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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 -
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 -
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 -
BMC Genomics Aug 2016Restriction site associated DNA sequencing (RAD-seq), a next-generation sequencing technology, has greatly facilitated genetic linkage mapping studies in outbred...
BACKGROUND
Restriction site associated DNA sequencing (RAD-seq), a next-generation sequencing technology, has greatly facilitated genetic linkage mapping studies in outbred species. RAD-seq is capable of discovering thousands of genetic markers for linkage mapping across many individuals, and can be applied in species with or without a reference genome. Although several analytical tools are available for RAD-seq data, alternative strategies are necessary for improving the marker quality and hence the genetic mapping accuracy.
RESULTS
We demonstrate a strategy for constructing dense genetic linkage maps in hybrid forest trees by combining RAD-seq and whole-genome sequencing technologies. We performed RAD-seq of 150 progeny and whole-genome sequencing of the two parents in an F1 hybrid population of Populus deltoides × P. simonii. Two rough references were assembled from the whole-genome sequencing reads of the two parents separately. Based on the parental reference sequences, 3442 high-quality single nucleotide polymorphisms (SNPs) were identified that segregate in the ratio of 1:1. The maternal linkage map of P. deltoides was constructed with 2012 SNPs, containing 19 linkage groups and spanning 4067.16 cM of the genome with an average distance of 2.04 cM between adjacent markers, while the male map of P. simonii consisted of 1430 SNPs and the same number of linkage groups with a total length of 4356.04 cM and an average interval distance of 3.09 cM. Collinearity between the parental linkage maps and the reference genome of P. trichocarpa was also investigated. Compared with the result on the basis of the existing reference genome, our strategy identified more high-quality SNPs and generated parental linkage groups that nicely match the karyotype of Populus.
CONCLUSIONS
The strategy of simultaneously using RAD and whole-genome sequencing technologies can be applied to constructing high-density genetic maps in forest trees regardless of whether a reference genome exists. The two parental linkage maps constructed here provide more accurate genetic resources for unraveling quantitative trait loci and accelerating molecular breeding programs, as well as for comparative genomics in Populus.
Topics: Chimera; Chromosome Mapping; Chromosomes, Plant; Genetic Linkage; Genome, Plant; Polymorphism, Single Nucleotide; Populus; Quantitative Trait Loci; Restriction Mapping; Sequence Analysis, DNA
PubMed: 27538483
DOI: 10.1186/s12864-016-3003-9 -
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 -
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 -
Revue Scientifique Et Technique... Dec 2006To determine the genomic variation of equine herpesviruses (EHVs) isolated in Argentina between 1979 and the first half of 2004, DNA sequences from all 69 strains... (Review)
Review
To determine the genomic variation of equine herpesviruses (EHVs) isolated in Argentina between 1979 and the first half of 2004, DNA sequences from all 69 strains isolated were analysed. Sixty strains were recovered from aborted fetuses, one from leucocyte-rich plasma from a horse with respiratory signs and eight from cases of neonatal disease. The DNA was extracted from rabbit kidney epithelial (RK13) cells infected with each strain and digested with three restriction endonucleases (BamHI, Bg/II and KpnI). Two strains could be differentiated using BamHI restriction and were assigned to the EHV-1 1B prototype group. Only one of these two strains was typed EHV-1 1B with Bg/II. DNA digestion with KpnI was ineffective. The results obtained in this study demonstrate that the EHV-1 1B genome has been present in Argentina since at least 1996. The finding of two strains with this electropherotype suggests that there is genomic heterogeneity among Argentinian isolates.
Topics: Abortion, Veterinary; Animals; Argentina; Base Sequence; DNA Restriction Enzymes; DNA, Viral; Female; Genetic Variation; Genome; Herpesviridae Infections; Herpesvirus 1, Equid; Horse Diseases; Horses; Pregnancy; Pregnancy Complications, Infectious; Restriction Mapping
PubMed: 17361771
DOI: No ID Found