scholarly journals How to Isolate a Plant’s Hypomethylome in One Shot

2015 ◽  
Vol 2015 ◽  
pp. 1-12 ◽  
Author(s):  
Elisabeth Wischnitzki ◽  
Eva Maria Sehr ◽  
Karin Hansel-Hohl ◽  
Maria Berenyi ◽  
Kornel Burg ◽  
...  
Keyword(s):  
De Novo ◽  
Cost Effective ◽  
Direct Analysis ◽  
Regulatory Elements ◽  
Crocus Sativus ◽  
Whole Genome ◽  
Gene Space ◽  
Wide Range ◽  
Flanking Regions ◽  
Generation Sequencing

Genome assembly remains a challenge for large and/or complex plant genomes due to their abundant repetitive regions resulting in studies focusing on gene space instead of the whole genome. Thus, DNA enrichment strategies facilitate the assembly by increasing the coverage and simultaneously reducing the complexity of the whole genome. In this paper we provide an easy, fast, and cost-effective variant of MRE-seq to obtain a plant’s hypomethylome by an optimized methyl filtration protocol followed by next generation sequencing. The method is demonstrated on three plant species with knowingly large and/or complex (polyploid) genomes:Oryza sativa,Picea abies, andCrocus sativus. The identified hypomethylomes show clear enrichment for genes and their flanking regions and clear reduction of transposable elements. Additionally, genomic sequences around genes are captured including regulatory elements in introns and up- and downstream flanks. High similarity of the results obtained by ade novoassembly approach with a reference based mapping in rice supports the applicability for studying and understanding the genomes of nonmodel organisms. Hence we show the high potential of MRE-seq in a wide range of scenarios for the direct analysis of methylation differences, for example, between ecotypes, individuals, within or across species harbouring large, and complex genomes.

scholarly journals Whole genome de novo assemblies of three divergent strains of rice, Oryza sativa , document novel gene space of aus and indica

2014 ◽  
Vol 15 (11) ◽  
pp. 506 ◽  
Author(s):  
Michael C Schatz ◽  
Lyza G Maron ◽  
Joshua C Stein ◽  
Alejandro Wences ◽  
James Gurtowski ◽  
...  
Keyword(s):  
Oryza Sativa ◽  
De Novo ◽  
Whole Genome ◽  
Novel Gene ◽  
Gene Space

scholarly journals Targeted transcriptomics reveals signatures of large-scale independent origins and concerted regulation of effector genes in Radopholus similis

2021 ◽  
Vol 17 (11) ◽  
pp. e1010036
Author(s):  
Paulo Vieira ◽  
Roxana Y. Myers ◽  
Clement Pellegrin ◽  
Catherine Wram ◽  
Cedar Hesse ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Large Scale ◽  
De Novo ◽  
Economic Damage ◽  
Radopholus Similis ◽  
Effector Genes ◽  
Wide Range ◽  
Burrowing Nematode ◽  
Generation Sequencing

The burrowing nematode, Radopholus similis, is an economically important plant-parasitic nematode that inflicts damage and yield loss to a wide range of crops. This migratory endoparasite is widely distributed in warmer regions and causes extensive destruction to the root systems of important food crops (e.g., citrus, banana). Despite the economic importance of this nematode, little is known about the repertoire of effectors owned by this species. Here we combined spatially and temporally resolved next-generation sequencing datasets of R. similis to select a list of candidates for the identification of effector genes for this species. We confirmed spatial expression of transcripts of 30 new candidate effectors within the esophageal glands of R. similis by in situ hybridization, revealing a large number of pioneer genes specific to this nematode. We identify a gland promoter motif specifically associated with the subventral glands (named Rs-SUG box), a putative hallmark of spatial and concerted regulation of these effectors. Nematode transcriptome analyses confirmed the expression of these effectors during the interaction with the host, with a large number of pioneer genes being especially abundant. Our data revealed that R. similis holds a diverse and emergent repertoire of effectors, which has been shaped by various evolutionary events, including neofunctionalization, horizontal gene transfer, and possibly by de novo gene birth. In addition, we also report the first GH62 gene so far discovered for any metazoan and putatively acquired by lateral gene transfer from a bacterial donor. Considering the economic damage caused by R. similis, this information provides valuable data to elucidate the mode of parasitism of this nematode.

scholarly journals CRISPR-bind: a simple, custom CRISPR/dCas9-mediated labeling of genomic DNA for mapping in nanochannel arrays

2018 ◽  
Author(s):  
Denghong Zhang ◽  
Saki Chan ◽  
Kenneth Sugerman ◽  
Joyce Lee ◽  
Ernest T. Lam ◽  
...  
Keyword(s):  
De Novo ◽  
Genome Mapping ◽  
Cost Effective ◽  
Target Sequence ◽  
Whole Genome ◽  
Nick Translation ◽  
Double Knockout ◽  
Genomic Structural Variation ◽  
Additional Information ◽  
Whole Genome Mapping

AbstractBionano genome mapping is a robust optical mapping technology used for de novo construction of whole genomes using ultra-long DNA molecules, able to efficiently interrogate genomic structural variation. It is also used for functional analysis such as epigenetic analysis and DNA replication mapping and kinetics. Genomic labeling for genome mapping is currently specified by a single strand nicking restriction enzyme followed by fluorophore incorporation by nick-translation (NLRS), or by a direct label and stain (DLS) chemistry which conjugates a fluorophore directly to an enzyme-defined recognition site. Although these methods are efficient and produce high quality whole genome mapping data, they are limited by the number of available enzymes—and thus the number of recognition sequences—to choose from. The ability to label other sequences can provide higher definition in the data and may be used for countless additional applications. Previously, custom labeling was accomplished via the nick-translation approach using CRISPR-Cas9, leveraging Cas9 mutant D10A which has one of its cleavage sites deactivated, thus effectively converting the CRISPR-Cas9 complex into a nickase with customizable target sequences. Here we have improved upon this approach by using dCas9, a nuclease-deficient double knockout Cas9 with no cutting activity, to directly label DNA with a fluorescent CRISPR-dCas9 complex (CRISPR-bind). Unlike labeling with CRISPR-Cas9 D10A nickase, in which nicking, labeling, and repair by ligation, all occur as separate steps, the new assay has the advantage of labeling DNA in one step, since the CRISPR-dCas9 complex itself is fluorescent and remains bound during imaging. CRISPR-bind can be added directly to a sample that has already been labeled using DLS or NLRS, thus overlaying additional information onto the same molecules. Using the dCas9 protein assembled with custom target crRNA and fluorescently labeled tracrRNA, we demonstrate rapid labeling of repetitive DUF1220 elements. We also combine NLRS-based whole genome mapping with CRISPR-bind labeling targeting Alu loci. This rapid, convenient, non-damaging, and cost-effective technology is a valuable tool for custom labeling of any CRISPR-Cas9 amenable target sequence.

scholarly journals Genome assembly of Vitis rotundifolia Michx. using third-generation sequencing (Oxford Nanopore Technologies)

2021 ◽  
Vol 182 (2) ◽  
pp. 63-71
Author(s):  
M. M. Agakhanov ◽  
E. A. Grigoreva ◽  
E. K. Potokina ◽  
P. S. Ulianich ◽  
Y. V. Ukhatova
Keyword(s):  
Genome Sequence ◽  
Genome Assembly ◽  
De Novo ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
Third Generation ◽  
Vitis Rotundifolia ◽  
Third Generation Sequencing ◽  
Genome Sequence Assembly ◽  
Generation Sequencing

The immune North American grapevine species Vitis rotundifolia Michaux (subgen. Muscadinia Planch.) is regarded as a potential donor of disease resistance genes, withstanding such dangerous diseases of grapes as powdery and downy mildews. The cultivar ‘Dixie’ is the only representative of this species preserved ex situ in Russia: it is maintained by the N.I. Vavilov All-Russian Institute of Plant Genetic Resources (VIR) in the orchards of its branch, Krymsk Experiment Breeding Station. Third-generation sequencing on the MinION platform was performed to obtain information on the primary structure of the cultivar’s genomic DNA, employing also the results of Illumina sequencing available in databases. A detailed description of the technique with modifications at various stages is presented, as it was used for grapevine genome sequencing and whole-genome sequence assembly. The modified technique included the main stages of the original protocol recommended by the MinION producer: 1) DNA extraction; 2) preparation of libraries for sequencing; 3) MinION sequencing and bioinformatic data processing; 4) de novo whole-genome sequence assembly using only MinION data or hybrid assembly (MinION+Illumina data); and 5) functional annotation of the whole-genome assembly. Stage 4 included not only de novo sequencing, but also the analysis of the available bioinformatic data, thus minimizing errors and increasing precision during the assembly of the studied genome. The DNA isolated from the leaves of cv. ‘Dixie’ was sequenced using two MinION flow cells (R9.4.1).

scholarly journals Characterizing Genetic Regulatory Elements in Ovine Tissues

2021 ◽  
Vol 12 ◽  
Author(s):  
Kimberly M. Davenport ◽  
Alisha T. Massa ◽  
Suraj Bhattarai ◽  
Stephanie D. McKay ◽  
Michelle R. Mousel ◽  
...  
Keyword(s):  
Dna Methylation ◽  
De Novo ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Ctcf Binding ◽  
Domestic Sheep ◽  
Whole Genome ◽  
Production Traits ◽  
Chromatin States ◽  
Genome Bisulfite Sequencing

The Ovine Functional Annotation of Animal Genomes (FAANG) project, part of the broader livestock species FAANG initiative, aims to identify and characterize gene regulatory elements in domestic sheep. Regulatory element annotation is essential for identifying genetic variants that affect health and production traits in this important agricultural species, as greater than 90% of variants underlying genetic effects are estimated to lie outside of transcribed regions. Histone modifications that distinguish active or repressed chromatin states, CTCF binding, and DNA methylation were used to characterize regulatory elements in liver, spleen, and cerebellum tissues from four yearling sheep. Chromatin immunoprecipitation with sequencing (ChIP-seq) was performed for H3K4me3, H3K27ac, H3K4me1, H3K27me3, and CTCF. Nine chromatin states including active promoters, active enhancers, poised enhancers, repressed enhancers, and insulators were characterized in each tissue using ChromHMM. Whole-genome bisulfite sequencing (WGBS) was performed to determine the complement of whole-genome DNA methylation with the ChIP-seq data. Hypermethylated and hypomethylated regions were identified across tissues, and these locations were compared with chromatin states to better distinguish and validate regulatory elements in these tissues. Interestingly, chromatin states with the poised enhancer mark H3K4me1 in the spleen and cerebellum and CTCF in the liver displayed the greatest number of hypermethylated sites. Not surprisingly, active enhancers in the liver and spleen, and promoters in the cerebellum, displayed the greatest number of hypomethylated sites. Overall, chromatin states defined by histone marks and CTCF occupied approximately 22% of the genome in all three tissues. Furthermore, the liver and spleen displayed in common the greatest percent of active promoter (65%) and active enhancer (81%) states, and the liver and cerebellum displayed in common the greatest percent of poised enhancer (53%), repressed enhancer (68%), hypermethylated sites (75%), and hypomethylated sites (73%). In addition, both known and de novo CTCF-binding motifs were identified in all three tissues, with the highest number of unique motifs identified in the cerebellum. In summary, this study has identified the regulatory regions of genes in three tissues that play key roles in defining health and economically important traits and has set the precedent for the characterization of regulatory elements in ovine tissues using the Rambouillet reference genome.

scholarly journals A weighted sequence alignment strategy for gene structure annotation lift over from reference genome to a newly sequenced individual

2019 ◽  
Author(s):  
Baoxing Song ◽  
Qing Sang ◽  
Hai Wang ◽  
Huimin Pei ◽  
Fen Wang ◽  
...  
Keyword(s):  
Reference Gene ◽  
Genome Sequence ◽  
Sequence Alignment ◽  
Gene Structure ◽  
Natural Variation ◽  
De Novo ◽  
Whole Genome ◽  
Genomic Variants ◽  
Wide Range ◽  
Structure Annotation

AbstractGenome sequences and gene structure annotation are very important for genomic analysis, while only the reference gene structure annotation is widely used for a wide range of investigations of different natural variation individuals. Herein, we are reporting the software GEAN which could lift over the reference gene structure annotation to other individuals belonging to the same or closely related species whose genome sequence was determined by whole-genome resequencing or de novo assembly. We found that inconsistent sequence alignment makes the coordinate lift over between different individual genomes unreliable, thus obscuring the lift over of gene structure annotations and genomic variants functional prediction. We designed a zebraic dynamic programming (ZDP) algorithm by providing different weights to different genetic features to refine the gene structure lift over. Using the lift over gene structure annotation as anchors, a base-pair resolution whole-genome-wide sequence alignment and variant calling pipeline for de novo assembly have been implemented. Taking Arabidopsis thaliana as example, we show that the natural variation alleles expression level of apoptosis death and defence response related genes might could be better quantified using GEAN. And GEAN could be used to refine the functional annotation of genetic variants, annotate de novo assembly genome sequence, detect syntenic blocks, improve the quantification of gene expression levels using RNA-seq data and genomic variants encoding for population genetic analysis. We expect that GEAN will be a standard gene structure annotation lift over and genome sequence alignment tool for the coming age of de novo assembly population genetics analysis.

scholarly journals Regulatory remodeling in the allo-tetraploid frog Xenopus laevis

2017 ◽  
Author(s):  
Dei M. Elurbe ◽  
Sarita S. Paranjpe ◽  
Georgios Georgiou ◽  
Ila van Kruijsbergen ◽  
Ozren Bogdanovic ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Whole Genome Duplication ◽  
Genome Duplication ◽  
Regulatory Elements ◽  
Whole Genome ◽  
Dna Repeats ◽  
Vertebrate Genome ◽  
Closely Related Species ◽  
Flanking Regions ◽  
Regulatory Landscape

AbstractBackgroundGenome duplication has played a pivotal role in the evolution of many eukaryotic lineages, including the vertebrates. The most recent vertebrate genome duplication is that in Xenopus laevis, resulting from the hybridization of two closely related species about 17 million years ago [1]. However, little is known about the consequences of this duplication at the level of the genome, the epigenome and gene expression.ResultsOf the parental subgenomes, S chromosomes have degraded faster than L chromosomes ever since the genome duplication and until the present day. Deletions appear to have the largest effect on pseudogene formation and loss of regulatory regions. Deleted regions are enriched for long DNA repeats and the flanking regions have high alignment scores, suggesting that non-allelic homologous recombination (NAHR) has played a significant role in the loss of DNA. To assess innovations in the X. laevis subgenomes we examined p300 (Ep300)-bound enhancer peaks that are unique to one subgenome and absent from X. tropicalis. A large majority of new enhancers are comprised of transposable elements. Finally, to dissect early and late events following interspecific hybridization, we examined the epigenome and the enhancer landscape in X. tropicalis × X. laevis hybrid embryos. Strikingly, young X. tropicalis DNA transposons are derepressed and recruit p300 in hybrid embryos.ConclusionsThe results show that erosion of X. laevis genes and functional regulatory elements is associated with repeats and NAHR, and furthermore that young repeats have also contributed to the p300-bound regulatory landscape following hybridization and whole genome duplication.

scholarly journals Next Gen Pop Gen: implementing a high-throughput approach to population genetics in boarfish ( Capros aper )

2016 ◽  
Vol 3 (12) ◽  
pp. 160651 ◽  
Author(s):  
Edward D. Farrell ◽  
Jeanette E. L. Carlsson ◽  
Jens Carlsson
Keyword(s):  
De Novo ◽  
Genotyping By Sequencing ◽  
Cost Effective ◽  
Species Range ◽  
Genetic Population ◽  
Genetic Studies ◽  
Scale Population ◽  
Management And Conservation ◽  
Generation Sequencing ◽  
Insular Populations

The recently developed approach for microsatellite genotyping by sequencing (GBS) using individual combinatorial barcoding was further improved and used to assess the genetic population structure of boarfish ( Capros aper ) across the species' range. Microsatellite loci were developed de novo and genotyped by next-generation sequencing. Genetic analyses of the samples indicated that boarfish can be subdivided into at least seven biological units (populations) across the species' range. Furthermore, the recent apparent increase in abundance in the northeast Atlantic is better explained by demographic changes within this area than by influx from southern or insular populations. This study clearly shows that the microsatellite GBS approach is a generic, cost-effective, rapid and powerful method suitable for full-scale population genetic studies—a crucial element for assessment, sustainable management and conservation of valuable biological resources.

scholarly journals Reduction of the Rate of Poliovirus Protein Synthesis through Large-Scale Codon Deoptimization Causes Attenuation of Viral Virulence by Lowering Specific Infectivity

2006 ◽  
Vol 80 (19) ◽  
pp. 9687-9696 ◽  
Author(s):  
Steffen Mueller ◽  
Dimitris Papamichail ◽  
J. Robert Coleman ◽  
Steven Skiena ◽  
Eckard Wimmer
Keyword(s):  
Large Scale ◽  
De Novo ◽  
Synonymous Codon ◽  
Direct Analysis ◽  
Synonymous Codon Usage ◽  
Wild Type ◽  
Coding Region ◽  
Virus Particles ◽  
Viral Virulence ◽  
Wide Range

ABSTRACT Exploring the utility of de novo gene synthesis with the aim of designing stably attenuated polioviruses (PV), we followed two strategies to construct PV variants containing synthetic replacements of the capsid coding sequences either by deoptimizing synonymous codon usage (PV-AB) or by maximizing synonymous codon position changes of the existing wild-type (wt) poliovirus codons (PV-SD). Despite 934 nucleotide changes in the capsid coding region, PV-SD RNA produced virus with wild-type characteristics. In contrast, no viable virus was recovered from PV-AB RNA carrying 680 silent mutations, due to a reduction of genome translation and replication below a critical level. After subcloning of smaller portions of the AB capsid coding sequence into the wt background, several viable viruses were obtained with a wide range of phenotypes corresponding to their efficiency of directing genome translation. Surprisingly, when inoculated with equal infectious doses (PFU), even the most replication-deficient viruses appeared to be as pathogenic in PV-sensitive CD155tg (transgenic) mice as the PV(M) wild type. However, infection with equal amounts of virus particles revealed a neuroattenuated phenotype over 100-fold. Direct analysis indicated a striking reduction of the specific infectivity of PV-AB-type virus particles. Due to the distribution effect of many silent mutations over large genome segments, codon-deoptimized viruses should have genetically stable phenotypes, and they may prove suitable as attenuated substrates for the production of poliovirus vaccines.

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