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PloS One 2021In genomics, optical mapping technology provides long-range contiguity information to improve genome sequence assemblies and detect structural variation. Originally a...
In genomics, optical mapping technology provides long-range contiguity information to improve genome sequence assemblies and detect structural variation. Originally a laborious manual process, Bionano Genomics platforms now offer high-throughput, automated optical mapping based on chips packed with nanochannels through which unwound DNA is guided and the fluorescent DNA backbone and specific restriction sites are recorded. Although the raw image data obtained is of high quality, the processing and assembly software accompanying the platforms is closed source and does not seem to make full use of data, labeling approximately half of the measured signals as unusable. Here we introduce two new software tools, independent of Bionano Genomics software, to extract and process molecules from raw images (OptiScan) and to perform molecule-to-molecule and molecule-to-reference alignments using a novel signal-based approach (OptiMap). We demonstrate that the molecules detected by OptiScan can yield better assemblies, and that the approach taken by OptiMap results in higher use of molecules from the raw data. These tools lay the foundation for a suite of open-source methods to process and analyze high-throughput optical mapping data. The Python implementations of the OptiTools are publicly available through http://www.bif.wur.nl/.
Topics: Chromosome Mapping; Genomics; High-Throughput Nucleotide Sequencing; Optical Restriction Mapping; Sequence Analysis, DNA
PubMed: 34591846
DOI: 10.1371/journal.pone.0253102 -
Nature Reviews. Genetics Feb 2016High-throughput techniques based on restriction site-associated DNA sequencing (RADseq) are enabling the low-cost discovery and genotyping of thousands of genetic... (Review)
Review
High-throughput techniques based on restriction site-associated DNA sequencing (RADseq) are enabling the low-cost discovery and genotyping of thousands of genetic markers for any species, including non-model organisms, which is revolutionizing ecological, evolutionary and conservation genetics. Technical differences among these methods lead to important considerations for all steps of genomics studies, from the specific scientific questions that can be addressed, and the costs of library preparation and sequencing, to the types of bias and error inherent in the resulting data. In this Review, we provide a comprehensive discussion of RADseq methods to aid researchers in choosing among the many different approaches and avoiding erroneous scientific conclusions from RADseq data, a problem that has plagued other genetic marker types in the past.
Topics: Biological Evolution; Genomics; High-Throughput Nucleotide Sequencing; Humans; Metagenomics; Restriction Mapping
PubMed: 26729255
DOI: 10.1038/nrg.2015.28 -
Anticancer Research 2008Many different single nucleotide polymorphisms (SNPs) genotyping methods have been developed recently. However, most of them are expensive. Using restriction enzymes for... (Review)
Review
Many different single nucleotide polymorphisms (SNPs) genotyping methods have been developed recently. However, most of them are expensive. Using restriction enzymes for SNP genotyping is a cost-effective method. However, restriction enzyme mining for SNPs in a genome sequence is still challenging for researchers who do not have a background in genomics and bioinformatics. In this review, the basic bioinformatics tools used for restriction enzyme mining for SNP genotyping are summarized and described. The objectives of this paper include: i) the introduction of SNPs, genotyping and PCR-restriction fragment length polymorphism (RFLP); ii) a review of components for genotyping software, including tools for primer design only or restriction enzyme mining only; iii) a review of software providing the flanking sequence for primer design; iv) recent advances in PCR-RFLP tools and natural and mutagenic PCR-RFLP; v) highlighting the strategy for restriction enzyme mining for SNP genotyping; vi) a discussion of potential problems for multiple PCR-RFLP. The different implications for restriction enzymes on sense and antisense strands are also discussed. Our PCR-RFLP freeware, SNP-RFLPing, is included in this review to illustrate many characteristics of PCR-RFLP software design. Future developments will include further sophistication of PCR-RFLP software in order to provide better visualization and a more interactive environment for SNP genotyping and to integrate the software with other tools used in association studies.
Topics: Animals; DNA Restriction Enzymes; Genome; Genotype; Humans; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Polymorphism, Single Nucleotide; Restriction Mapping
PubMed: 18649739
DOI: No ID Found -
Genetics Jul 2017Assembly of complex genomes using short reads remains a major challenge, which usually yields highly fragmented assemblies. Generation of ultradense linkage maps is...
Assembly of complex genomes using short reads remains a major challenge, which usually yields highly fragmented assemblies. Generation of ultradense linkage maps is promising for anchoring such assemblies, but traditional linkage mapping methods are hindered by the infrequency and unevenness of meiotic recombination that limit attainable map resolution. Here we develop a sequencing-based "" linkage mapping approach (called RadMap), where chromosome breakage and segregation are realized by generating hundreds of "subhaploid" fosmid/bacterial-artificial-chromosome clone pools, and by restriction site-associated DNA sequencing of these clone pools to produce an ultradense whole-genome restriction map to facilitate genome scaffolding. A bootstrap-based minimum spanning tree algorithm is developed for grouping and ordering of genome-wide markers and is implemented in a user-friendly, integrated software package (AMMO). We perform extensive analyses to validate the power and accuracy of our approach in the model plant and human. We also demonstrate the utility of RadMap for enhancing the contiguity of a variety of whole-genome shotgun assemblies generated using either short Illumina reads (300 bp) or long PacBio reads (6-14 kb), with up to 15-fold improvement of N50 (∼816 kb-3.7 Mb) and high scaffolding accuracy (98.1-98.5%). RadMap outperforms BioNano and Hi-C when input assembly is highly fragmented (contig N50 = 54 kb). RadMap can capture wide-range contiguity information and provide an efficient and flexible tool for high-resolution physical mapping and scaffolding of highly fragmented assemblies.
Topics: Arabidopsis; Genetic Linkage; Genome, Plant; Restriction Mapping; Software
PubMed: 28468906
DOI: 10.1534/genetics.117.200303 -
Nucleic Acids Research Apr 2021DNA phosphorothioate (PT) modifications, with the nonbridging phosphate oxygen replaced by sulfur, governed by DndABCDE or SspABCD, are widely distributed in prokaryotes...
DNA phosphorothioate (PT) modifications, with the nonbridging phosphate oxygen replaced by sulfur, governed by DndABCDE or SspABCD, are widely distributed in prokaryotes and have a highly unusual feature of occupying only a small portion of available consensus sequences in a genome. Despite the presence of plentiful non-PT-protected consensuses, DNA PT modification is still employed as a recognition tag by the restriction cognate, for example, DndFGH or SspE, to discriminate and destroy PT-lacking foreign DNA. This raises a fundamental question about how PT modifications are distributed along DNA molecules to keep the restriction components in check. Here, we present two single-molecule strategies that take advantage of the nucleophilicity of PT in combination with fluorescent markers for optical mapping of both single- and double-stranded PT modifications across individual DNA molecules. Surprisingly, PT profiles vary markedly from molecule to molecule, with different PT locations and spacing distances between PT pairs, even in the presence of DndFGH or SspE. The results revealed unprecedented PT modification features previously obscured by ensemble averaging, providing novel insights into the riddles regarding unusual target selection by PT modification and restriction components.
Topics: Bacterial Proteins; DNA, Bacterial; Epigenesis, Genetic; Escherichia coli; Genome, Bacterial; Optical Restriction Mapping; Phosphorothioate Oligonucleotides
PubMed: 33764453
DOI: 10.1093/nar/gkab169 -
GigaScience 2015Optical mapping is a technology that gathers long-range information on genome sequences similar to ordered restriction digest maps. Because it is not subject to cloning,... (Review)
Review
Optical mapping is a technology that gathers long-range information on genome sequences similar to ordered restriction digest maps. Because it is not subject to cloning, amplification, hybridisation or sequencing bias, it is ideally suited to the improvement of fragmented genome assemblies that can no longer be improved by classical methods. In addition, its low cost and rapid turnaround make it equally useful during the scaffolding process of de novo assembly from high throughput sequencing reads. We describe how optical mapping has been used in practice to produce high quality vertebrate genome assemblies. In particular, we detail the efforts undertaken by the Genome Reference Consortium (GRC), which maintains the reference genomes for human, mouse, zebrafish and chicken, and uses different optical mapping platforms for genome curation.
Topics: Animals; Genome; Genomics; Restriction Mapping; Sequence Analysis; Vertebrates
PubMed: 25789164
DOI: 10.1186/s13742-015-0052-y -
Bioinformatics (Oxford, England) Apr 2016The Optical Mapping System discovers structural variants and potentiates sequence assembly of genomes via scaffolding and comparisons that globally validate or correct...
MOTIVATION
The Optical Mapping System discovers structural variants and potentiates sequence assembly of genomes via scaffolding and comparisons that globally validate or correct sequence assemblies. Despite its utility, there are few publicly available tools for aligning optical mapping datasets.
RESULTS
Here we present software, named 'Maligner', for the alignment of both single molecule restriction maps (Rmaps) and in silico restriction maps of sequence contigs to a reference. Maligner provides two modes of alignment: an efficient, sensitive dynamic programming implementation that scales to large eukaryotic genomes, and a faster indexed based implementation for finding alignments with unmatched sites in the reference but not the query. We compare our software to other publicly available tools on Rmap datasets and show that Maligner finds more correct alignments in comparable runtime. Lastly, we introduce the M-Score statistic for normalizing alignment scores across restriction maps and demonstrate its utility for selecting high quality alignments.
AVAILABILITY AND IMPLEMENTATION
The Maligner software is written in C ++ and is available at https://github.com/LeeMendelowitz/maligner under the GNU General Public License.
CONTACT
Topics: Algorithms; Computer Simulation; Genome; Restriction Mapping; Sequence Alignment; Sequence Analysis, DNA; Software
PubMed: 26637292
DOI: 10.1093/bioinformatics/btv711 -
Experimental Animals Jan 1998A genetic typing method for the mouse and rat nude mutations by PCR and restriction fragment length polymorphism (RFLP) analysis was developed. Since restriction sites... (Review)
Review
A genetic typing method for the mouse and rat nude mutations by PCR and restriction fragment length polymorphism (RFLP) analysis was developed. Since restriction sites useful for RFLP analysis do not exist in the mouse nu and rat rnu mutations, artificial restriction sites were introduced by PCR with modified primers. Three genotypes in the mouse (nu/nu, nu/+ and +/+) or rat (rnu/rnu, rnu/+ and +/+) are rapidly differentiated with the PCR-RFLP assay. In addition, congenic nude strains can be efficiently established by using this assay. Finally, genetic mapping of the rnu locus was performed with microsatellite markers. The locus order on rat chromosome 10 was D10Mgh14-(2.0cM)-D10Mit2-(1.4cM)-rnu-(0.7cM++ +)-D10Mgh6-(2.7cM)-D10Mit8.
Topics: Animals; Genotype; Mice; Mice, Inbred BALB C; Mice, Nude; Microsatellite Repeats; Mutation; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Rats; Rats, Nude; Rats, Wistar; Restriction Mapping
PubMed: 9498115
DOI: 10.1538/expanim.47.63 -
BioTechniques Sep 2002Ever since methylcytosine was found in genomic DNA, this epigenetic alteration has become a center of scientific attraction, especially because of its relation to gene... (Review)
Review
Ever since methylcytosine was found in genomic DNA, this epigenetic alteration has become a center of scientific attraction, especially because of its relation to gene silencing in disease. There is currently a wide range of methods designed to yield quantitative and qualitative information on genomic DNA methylation. The earliest approaches were concentrated on the study of overall levels of methylcytosine, but more recent efforts havefocused on the study ofthe methylation status of specific DNA sequences. Particularly, optimization of the methods based on bisulfite modification of DNA permits the analysis of limited CpGs in restriction enzyme sites (e.g., combined bisulfite restriction analyses and methylation-sensitive single nucleotide primer extension) and the overall characterization based on differential methylation states (e.g., methylation-specific PCR, MethyLight, and methylation-sensitive single-stranded conformational polymorphism) and allows very specific patterns of methylation to be revealed (bisulfite DNA sequencing). In addition, novel methods designed to search for new methylcytosine hot spots have yielded further data without requiring prior knowledge of the DNA sequence. We hope this review will be a valuable tool in selecting the best techniques to address particular questions concerning the cytosine methylation status of genomic DNA.
Topics: 5-Methylcytosine; Chromatography, High Pressure Liquid; Cytosine; DNA; DNA Methylation; Electrophoresis, Capillary; Genome; Microchemistry; Polymerase Chain Reaction; Restriction Mapping; Sequence Analysis, DNA; Sulfites
PubMed: 12238773
DOI: 10.2144/02333rv01 -
Revue Scientifique Et Technique... Sep 1990The potential contributions of techniques, such as restriction enzyme analysis, nucleic acid detection, the polymerase chain reaction and competitive inhibitive tests,... (Review)
Review
The potential contributions of techniques, such as restriction enzyme analysis, nucleic acid detection, the polymerase chain reaction and competitive inhibitive tests, are only beginning to be defined. The extraordinary promise of these procedures has yet to be fully realized. However, before these techniques are accepted and widely used, they should be shown to have sensitivity and specificity comparable to those of current tests. Finally, they should be safe, easy to conduct and automated to facilitate the study of large numbers of specimens.
Topics: Animals; Antibodies, Monoclonal; Antigens; Bacterial Infections; DNA Probes; Polymerase Chain Reaction; Recombinant Proteins; Restriction Mapping; Virus Diseases
PubMed: 2132702
DOI: 10.20506/rst.9.3.515