of a Tree

We study the problem of finding the maximum interval subgraph in a tree. This problem is related to the Double Digestion Problem of DNA physical mapping. We show that the complexity of an algorithm of Wang is O(n). We also present a linear algorithm of our own. We study the case when the edges of the tree is weighted as well. An algorithm with complexity O(n³) is presented.

Complexity issues for the sandwich homogeneous set problem

Graph sandwich problems were introduced by Golumbic et al. (1994) in [12] for DNA physical mapping problems and can be described as follows. Given a property Π of graphs and two disjoint sets of edges E 1 , E 2 with E 1 ⊆ E 2 on a vertex set V , the problem is to find a graph G on V with edge set E s having property Π and such that E 1 ⊆ E s ⊆ E 2 . In this paper, we exhibit a quasi-linear reduction between the problem of finding an independent set of size k ≥ 2 in a graph and the problem of finding a sandwich homogeneous set of the same size k . Using this reduction, we prove that a number of natural (decision and counting) problems related to sandwich homogeneous sets are hard in general. We then exploit a little further the reduction and show that finding efficient algorithms to compute small sandwich homogeneous sets would imply substantial improvement for computing triangles in graphs.

The Double Digest Problem: finding all solutions

The strongly NP-complete Double Digest Problem (DDP), for physical mapping of DNA, is now used for efficient genotyping. Existing methods: are inefficient in tackling large instances; produce only one solution while an instance may have multiple distinct solutions. In this paper, we employ the notion of equivalence among the distinct solutions to obtain almost all of them. Our method comprises two phases: finding a representative from each equivalence class using an elitist Genetic Algorithm (GA); for each representative generating the entire class efficiently. Experimental results tally for known instances. Significant reduction in search space provides notable efficiency.

A characterization of cycle-free unit probe interval graphs

A graph is a probe interval graph (PIG) if its vertices can be partitioned into probes and nonprobes with an interval assigned to each vertex so that vertices are adjacent if and only if their corresponding intervals overlap and at least one of them is a probe. PIGs are a generalization of interval graphs introduced by Zhang for an application concerning the physical mapping of DNA in the human genome project. PIGs have been characterized in the cycle-free case by Sheng, and other miscellaneous results are given by McMorris, Wang, and Zhang. Johnson and Spinrad give a polynomial time recognition algorithm for when the partition of vertices into probes and nonprobes is given. The complexity for the general recognition problem is not known. Here, we restrict attention to the case where all intervals have the same length, that is, we study the unit probe interval graphs and characterize the cycle-free graphs that are unit probe interval graphs via a list of forbidden induced subgraphs.

A Detailed Larval Salivary Gland Polytene Chromosome Photomap for &lt;I&gt;Culex quinquefasciatus&lt;/I&gt; (Diptera: Culicidae) from Johannesburg, South Africa

The suitability of polytene chromosomes for population genetic and in situ DNA physical mapping was investigated in Johannesburg, South Africa, Culex quinquefasciatus Say and Culex pipiens L.. The most defined and consistent polytene spreads were obtained from salivary glands of early fourth instars from Cx. quinquefasciatus. A photomap is provided for a Cx. quinquefasciatus colony originating from Johannesburg. Variations in banding patterns, chromosome aberrations, and paracentric inversions within the colony and comparisons with previous published descriptions are discussed. Chromosomes of sufficient quality were obtained such that a DNA coding for an odorant binding protein could be mapped by colorimetric in situ hybridization.