High-resolution Restriction Maps Research Articles

Determining optical mapping errors by simulations.

Optical mapping is a complementary technology to traditional DNA sequencing technologies, such as next-generation sequencing (NGS). It provides genome-wide, high-resolution restriction maps from single, stained molecules of DNA. It can be used to detect large and small structural variants, copy number variations and complex rearrangements. Optical mapping is affected by different kinds of errors in comparison with traditional DNA sequencing technologies. It is important to understand the source of these errors and how they affect the obtained data. This article proposes a novel approach to modeling errors in the data obtained from the Bionano Genomics Inc. Saphyr system with Direct Label and Stain (DLS) chemistry. Some studies have already addressed this issue for older instruments with nicking enzymes, but we are unaware of a study that addresses this new system. The main result is a framework for studying errors in the data obtained from the Saphyr instrument with DLS chemistry. The framework's main component is a simulation that computes how major sources of errors for this instrument (a false site, a missing site and resolution errors) affect the distribution of fragment lengths in optical maps. The simulation is parametrized by variables describing these errors and we are using a differential evolution algorithm to evaluate parameters that best fit the data from the instrument. Results of the experiments manifest that this approach can be used to study errors in the optical mapping data analysis. Source codes supporting the presented results are available at: https://github.com/mvasinek/olgen-om-error-prediction. The data underlying this article are available on the Bionano Genomics Inc. website, at: https://bionanogenomics.com/library/datasets/. Supplementary data are available at Bioinformatics online.

Aligning optical maps to de Bruijn graphs.

Optical maps are high-resolution restriction maps (Rmaps) that give a unique numeric representation to a genome. Used in concert with sequence reads, they provide a useful tool for genome assembly and for discovering structural variations and rearrangements. Although they have been a regular feature of modern genome assembly projects, optical maps have been mainly used in post-processing step and not in the genome assembly process itself. Several methods have been proposed for pairwise alignment of single molecule optical maps-called Rmaps, or for aligning optical maps to assembled reads. However, the problem of aligning an Rmap to a graph representing the sequence data of the same genome has not been studied before. Such an alignment provides a mapping between two sets of data: optical maps and sequence data which will facilitate the usage of optical maps in the sequence assembly step itself. We define the problem of aligning an Rmap to a de Bruijn graph and present the first algorithm for solving this problem which is based on a seed-and-extend approach. We demonstrate that our method is capable of aligning 73% of Rmaps generated from the Escherichia coli genome to the de Bruijn graph constructed from short reads generated from the same genome. We validate the alignments and show that our method achieves an accuracy of 99.6%. We also show that our method scales to larger genomes. In particular, we show that 76% of Rmaps can be aligned to the de Bruijn graph in the case of human data. The software for aligning optical maps to de Bruijn graph, omGraph is written in C++ and is publicly available under GNU General Public License at https://github.com/kingufl/omGraph. Supplementary data are available at Bioinformatics online.

Whole genome mapping as a fast-track tool to assess genomic stability of sequenced Staphylococcus aureus strains.

BackgroundWhole genome (optical) mapping (WGM), a state-of-the-art mapping technology based on the generation of high resolution restriction maps, has so far been used for typing clinical outbreak strains and for mapping de novo sequence contigs in genome sequencing projects. We employed WGM to assess the genomic stability of previously sequenced Staphylococcus aureus strains that are commonly used in laboratories as reference standards.ResultsS. aureus strains (n = 12) were mapped on the Argus™ Optical Mapping System (Opgen Inc, Gaithersburg, USA). Assembly of NcoI-restricted DNA molecules, visualization, and editing of whole genome maps was performed employing MapManager and MapSolver softwares (Opgen Inc). In silico whole genome NcoI-restricted maps were also generated from available sequence data, and compared to the laboratory-generated maps. Strains showing differences between the two maps were resequenced using Nextera XT DNA Sample Preparation Kit and Miseq Reagent Kit V2 (MiSeq, Illumina) and de novo assembled into sequence contigs using the Velvet assembly tool. Sequence data were correlated with corresponding whole genome maps to perform contig mapping and genome assembly using MapSolver. Of the twelve strains tested, one (USA300_FPR3757) showed a 19-kbp deletion on WGM compared to its in silico generated map and reference sequence data. Resequencing of the USA300_FPR3757 identified the deleted fragment to be a 13kbp-long integrative conjugative element ICE6013.ConclusionsFrequent subculturing and inter-laboratory transfers can induce genomic and therefore, phenotypic changes that could compromise the utility of standard reference strains. WGM can thus be used as a rapid genome screening method to identify genomic rearrangements whose size and type can be confirmed by sequencing.Electronic supplementary materialThe online version of this article (doi:10.1186/1756-0500-7-704) contains supplementary material, which is available to authorized users.

Multidrug-resistant (MDR) Klebsiella pneumoniae clinical isolates: a zone of high heterogeneity (HHZ) as a tool for epidemiological studies

Comparison of genome-wide, high-resolution restriction maps of Klebsiella pneumoniae clinical isolates, including an NDM-1 producer, and in silico-generated restriction maps of sequenced genomes revealed a highly heterogeneous region we designated the 'high heterogeneity zone' (HHZ). The HHZ consists of several regions, including a 'hot spot' prone to insertions and other rearrangements. The HHZ is a characteristic genomic area that can be used in the identification and tracking of outbreak-causing strains.

Pattern of segmental recombination in the distal inversion of mouse t haplotypes.

To examine genetic exchange between t haplotypes and their wild-type homologs, four previously identified mouse Chromosome (Chr) 17 variants termed mosaic haplotypes were analyzed in detail. Three of these haplotypes-one from a Mus musculus population in Bulgaria, one from a Mus domesticus population in Chile, and one from a M. domesticus population in Germany-display properties indicative of the t complex. All four haplotypes are exceptional because they are characterized by the presence of a few wild-type DNA markers in the distal inversion [In(17)4] of a t haplotype chromosome: thus, they are classified as mosaic t haplotypes. The mosaic pattern for each haplotype is distinct, however. We compared the mosaic haplotypes with each other, and with several well-characterized laboratory t haplotypes, by analyzing several DNA markers in the In(17)4 region of the t complex, where all of the mosaicism occurs. We used a combination of high-resolution restriction mapping, DNA sequencing, and analysis of new DNA markers to classify the haplotypes. This analysis shows that segmental exchange, either by gene conversion or double crossing-over, has occurred at molecular markers in the vicinity of a gene, Dnahc8, that is a candidate for the t complex distorter locus Tcd2. While it is unclear whether segmental exchanges have included the Tcd2 gene, it is apparent that several independent recombination events have occurred in In(17)4 during the recent evolution of t haplotypes.

Fine physical and transcript mapping of a 1.8 Mb region spanning the locus for childhood acute lymphoblastic leukemia on chromosome 12p12. 3.

Rearrangements of the short arm of chromosome 12 are frequently observed in hematological disorders. Previous studies of loss of heterozygosity identified a small genetic interval on chromosome 12p12.3 that is frequently deleted in childhood acute lymphoblastic leukemia (ALL). Two genes, ETV6/TEL and p27/KIP1, are located in this interval. Evidence has accumulated that an as-yet unidentified tumor suppressor gene is closely linked to these. To facilitate the identification of candidate genes, a long-range high-resolution restriction map of the ALL locus was constructed using a contig of YAC clones. Several marker loci, including 11 STS, three newly developed YAC end-based STS, six EST, and seven genes were unambiguously positioned in the new map. The map covers 1.8Mb and extends from the distal salivary proline-rich protein gene cluster to the proximal p27/KIP1 gene. The data confirmed the order tel-D12S358-p27/KIP1-cen and excluded p27/KIP1 as a candidate tumor suppressor gene. The critical region delimited by D12S89 and D12S358 is a 750kb CpG-island rich region that includes the 240kb TEL/ETV6 gene as well as CLAPS3 (clathrin-adaptor small chain 3). The new map provides a molecular framework for the identification of novel genes and transcriptional units in the ALL interval.

Optical Mapping of Plasmodium falciparum Chromosome 2

Detailed restriction maps of microbial genomes are a valuable resource in genome sequencing studies but are toilsome to construct by contig construction of maps derived from cloned DNA. Analysis of genomic DNA enables large stretches of the genome to be mapped and circumvents library construction and associated cloning artifacts. We used pulsed-field gel electrophoresis purified Plasmodium falciparum chromosome 2 DNA as the starting material for optical mapping, a system for making ordered restriction maps from ensembles of individual DNA molecules. DNA molecules were bound to derivatized glass surfaces, cleaved with NheI or BamHI, and imaged by digital fluorescence microscopy. Large pieces of the chromosome containing ordered DNA restriction fragments were mapped. Maps were assembled from 50 molecules producing an average contig depth of 15 molecules and high-resolution restriction maps covering the entire chromosome. Chromosome 2 was found to be 976 kb by optical mapping with NheI, and 946 kb with BamHI, which compares closely to the published size of 947 kb from large-scale sequencing. The maps were used to further verify assemblies from the plasmid library used for sequencing. Maps generated in silico from the sequence data were compared to the optical mapping data, and good correspondence was found. Such high-resolution restriction maps may become an indispensable resource for large-scale genome sequencing projects.

Automated high resolution optical mapping using arrayed, fluid-fixed DNA molecules.

New mapping approaches construct ordered restriction maps from fluorescence microscope images of individual, endonuclease-digested DNA molecules. In optical mapping, molecules are elongated and fixed onto derivatized glass surfaces, preserving biochemical accessibility and fragment order after enzymatic digestion. Measurements of relative fluorescence intensity and apparent length determine the sizes of restriction fragments, enabling ordered map construction without electrophoretic analysis. The optical mapping system reported here is based on our physical characterization of an effect using fluid flows developed within tiny, evaporating droplets to elongate and fix DNA molecules onto derivatized surfaces. Such evaporation-driven molecular fixation produces well elongated molecules accessible to restriction endonucleases, and notably, DNA polymerase I. We then developed the robotic means to grid DNA spots in well defined arrays that are digested and analyzed in parallel. To effectively harness this effect for high-throughput genome mapping, we developed: (i) machine vision and automatic image acquisition techniques to work with fixed, digested molecules within gridded samples, and (ii) Bayesian inference approaches that are used to analyze machine vision data, automatically producing high-resolution restriction maps from images of individual DNA molecules. The aggregate significance of this work is the development of an integrated system for mapping small insert clones allowing biochemical data obtained from engineered ensembles of individual molecules to be automatically accumulated and analyzed for map construction. These approaches are sufficiently general for varied biochemical analyses of individual molecules using statistically meaningful population sizes.

Smith/Birnstiel mapping of genome rearrangements in Pseudomonas aeruginosa.

A novel application of the Smith/Birnstiel technique is presented for the analysis of intraspecies genomic diversity in small genomes. Rare-cutter total/N frequent-cutter partial restriction digestions are separated in N separate lanes by pulsed-field gel electrophoresis, blotted and hybridized with rare-cutter fragment ends as probes. The evaluation of the autoradiogram results in high-resolution restriction maps of 10-200 kbp regions. The technique was applied to the analysis of genome rearrangements in Pseudomonas aeruginosa strains. Comparison of the region encoding the tryptophan biosynthesis genes in the PAO and the IATS serotype 5 strains revealed that shared sequence characterized by almost identical restriction fingerprints was interrupted in the serotype 5 strain by small islands displaying strain-specific restriction site signatures. A multistep rearrangement in a hypervariable chromosome region downstream of the phn locus was detected in serial airway isolates from a patient with cystic fibrosis.

High-resolution restriction maps of bacterial artificial chromosomes constructed by optical mapping.

Large insert clone libraries have been the primary resource used for the physical mapping of the human genome. Research directions in the genome community now are shifting direction from purely mapping to large-scale sequencing, which in turn, require new standards to be met by physical maps and large insert libraries. Bacterial artificial chromosome libraries offer enormous potential as the chosen substrate for both mapping and sequencing studies. Physical mapping, however, has come under some scrutiny as being "redundant" in the age of large-scale automated sequencing. We report the development and applications of nonelectrophoretic, optical approaches for high-resolution mapping of bacterial artificial chromosome that offer the potential to complement and thereby advance large-scale sequencing projects.

A 37-kb restriction map of the human immunoglobulin lambda variable locus, VB cluster, harboring four functional genes and two non-coding V<FONT FACE=Symbol>l</font> sequences

The human immunoglobulin lambda variable locus (IGLV) is mapped at chromosome 22 band q11.1-q11.2. The 30 functional germline v-lambda genes sequenced untill now have been subgrouped into 10 families (V<FONT FACE="Symbol">l</font>1 to V<FONT FACE="Symbol">l</font>10). The number of V<FONT FACE="Symbol">l</font> genes has been estimated at approximately 70. This locus is formed by three gene clusters (VA, VB and VC) that encompass the variable coding genes (V) responsible for the synthesis of lambda-type Ig light chains, and the J<FONT FACE="Symbol">l</font>-C<FONT FACE="Symbol">l</font> cluster with the joining segments and the constant genes. Recently the entire variable lambda gene locus was mapped by contig methodology and its one- megabase DNA totally sequenced. All the known functional V-lambda genes and pseudogenes were located. We screened a human genomic DNA cosmid library and isolated a clone with an insert of 37 kb (cosmid 8.3) encompassing four functional genes (IGLV7S1, IGLV1S1, IGLV1S2 and IGLV5a), a pseudogene (V<FONT FACE="Symbol">l</font>A) and a vestigial sequence (vg1) to study in detail the positions of the restriction sites surrounding the V<FONT FACE="Symbol">l</font> genes. We generated a high resolution restriction map, locating 31 restriction sites in 37 kb of the VB cluster, a region rich in functional V<FONT FACE="Symbol">l</font> genes. This mapping information opens the perspective for further RFLP studies and sequencing

A YAC contig and high resolution restriction map of chromosome 3 from Plasmodium falciparum

A YAC contig and high resolution restriction map of chromosome 3 from Plasmodium falciparum

Construction of a 1.2-Mb Contig Surrounding, and Molecular Analysis of, the Human CREB-Binding Protein (CBP/CREBBP) Gene on Chromosome 16p13.3

In the interest of cloning and analyzing the genes responsible for two very different diseases, the Rubinstein–Taybi syndrome (RTS) and acute myeloid leukemia (AML) associated with the somatic translocation t(8;16)(p11;p13.3), we constructed a high-resolution restriction map of contiguous cosmids (contig) covering 1.2 Mb of chromosome 16p13.3. By fluorescencein situhybridization and Southern blot analysis, we assigned all tested RTS and t(8;16) translocation breakpoints to a 100-kb region. We have previously reported exact physical locations of these 16p breakpoints, which all disrupt one gene we mapped to this interval: the CREB-binding protein (CBP or CREBBP) gene. Intriguingly, mutations in the CBP gene are responsible for RTS as well as the t(8;16)-associated AML. CBP functions as an integrator in the assembly of various multiprotein regulatory complexes and is thus necessary for transcription in a broad range of transduction pathways. We report here the cloning, physical mapping, characterization, and full cDNA nucleotide sequence of the human CBP gene.

Assembly of High-Resolution Restriction Maps Based on Multiple Complete Digests of a Redundant Set of Overlapping Clones

An approach to restriction-site mapping and contig building that uses fragment-size data from multiple complete digests of a set of clones that oversample a genomic region is presented. Maps containing both fragment-length data and clone-end data are maintained for each restriction enzyme. Synchroniza- tion between the maps for the different enzymes is achieved by requiring the clone-end maps for all enzymes to be compatible. Basic concepts that under- lie multiple-complete-digest mapping—including the match/merge approach to map incorporation, extension vs assimilation, ambiguity, and clone-end compatibility—are presented. An initial application of multiple-complete-digest mapping to real data on a set of cosmid clones suggests that this mapping method has exceptional power to produce accurate maps that are well suited to the needs of large-scale DNA-sequencing projects.

High resolution restriction mapping of YACs using chromosome fragmentation

High resolution restriction mapping of YACs using chromosome fragmentation

A 1.6-Mb P1-Based Physical Map of the Down Syndrome Region on Chromosome 21

The Down syndrome (DS) region on chromosome 21, which is responsible for the main features of DS such as characteristic facial features, a congenital heart defect, and mental retardation, has been defined by molecular analysis of DS patients with partial trisomy 21. The 2.5-Mb region around the markerD21S55betweenD21S17andERGin 21q22 is thought to be important, although contributions of other regions cannot be excluded. In this region, we focused on a 1.6-Mb region between aNotI site, LA68 (D21S396,which is mapped distal toD21S17) andERG,because analysis of a Japanese DS family with partial trisomy 21 revealed that the proximal border of its triplicated region was distal to LA68. We constructed P1 contigs with 46 P1 clones covering more than 95% of the 1.6-Mb region. A high-resolution restriction map usingBamHI was also constructed for more detailed analysis. Our P1 contig map supplements other physical maps previously reported and provides useful materials for further analysis including gene isolation and sequencing of the DS region.

Construction of a 750-kb bacterial clone contig and restriction map in the region of human chromosome 21 containing the progressive myoclonus epilepsy gene.

The gene responsible for progressive myoclonus epilepsy of the Unverricht-Lundborg type (EPM1) is located on human chromosome 21q22.3 in a region defined by recombination breakpoints and linkage disequilibrium. As part of an effort to clone the EPM1 gene on the basis of its chromosomal location, we have constructed a 753-kb bacterial clone contig that encompasses the region containing the gene. Because DNA markers from the region did not identify intact yeast artificial chromosome (YAC) clones after screening several libraries, we built the contig from cosmid clones and used bacterial artificial chromosome (BAC) and bacteriophage P1 clones to fill gaps. In addition to constructing the clone contig, we determined the locations of the EcoRI, SacII, EagI, and NotI restriction sites in the clones, resulting in a high-resolution restriction map of the region. Most of the contig is represented by a level of redundancy that allows the orders of most restriction sites to be determined, provides multiple data points supporting the clone orders and orientations, and allows a set of clones with a minimum degree of overlap to be chosen for efficient additional analysis. The clone and restriction maps are in excellent agreement with maps generated of the region by other methods. These ordered bacterial clones and the mapping information obtained from them provide valuable reagents for isolating candidate genes for EPM1, as well as for determining the nucleotide sequence of a 750 kb region of the human genome.

High-Resolution Mapping of a 130-kb Core Region of theMYCNAmplicon in Neuroblastomas

TheMYCNproto-oncogene is amplified in 25% of neuroblastomas, and amplification is strongly correlated with advanced disease stage and with rapid tumor progression. We have generated a high-resolution restriction map of nearly 500 kb spanning theMYCNlocus by subcloning yeast artificial chromosomes into cosmids. Cosmids plus additional amplified probes were hybridized to DNA from 33MYCNamplified neuroblastomas, and we determined that the amplicons range from 350 kb to over 1 Mb. Deletions and rearrangements of theMYCNamplicon occurred less frequently in primary tumors than in cell lines. We have defined a 130-kb region that was amplified in 32 of 33 tumors. One additional tumor deleted 65 kb of this region from its amplicon, while amplifying a large amount of flanking DNA. The only CpG island found in this region was within theMYCNgene. In conclusion, our data demonstrate that, despite the large size of most amplicons, the core domain that is consistently amplified in neuroblastomas probably contains little more than theMYCNgene.

Cloning and applications of the two/three-base restriction endonuclease R· CviJI from IL-3A virus-infected chlorella

The gene ( cviJIR) encoding the two/three-base R· CviJI eukaryotic restriction endonuclease (ENase) from IL-3A virus-infected Chlorella was cloned into Escherichia coli. A high frequency of DNA cleavage by R· CviJI required overexpression of the gene encoding the M· CviJI methyltransferase prior to cloning the gene for the ENase. Both genes were sequenced and their organization was determined to be in head-to-tail order. The open reading frame coding for R· CviJI can potentially translate a 41.4-kDa protein; however, in the E. coli host, a truncated version of the enzyme is produced (32.5 kDa). The recombinant ENase does not exhibit ATP-induced ‘star’ activity (R· CviJI cleaves at RGCY, while R·CviJI∗ also cleaves at RGCR and YGCY, but not at YGCR), as is characteristic for native R· CviJI. The very high frequency of DNA cleavage by R· CviJI∗ was exploited in the development of a quasi-random shotgun library method. R· CviJI∗-generated oligodeoxyribonucleotides were applied to improve certain molecular biology applications, i.e., DNA labeling, detection, high-resolution restriction mapping, amplification and epitope mapping.

The PML gene is linked to a megabase-scale insertion/deletion restriction fragment length polymorphism

The PML gene located on chromosome band 15q22 is involved with the RARα locus (17q21) in a balanced reciprocal translocation uniquely observed in acute promyelocytic leukemia. Physical mapping studies by pulsed-field gel electrophoresis revealed that the PML gene is flanked by two CpG islands that are separated by a variable distance in normal individuals. Several lines of evidence demonstrate that this is the consequence of a large insertion/deletion polymorphism linked to the PML locus: (1) overlapping fragments obtained with a variety of rare-cutting restriction enzymes demonstrated the same variability in distance between the flanking CpG islands; (2) mapping with restriction enzymes insensitive to CpG methylation confirmed that the findings were not a consequence of variable methylation of CpG dinucleotides; (3) the polymorphism followed a Mendelian inheritance pattern. This polymorphism is localized 3′ to the PML locus. There are five common alleles, described on the basis of BssHII fragments, ranging from 220 to 350 kb with increments of approximately 30 kb between alleles. Both heterozygous (61%) and homozygous (39%) patterns were observed in normal individuals. Megabase-scale insertion/deletion restriction fragment length polymorphisms are very rare and have been described initially in the context of multigene families. Such structures have been also reported as likely regions of genetic instability. High-resolution restriction mapping of this particular structure linked to the PML locus is underway.