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April 21, 2020

Single-Molecule Sequencing: Towards Clinical Applications.

In the past several years, single-molecule sequencing platforms, such as those by Pacific Biosciences and Oxford Nanopore Technologies, have become available to researchers and are currently being tested for clinical applications. They offer exceptionally long reads that permit direct sequencing through regions of the genome inaccessible or difficult to analyze by short-read platforms. This includes disease-causing long repetitive elements, extreme GC content regions, and complex gene loci. Similarly, these platforms enable structural variation characterization at previously unparalleled resolution and direct detection of epigenetic marks in native DNA. Here, we review how these technologies are opening up new clinical avenues that are being applied to pathogenic microorganisms and viruses, constitutional disorders, pharmacogenomics, cancer, and more.Copyright © 2018 Elsevier Ltd. All rights reserved.

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April 21, 2020

Complete Genome Sequence of the Wolbachia wAlbB Endosymbiont of Aedes albopictus.

Wolbachia, an alpha-proteobacterium closely related to Rickettsia, is a maternally transmitted, intracellular symbiont of arthropods and nematodes. Aedes albopictus mosquitoes are naturally infected with Wolbachia strains wAlbA and wAlbB. Cell line Aa23 established from Ae. albopictus embryos retains only wAlbB and is a key model to study host-endosymbiont interactions. We have assembled the complete circular genome of wAlbB from the Aa23 cell line using long-read PacBio sequencing at 500× median coverage. The assembled circular chromosome is 1.48 megabases in size, an increase of more than 300 kb over the published draft wAlbB genome. The annotation of the genome identified 1,205 protein coding genes, 34 tRNA, 3 rRNA, 1 tmRNA, and 3 other ncRNA loci. The long reads enabled sequencing over complex repeat regions which are difficult to resolve with short-read sequencing. Thirteen percent of the genome comprised insertion sequence elements distributed throughout the genome, some of which cause pseudogenization. Prophage WO genes encoding some essential components of phage particle assembly are missing, while the remainder are found in five prophage regions/WO-like islands or scattered around the genome. Orthology analysis identified a core proteome of 535 orthogroups across all completed Wolbachia genomes. The majority of proteins could be annotated using Pfam and eggNOG analyses, including ankyrins and components of the Type IV secretion system. KEGG analysis revealed the absence of five genes in wAlbB which are present in other Wolbachia. The availability of a complete circular chromosome from wAlbB will enable further biochemical, molecular, and genetic analyses on this strain and related Wolbachia. © The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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April 21, 2020

Tools and Strategies for Long-Read Sequencing and De Novo Assembly of Plant Genomes.

The commercial release of third-generation sequencing technologies (TGSTs), giving long and ultra-long sequencing reads, has stimulated the development of new tools for assembling highly contiguous genome sequences with unprecedented accuracy across complex repeat regions. We survey here a wide range of emerging sequencing platforms and analytical tools for de novo assembly, provide background information for each of their steps, and discuss the spectrum of available options. Our decision tree recommends workflows for the generation of a high-quality genome assembly when used in combination with the specific needs and resources of a project.Copyright © 2019 Elsevier Ltd. All rights reserved.

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April 21, 2020

Dnase1l3 deletion causes aberrations in length and end-motif frequencies in plasma DNA.

Circulating DNA in plasma consists of short DNA fragments. The biological processes generating such fragments are not well understood. DNASE1L3 is a secreted DNASE1-like nuclease capable of digesting DNA in chromatin, and its absence causes anti-DNA responses and autoimmunity in humans and mice. We found that the deletion of Dnase1l3 in mice resulted in aberrations in the fragmentation of plasma DNA. Such aberrations included an increase in short DNA molecules below 120 bp, which was positively correlated with anti-DNA antibody levels. We also observed an increase in long, multinucleosomal DNA molecules and decreased frequencies of the most common end motifs found in plasma DNA. These aberrations were independent of anti-DNA response, suggesting that they represented a primary effect of DNASE1L3 loss. Pregnant Dnase1l3-/- mice carrying Dnase1l3+/- fetuses showed a partial restoration of normal frequencies of plasma DNA end motifs, suggesting that DNASE1L3 from Dnase1l3-proficient fetuses could enter maternal systemic circulation and affect both fetal and maternal DNA fragmentation in a systemic as well as local manner. However, the observed shortening of circulating fetal DNA relative to maternal DNA was not affected by the deletion of Dnase1l3 Collectively, our findings demonstrate that DNASE1L3 plays a role in circulating plasma DNA homeostasis by enhancing fragmentation and influencing end-motif frequencies. These results support a distinct role of DNASE1L3 as a regulator of the physical form and availability of cell-free DNA and may have important implications for the mechanism whereby this enzyme prevents autoimmunity. Copyright © 2019 the Author(s). Published by PNAS.

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April 21, 2020

Genome assembly and gene expression in the American black bear provides new insights into the renal response to hibernation.

The prevalence of chronic kidney disease (CKD) is rising worldwide and 10-15% of the global population currently suffers from CKD and its complications. Given the increasing prevalence of CKD there is an urgent need to find novel treatment options. The American black bear (Ursus americanus) copes with months of lowered kidney function and metabolism during hibernation without the devastating effects on metabolism and other consequences observed in humans. In a biomimetic approach to better understand kidney adaptations and physiology in hibernating black bears, we established a high-quality genome assembly. Subsequent RNA-Seq analysis of kidneys comparing gene expression profiles in black bears entering (late fall) and emerging (early spring) from hibernation identified 169 protein-coding genes that were differentially expressed. Of these, 101 genes were downregulated and 68 genes were upregulated after hibernation. Fold changes ranged from 1.8-fold downregulation (RTN4RL2) to 2.4-fold upregulation (CISH). Most notable was the upregulation of cytokine suppression genes (SOCS2, CISH, and SERPINC1) and the lack of increased expression of cytokines and genes involved in inflammation. The identification of these differences in gene expression in the black bear kidney may provide new insights in the prevention and treatment of CKD. © The Author(s) 2018. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

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April 21, 2020

RADAR-seq: A RAre DAmage and Repair sequencing method for detecting DNA damage on a genome-wide scale.

RAre DAmage and Repair sequencing (RADAR-seq) is a highly adaptable sequencing method that enables the identification and detection of rare DNA damage events for a wide variety of DNA lesions at single-molecule resolution on a genome-wide scale. In RADAR-seq, DNA lesions are replaced with a patch of modified bases that can be directly detected by Pacific Biosciences Single Molecule Real-Time (SMRT) sequencing. RADAR-seq enables dynamic detection over a wide range of DNA damage frequencies, including low physiological levels. Furthermore, without the need for DNA amplification and enrichment steps, RADAR-seq provides sequencing coverage of damaged and undamaged DNA across an entire genome. Here, we use RADAR-seq to measure the frequency and map the location of ribonucleotides in wild-type and RNaseH2-deficient E. coli and Thermococcus kodakarensis strains. Additionally, by tracking ribonucleotides incorporated during in vivo lagging strand DNA synthesis, we determined the replication initiation point in E. coli, and its relation to the origin of replication (oriC). RADAR-seq was also used to map cyclobutane pyrimidine dimers (CPDs) in Escherichia coli (E. coli) genomic DNA exposed to UV-radiation. On a broader scale, RADAR-seq can be applied to understand formation and repair of DNA damage, the correlation between DNA damage and disease initiation and progression, and complex biological pathways, including DNA replication.Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.

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April 21, 2020

Genomic investigation of Staphylococcus aureus recovered from Gambian women and newborns following an oral dose of intra-partum azithromycin.

Oral azithromycin given during labour reduces carriage of bacteria responsible for neonatal sepsis, including Staphylococcus aureus. However, there is concern that this may promote drug resistance.Here, we combine genomic and epidemiological data on S. aureus isolated from mothers and babies in a randomized intra-partum azithromycin trial (PregnAnZI) to describe bacterial population dynamics and resistance mechanisms.Participants from both arms of the trial, who carried S. aureus in day 3 and day 28 samples post-intervention, were included. Sixty-six S. aureus isolates (from 7 mothers and 10 babies) underwent comparative genome analyses and the data were then combined with epidemiological data. Trial registration (main trial): ClinicalTrials.gov Identifier NCT01800942.Seven S. aureus STs were identified, with ST5 dominant (n?=?40, 61.0%), followed by ST15 (n?=?11, 17.0%). ST5 predominated in the placebo arm (73.0% versus 49.0%, P?=?0.039) and ST15 in the azithromycin arm (27.0% versus 6.0%, P?=?0.022). In azithromycin-resistant isolates, msr(A) was the main macrolide resistance gene (n?=?36, 80%). Ten study participants, from both trial arms, acquired azithromycin-resistant S. aureus after initially harbouring a susceptible isolate. In nine (90%) of these cases, the acquired clone was an msr(A)-containing ST5 S. aureus. Long-read sequencing demonstrated that in ST5, msr(A) was found on an MDR plasmid.Our data reveal in this Gambian population the presence of a dominant clone of S. aureus harbouring plasmid-encoded azithromycin resistance, which was acquired by participants in both arms of the study. Understanding these resistance dynamics is crucial to defining the public health drug resistance impacts of azithromycin prophylaxis given during labour in Africa. © The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.

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April 21, 2020

Alternative Splicing of the Delta-Opioid Receptor Gene Suggests Existence of New Functional Isoforms.

The delta-opioid receptor (DOPr) participates in mediating the effects of opioid analgesics. However, no selective agonists have entered clinical care despite potential to ameliorate many neurological and psychiatric disorders. In an effort to address the drug development challenges, the functional contribution of receptor isoforms created by alternative splicing of the three-exonic coding gene, OPRD1, has been overlooked. We report that the gene is transcriptionally more diverse than previously demonstrated, producing novel protein isoforms in humans and mice. We provide support for the functional relevance of splice variants through context-dependent expression profiling (tissues, disease model) and conservation of the transcriptional landscape in closely related vertebrates. The conserved alternative transcriptional events have two distinct patterns. First, cassette exon inclusions between exons 1 and 2 interrupt the reading frame, producing truncated receptor fragments comprising only the first transmembrane (TM) domain, despite the lack of exact exon orthologues between distant species. Second, a novel promoter and transcriptional start site upstream of exon 2 produces a transcript of an N-terminally truncated 6TM isoform. However, a fundamental difference in the exonic landscaping as well as translation and translation products poses limits for modelling the human DOPr receptor system in mice.

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April 21, 2020

Evolution of Goat’s Rue Rhizobia (Neorhizobium galegae): Analysis of Polymorphism of the Nitrogen Fixation and Nodule Formation Genes

The goat’s rue rhizobia (Neorhizobium galegae) represent a convenient model to study the evolution and speciation of symbiotic bacteria. This rhizobial species is composed of two biovars (bv. orientalis and bv. officinalis), which form N2-fixing nodules with certain species of goat’s rue (Galega orientalis and G. officinalis). The cross-inoculation between them results in the formation of nodules unable to fix nitrogen. On the basis of the data on the whole-genome sequencing, we studied the nucleotide polymorphism of 11 N. galegae strains isolated from the North Caucasus ecosystems, where G. orientalis has higher diversity than G. officinalis. The low level of differences in the polymorphism within the group of the sym genes in comparison with the nonsymbiotic genes can be associated with the active participation of host plants in the evolution of rhizobia. The intragenic polymorphism of bv. orientalis proved to be significantly higher than that of bv. officinalis. The level of polymorphism of nonsymbiotic genes was lower than that of the symbiotic genes, which are functionally more homogeneous. The divergence of the nitrogen fixation genes (nif/fix) is more pronounced than that of the nodule formation genes (nod) in the N. galegae biovars. These facts indicate the leading role of the host-specific nitrogen fixation in the evolution of the studied rizhobial species.

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April 21, 2020

BREX system of Escherichia coli distinguishes self from non-self by methylation of a specific DNA site.

Prokaryotes evolved numerous systems that defend against predation by bacteriophages. In addition to well-known restriction-modification and CRISPR-Cas immunity systems, many poorly characterized systems exist. One class of such systems, named BREX, consists of a putative phosphatase, a methyltransferase and four other proteins. A Bacillus cereus BREX system provides resistance to several unrelated phages and leads to modification of specific motif in host DNA. Here, we study the action of BREX system from a natural Escherichia coli isolate. We show that while it makes cells resistant to phage ? infection, induction of ? prophage from cells carrying BREX leads to production of viruses that overcome the defense. The induced phage DNA contains a methylated adenine residue in a specific motif. The same modification is found in the genome of BREX-carrying cells. The results establish, for the first time, that immunity to BREX system defense is provided by an epigenetic modification.

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April 21, 2020

DNA Methylation at the Schizophrenia and Intelligence GWAS-Implicated MIR137HG Locus May Be Associated with Disease and Cognitive Functions

The largest genome-wide association studies have identified schizophrenia and intelligence associated variants in the MIR137HG locus containing genes encoding microRNA-137 and microRNA-2682. In the present study, we investigated DNA methylation in the MIR137HG intragenic CpG island (CGI) in the peripheral blood of 44 patients with schizophrenia and 50 healthy controls. The CGI included the entire MIR137 gene and the region adjacent to the 5′-end of MIR2682. The aim of the study was to examine the relationship of the CGI methylation with schizophrenia and cognitive functioning. The methylation level of 91 CpG located in the selected region was established for each participant by means of single-molecule real-time bisulfite sequencing. All subjects completed the battery of neuropsychological tests. We found that the CGI was hypomethylated in both groups, except for one site—CpG (chr1: 98?511?049), with significant interindividual variability in methylation. A higher level of methylation of this CpG was seen in male patients and was associated with a decrease in the cognitive index in the combined sample of patients and controls. Our data suggest that further investigation of mechanisms that regulate the MIR137 and MIR2682 genes expression might help to understand the molecular basis of cognitive deficits in schizophrenia.

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April 21, 2020

Reference genome sequences of two cultivated allotetraploid cottons, Gossypium hirsutum and Gossypium barbadense.

Allotetraploid cotton species (Gossypium hirsutum and Gossypium barbadense) have long been cultivated worldwide for natural renewable textile fibers. The draft genome sequences of both species are available but they are highly fragmented and incomplete1-4. Here we report reference-grade genome assemblies and annotations for G. hirsutum accession Texas Marker-1 (TM-1) and G. barbadense accession 3-79 by integrating single-molecule real-time sequencing, BioNano optical mapping and high-throughput chromosome conformation capture techniques. Compared with previous assembled draft genomes1,3, these genome sequences show considerable improvements in contiguity and completeness for regions with high content of repeats such as centromeres. Comparative genomics analyses identify extensive structural variations that probably occurred after polyploidization, highlighted by large paracentric/pericentric inversions in 14 chromosomes. We constructed an introgression line population to introduce favorable chromosome segments from G. barbadense to G. hirsutum, allowing us to identify 13 quantitative trait loci associated with superior fiber quality. These resources will accelerate evolutionary and functional genomic studies in cotton and inform future breeding programs for fiber improvement.

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April 21, 2020

The developmental dynamics of the Populus stem transcriptome.

The Populus shoot undergoes primary growth (longitudinal growth) followed by secondary growth (radial growth), which produces biomass that is an important source of energy worldwide. We adopted joint PacBio Iso-Seq and RNA-seq analysis to identify differentially expressed transcripts along a developmental gradient from the shoot apex to the fifth internode of Populus Nanlin895. We obtained 87 150 full-length transcripts, including 2081 new isoforms and 62 058 new alternatively spliced isoforms, most of which were produced by intron retention, that were used to update the Populus annotation. Among these novel isoforms, there are 1187 long non-coding RNAs and 356 fusion genes. Using this annotation, we found 15 838 differentially expressed transcripts along the shoot developmental gradient, of which 1216 were transcription factors (TFs). Only a few of these genes were reported previously. The differential expression of these TFs suggests that they may play important roles in primary and secondary growth. AP2, ARF, YABBY and GRF TFs are highly expressed in the apex, whereas NAC, bZIP, PLATZ and HSF TFs are likely to be important for secondary growth. Overall, our findings provide evidence that long-read sequencing can complement short-read sequencing for cataloguing and quantifying eukaryotic transcripts and increase our understanding of the vital and dynamic process of shoot development. © 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

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April 21, 2020

Mate Selection in Self-Compatible Wild Tobacco Results from Coordinated Variation in Homologous Self-Incompatibility Genes.

In flowering plants, intraspecific mate preference is frequently related to mating systems: the rejection of self pollen in self-incompatible (SI) plants that prevents inbreeding is one of the best described examples. However, in other mating systems, more nuanced patterns of pollen rejection occur. In the self-compatible (SC) Nicotiana attenuata, in which SI is not found and all crosses are compatible, certain pollen genotypes are consistently selected in mixed pollinations. However, the molecular mechanisms of this polyandrous mate selection remain unknown. Style-expressed NaS-like-RNases and pollen-expressed NaSLF-like genes, homologous to SI factors in Solanaceae, were identified and examined for a role in N. attenuata’s mate selection. A comparison of two NaS-like-RNases and six NaSLF-like genes among 26 natural accessions revealed specific combinations of co-expression and direct protein-protein interactions. To evaluate their role in mate selection, we silenced the expression of specific NaS-like-RNases and NaSLF-like proteins and conducted diagnostic binary mixed pollinations and mixed pollinations with 14 different non-self pollen donors. Styles expressing particular combinations of NaS-like-RNases selected mates from plants with corresponding NaS-like-RNase expression patterns, while styles lacking NaS-like-RNase expression were non-selective in their fertilizations, which reflected the genotype ratios of pollen mixtures deposited on the stigmas. DNA methylation could account for some of the observed variation in stylar NaS-like-RNase patterns. We conclude that the S-RNase-SLF recognition mechanism plays a central role in polyandrous mate selection in this self-compatible species. These results suggest that after the SI-SC transition, natural variation of SI homologous genes was repurposed to mediate intraspecific mate selection. Copyright © 2019 Elsevier Ltd. All rights reserved.

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April 21, 2020

Computational aspects underlying genome to phenome analysis in plants.

Recent advances in genomics technologies have greatly accelerated the progress in both fundamental plant science and applied breeding research. Concurrently, high-throughput plant phenotyping is becoming widely adopted in the plant community, promising to alleviate the phenotypic bottleneck. While these technological breakthroughs are significantly accelerating quantitative trait locus (QTL) and causal gene identification, challenges to enable even more sophisticated analyses remain. In particular, care needs to be taken to standardize, describe and conduct experiments robustly while relying on plant physiology expertise. In this article, we review the state of the art regarding genome assembly and the future potential of pangenomics in plant research. We also describe the necessity of standardizing and describing phenotypic studies using the Minimum Information About a Plant Phenotyping Experiment (MIAPPE) standard to enable the reuse and integration of phenotypic data. In addition, we show how deep phenotypic data might yield novel trait-trait correlations and review how to link phenotypic data to genomic data. Finally, we provide perspectives on the golden future of machine learning and their potential in linking phenotypes to genomic features. © 2018 The Authors The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.

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