scholarly journals Hybridization Capture Using RAD Probes (hyRAD), a New Tool for Performing Genomic Analyses on Collection Specimens

2015 ◽  
Author(s):  
Tomasz Suchan ◽  
Camille Pitteloud ◽  
Nadezhda S. Gerasimova ◽  
Anna Kostikova ◽  
Sarah Schmid ◽  
...  
Keyword(s):  
Restriction Site ◽  
Degraded Dna ◽  
Model Organisms ◽  
Butterfly Species ◽  
Large Set ◽  
Grasshopper Species ◽  
Size Selection ◽  
Restriction Sites ◽  
Museum Samples ◽  
Orthologous Loci

In the recent years, many protocols aimed at reproducibly sequencing reduced-genome subsets in non-model organisms have been published. Among them, RAD-sequencing is one of the most widely used. It relies on digesting DNA with specific restriction enzymes and performing size selection on the resulting fragments. Despite its acknowledged utility, this method is of limited use with degraded DNA samples, such as those isolated from museum specimens, as these samples are less likely to harbor fragments long enough to comprise two restriction sites making possible ligation of the adapter sequences (in the case of double-digest RAD) or performing size selection of the resulting fragments (in the case of single-digest RAD). Here, we address these limitations by presenting a novel method called hybridization RAD (hyRAD). In this approach, biotinylated RAD fragments, covering a random fraction of the genome, are used as baits for capturing homologous fragments from genomic shotgun sequencing libraries. This simple and cost-effective approach allows sequencing of orthologous loci even from highly degraded DNA samples, opening new avenues of research in the field of museum genomics. Not relying on the restriction site presence, it improves among-sample loci coverage. In a trial study, hyRAD allowed us to obtain a large set of orthologous loci from fresh and museum samples from a non-model butterfly species, with a high proportion of single nucleotide polymorphisms present in all eight analyzed specimens, including 58-year-old museum samples. The utility of the method was further validated using 49 museum and fresh samples of a Palearctic grasshopper species for which the spatial genetic structure was previously assessed using mtDNA amplicons. The application of the method is eventually discussed in a wider context. As it does not rely on the restriction site presence, it is therefore not sensitive to among-sample loci polymorphisms in the restriction sites that usually causes loci dropout. This should enable the application of hyRAD to analyses at broader evolutionary scales.

Removing the bad apples: a simple bioinformatic method to improve loci-recovery in de novo RADseq data for non-model organisms

2020 ◽  
Author(s):  
José Cerca ◽  
Marius F. Maurstad ◽  
Nicolas Rochette ◽  
Angel Rivera-Colón ◽  
Niraj Rayamajhi ◽  
...  
Keyword(s):  
Missing Data ◽  
Restriction Site ◽  
De Novo ◽  
Model Organisms ◽  
Library Preparation ◽  
Allele Dropout ◽  
Bioinformatic Tools ◽  
Restriction Sites ◽  
Genomic Markers ◽  
Wet Lab

The restriction site-associated DNA (RADseq) family of protocols involves digesting DNA and sequencing the region flanking the cut site, thus providing a cost and time efficient way for obtaining thousands of genomic markers. However, when working with non-model taxa with few genomic resources, optimization of RADseq wet-lab and bioinformatic tools may be challenging, often resulting in allele dropout – that is when a given RADseq locus is not sequenced in one or more individuals resulting in missing data. Additionally, as datasets include divergent taxa, rates of dropout will increase since restriction sites may be lost due to mutation. Mitigating the impacts of allele dropout is crucial, as missing data may lead to incorrect inferences in population genetics and phylogenetics. Here, we demonstrate a simple pipeline for the optimization of RADseq datasets which involves reducing and analysing datasets at a population or species level. By running the software Stacks at this level, we were able to reliably identify and remove individuals with high levels of missing data (bad apples) likely stemming from artefacts in library preparation, DNA quality or sequencing artefacts. Removal of the bad apples generally led to an increase of loci and decrease of missing data in the final datasets, thus improving the biological interpretability of the data.

scholarly journals Naturally occurring variation in the restriction map of the amy region of Drosophila melanogaster.

Genetics ◽  
1988 ◽  
Vol 119 (3) ◽  
pp. 619-629
Author(s):  
C H Langley ◽  
A E Shrimpton ◽  
T Yamazaki ◽  
N Miyashita ◽  
Y Matsuo ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Restriction Site ◽  
Natural Populations ◽  
Alpha Amylase ◽  
Low Frequencies ◽  
Linkage Disequilibria ◽  
Restriction Sites ◽  
Site Variation ◽  
Transcriptional Units ◽  
Polymorphic Restriction

Abstract The restriction maps of 85 alleles of the Amy region of Drosophila melanogaster from natural populations were surveyed. A subset of these were also scored for allozyme phenotype and adult enzyme activity of alpha-amylase. Large insertions were found in 12% of the alleles in a 15-kb region surrounding the two transcriptional units of the duplicated Amy locus. The low frequencies at which each of these large insertions were found are consistent with earlier reports of variation in other loci. Four small deletions were found in the region 5' to the Amy genes. Each was also rare in the population. Restriction site variation provided an estimate of per nucleotide heterozygosity of 0.006. Several statistically significant linkage disequilibria were observed between four polymorphic restriction sites and the allozymes. Adult alpha-amylase activity was correlated with the allozymes and with the polymorphism at one restriction site close to the transcriptional units.

A simplified procedure for cDNA and genomic library construction using nonpalindromic oligonucleotide adaptors

1989 ◽  
Vol 67 (4-5) ◽  
pp. 205-209 ◽  
Author(s):  
Cynthia R. D'Souza ◽  
Ken V. Deugau ◽  
John H. Spencer
Keyword(s):  
Genomic Dna ◽  
Restriction Site ◽  
Genomic Library ◽  
Restriction Endonuclease Site ◽  
Dna Libraries ◽  
Target Dna ◽  
Restriction Sites ◽  
Enzyme Cleavage ◽  
Simplified Procedure ◽  
Genomic Cdna

The properties and characteristics of oligonucleotide adaptors for use in a simplified procedure for the construction of cDNA and genomic DNA libraries are described. The adaptors are suitable for joining to blunt ended cDNA or sheared genomic DNA, and then to the cohesive ends of restriction sites in vectors. Each adaptor consists of two oligonucleotides with complementary but nonpalindromic sequences that include an internal restriction site, a 5′ phosphorylated blunt end, and an overlapping or staggered 5′ hydroxylated end corresponding to a restriction endonuclease site in a vector of choice. Ligation of the blunt end to high molecular weight target DNA proceeds efficiently and there is no tandem concatenation of the adaptor. Insertion into the appropriate vector only requires ligation of the cohesive ends. There is no requirement for methylation, restriction enzyme cleavage, G-C tailing, or denaturation after ligation of the adaptor to the target DNA, all characteristics of other procedures.Key words: library, genomic, cDNA, oligonucleotides, adaptors.

scholarly journals EVOLUTIONARY CHANGE OF RESTRICTION CLEAVAGE SITES AND PHYLOGENETIC INFERENCE

Genetics ◽  
1986 ◽  
Vol 113 (1) ◽  
pp. 187-213
Author(s):  
Wen-Hsiung Li
Keyword(s):  
Nucleotide Substitution ◽  
Restriction Site ◽  
Evolutionary Change ◽  
Cleavage Sites ◽  
Restriction Sites ◽  
Unequal Rates ◽  
Mathematical Formulas ◽  
Recent Species ◽  
Restriction Cleavage ◽  
Evolutionary Lineages

ABSTRACT Mathematical formulas are developed for the evolutionary change of restriction cleavage sites in a DNA sequence, allowing unequal rates between transitional and transversional types of nucleotide substitution. Formulas are also developed for the probability of having a particular pattern of site changes among evolutionary lineages, such as parallel gains or losses of sites, and for inferring the presence or absence of a restriction site in an ancestral sequence from data on the present-day sequences. The unordered compatibility method is proposed for inferring the phylogenetic relationships among relatively closely related organisms, treating restriction sites as cladistic characters. Formulas are derived for the probability (P  +) of obtaining the correct network for a given number (N) of informative sites for the cases of four and five species. These formulas are applied to evaluate the performance of the method and to estimate the N value required for P  + to be 95% or larger. The method performs well when the branches between ancestral nodes and the branches leading to the two most recent species are more or less equal in length, but performs poorly when the latter two branches are considerably longer than the former.

scholarly journals indCAPS: A tool for designing screening primers for CRISPR/Cas9 mutagenesis events

2017 ◽  
Author(s):  
Charles Hodgens ◽  
Zachary L. Nimchuk ◽  
Joseph J. Kieber
Keyword(s):  
Restriction Site ◽  
Genetic Manipulation ◽  
Pcr Primers ◽  
Null Alleles ◽  
Wild Type ◽  
Web Tool ◽  
Web Based ◽  
Restriction Sites ◽  
Polymerase Chain ◽  
Enzyme Recognition

AbstractGenetic manipulation of organisms using CRISPR/Cas9 technology generally produces small insertions/deletions (indels) that can be difficult to detect. Here, we describe a technique to easily and rapidly identify such indels. Sequence-identified mutations that alter a restriction enzyme recognition site can be easily distinguished from wild-type alleles using a cleaved amplified polymorphic sequence (CAPS) technique. If a restriction site is created or altered by the mutation such that only one allele contains the restriction site, a polymerase chain reaction (PCR) followed by a restriction digest can be used to distinguish the two alleles. However, in the case of most CRISPR-induced alleles, no such restriction sites are present in the target sequences. In this case, a derived CAPS (dCAPS) approach can be used in which mismatches are purposefully introduced in the oligonucleotide primers to create a restriction site in one, but not both, of the amplified templates. Web-based tools exist to aid dCAPS primer design, but when supplied sequences that include indels, the current tools often fail to suggest appropriate primers. Here, we report the development of a Python-based, species-agnostic web tool, called indCAPS, suitable for the design of PCR primers used in dCAPS assays that is compatible with indels. This tool should have wide utility for screening editing events following CRISPR/Cas9 mutagenesis as well as for identifying specific editing events in a pool of CRISPR-mediated mutagenesis events. This tool was field-tested in a CRISPR mutagenesis experiment targeting a cytokinin receptor (AHK3) in Arabidopsis thaliana. The tool suggested primers that successfully distinguished between wild-type and edited alleles of a target locus and facilitated the isolation of two novel ahk3 null alleles. Users can access indCAPS and design PCR primers to employ dCAPS to identify CRISPR/Cas9 alleles at http://indcaps.kieber.cloudapps.unc.edu/.

scholarly journals GBStools: A Unified Approach for Reduced Representation Sequencing and Genotyping

2015 ◽  
Author(s):  
Thomas F Cooke ◽  
Muh-Ching Yee ◽  
Marina Muzzio ◽  
Alexandra Sockell ◽  
Ryan Bell ◽  
...  
Keyword(s):  
Restriction Site ◽  
Variant Calling ◽  
Simulated Data ◽  
Error Rates ◽  
Genomic Diversity ◽  
Model Organisms ◽  
Data Sets ◽  
Reduced Representation ◽  
Restriction Site Polymorphisms ◽  
Reduced Representation Sequencing

Reduced representation sequencing methods such as genotyping-by-sequencing (GBS) enable low-cost measurement of genetic variation without the need for a reference genome assembly. These methods are widely used in genetic mapping and population genetics studies, especially with non-model organisms. Variant calling error rates, however, are higher in GBS than in standard sequencing, in particular due to restriction site polymorphisms, and few computational tools exist that specifically model and correct these errors. We developed a statistical method to remove errors caused by restriction site polymorphisms, implemented in the software package GBStools. We evaluated it in several simulated data sets, varying in number of samples, mean coverage and population mutation rate, and in two empirical human data sets (N = 8 and N = 63 samples). In our simulations, GBStools improved genotype accuracy more than commonly used filters such as Hardy-Weinberg equilibrium p-values. GBStools is most effective at removing genotype errors in data sets over 100 samples when coverage is 40X or higher, and the improvement is most pronounced in species with high genomic diversity. We also demonstrate the utility of GBS and GBStools for human population genetic inference in Argentine populations and reveal widely varying individual ancestry proportions and an excess of singletons, consistent with recent population growth.

scholarly journals Restriction digest screening facilitates efficient detection of site-directed mutations introduced by CRISPR in C. albicans UME6

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4920 ◽  
Author(s):  
Ben A. Evans ◽  
Olivia L. Smith ◽  
Ethan S. Pickerill ◽  
Mary K. York ◽  
Kristen J.P. Buenconsejo ◽  
...  
Keyword(s):  
Protein Function ◽  
Point Mutations ◽  
Pcr Amplification ◽  
Restriction Enzymes ◽  
Model Organisms ◽  
Efficient Manner ◽  
Restriction Digest ◽  
Efficient Detection ◽  
Wide Range ◽  
Restriction Sites

Introduction of point mutations to a gene of interest is a powerful tool when determining protein function. CRISPR-mediated genome editing allows for more efficient transfer of a desired mutation into a wide range of model organisms. Traditionally, PCR amplification and DNA sequencing is used to determine if isolates contain the intended mutation. However, mutation efficiency is highly variable, potentially making sequencing costly and time consuming. To more efficiently screen for correct transformants, we have identified restriction enzymes sites that encode for two identical amino acids or one or two stop codons. We used CRISPR to introduce these restriction sites directly upstream of the Candida albicans UME6 Zn2+-binding domain, a known regulator of C. albicans filamentation. While repair templates coding for different restriction sites were not equally successful at introducing mutations, restriction digest screening enabled us to rapidly identify isolates with the intended mutation in a cost-efficient manner. In addition, mutated isolates have clear defects in filamentation and virulence compared to wild type C. albicans. Our data suggest restriction digestion screening efficiently identifies point mutations introduced by CRISPR and streamlines the process of identifying residues important for a phenotype of interest.

scholarly journals Putative Nucleotide-Based Second Messengers in the Archaeal Model Organisms Haloferax volcanii and Sulfolobus acidocaldarius

2021 ◽  
Vol 12 ◽  
Author(s):  
Frank Braun ◽  
Alejandra Recalde ◽  
Heike Bähre ◽  
Roland Seifert ◽  
Sonja-Verena Albers
Keyword(s):  
Second Messengers ◽  
Building Blocks ◽  
Signaling Molecules ◽  
Haloferax Volcanii ◽  
Sulfolobus Acidocaldarius ◽  
Model Organisms ◽  
Large Set ◽  
Cell Extracts ◽  
Broad Variety ◽  
Modern Mass

Research on nucleotide-based second messengers began in 1956 with the discovery of cyclic adenosine monophosphate (3′,5′-cAMP) by Earl Wilbur Sutherland and his co-workers. Since then, a broad variety of different signaling molecules composed of nucleotides has been discovered. These molecules fulfill crucial tasks in the context of intracellular signal transduction. The vast majority of the currently available knowledge about nucleotide-based second messengers originates from model organisms belonging either to the domain of eukaryotes or to the domain of bacteria, while the archaeal domain is significantly underrepresented in the field of nucleotide-based second messenger research. For several well-stablished eukaryotic and/or bacterial nucleotide-based second messengers, it is currently not clear whether these signaling molecules are present in archaea. In order to shed some light on this issue, this study analyzed cell extracts of two major archaeal model organisms, the euryarchaeon Haloferax volcanii and the crenarchaeon Sulfolobus acidocaldarius, using a modern mass spectrometry method to detect a broad variety of currently known nucleotide-based second messengers. The nucleotides 3′,5′-cAMP, cyclic guanosine monophosphate (3′,5′-cGMP), 5′-phosphoadenylyl-3′,5′-adenosine (5′-pApA), diadenosine tetraphosphate (Ap4A) as well as the 2′,3′-cyclic isomers of all four RNA building blocks (2′,3′-cNMPs) were present in both species. In addition, H. volcanii cell extracts also contain cyclic cytosine monophosphate (3′,5′-cCMP), cyclic uridine monophosphate (3′,5′-cUMP) and cyclic diadenosine monophosphate (3′,5′-c-di-AMP). The widely distributed bacterial second messengers cyclic diguanosine monophosphate (3′,5′-c-di-GMP) and guanosine (penta-)/tetraphosphate [(p)ppGpp] could not be detected. In summary, this study gives a comprehensive overview on the presence of a large set of currently established or putative nucleotide-based second messengers in an eury- and a crenarchaeal model organism.

Molecular Genetic Divergence between Avian Sibling Species: King and Clapper Rails, Long-Billed and Short-Billed Dowitchers, Boat-Tailed and Great-Tailed Grackles, and Tufted and Black-Crested Titmice

The Auk ◽  
1988 ◽  
Vol 105 (3) ◽  
pp. 516-528 ◽  
Author(s):  
John C. Avise ◽  
Robert M. Zink
Keyword(s):  
Large Scale ◽  
Restriction Site ◽  
Molecular Genetic ◽  
Bird Species ◽  
Sequence Divergence ◽  
Genetic Distances ◽  
Resolving Power ◽  
Molecular Features ◽  
Restriction Sites ◽  
Parus Bicolor

Abstract Surveys of electrophoretic variation in proteins, and restriction site variation in mitochondrial DNA (mtDNA), were conducted to assess the resolving power of these molecular genetic techniques to distinguish four pairs of avian sibling taxa. Samples of rails (Rallus elegans and R. longirostris), dowitchers (Limnodromus scolopaceus and L. griseus), grackles (Quiscalus major and Q. mexicanus), and titmice (Parus bicolor bicolor and P. b. atricristatus) were assayed for allozymes encoded by 34-37 nuclear loci, and for an average of 77 mtDNA restriction sites per individual by 19 endonucleases. MtDNA's of the two rail species showed large-scale size polymorphism and individual heteroplasmy, the first such findings of these molecular features in an avian species. Genetic distances based on allozyme comparisons were small for all assayed taxa (Nei's D ≤ 0.063). The mtDNA assays offered consistently greater resolving power, providing at least five fixed restriction site differences for samples of any taxon pair. The Long-billed and Short-billed dowitchers were especially divergent, differing by at least 24 assayed mtDNA restriction sites and an estimated nucleotide sequence divergence of p = 0.082. We compared these results to previous reports of genetic distances within and among closely related bird species. The mtDNA divergence among dowitchers is near the high end of the scale of such estimates for avian congeners. The mtDNA distances between the pairs of rails (p = 0.006), titmice (p = 0.004), and grackles (p = 0.016) were typical for extremely closely related species, and overlap maximum values reported for some avian conspecifics.

Sign in / Sign up

Export Citation Format

Share Document