scholarly journals Genome-wide comparison of DNA methylation between life cycle stages of Drosophila melanogaster using high-throughput sequencing techniques

2020 ◽  
Author(s):  
Saniya Deshmukh ◽  
Varada Abhyankar ◽  
Shamsudheen Karuthedath Vellarikkal ◽  
Sridhar Sivasubbu ◽  
Vinod Scaria ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Drosophila Melanogaster ◽  
Life Cycle ◽  
High Throughput ◽  
Dna Methyltransferase ◽  
High Throughput Sequencing ◽  
Mitotic Cell ◽  
Chromosomal Distribution ◽  
Regulatory Processes ◽  
Life Cycle Stages

ABSTRACTDrosophila melanogaster undergoes holometabolous development, has very low levels of DNA methylation, and is known to possess a single known methyltransferase, dDNMT2. This study compares the DNA methylation patterns between the two life cycle stages of D. melanogaster using a combination of DNA immunoprecipitation and high throughput sequencing techniques.Our results indicate, a change in the chromosomal distribution of the sparse DNA methylation concerning genes and natural transposable elements between in the embryo and the adult stages of D. melanogaster. The differentially methylated regions localised on genes involved in the regulation of cell cycle processes of mitotic cell divisions and chromosomal segregation. dDNMT2 knockout flies exhibited altered patterns of DNA methylation. The observed differences in DNA methylation were in genes involved in cellular communication and cytoskeletal functions. The variation in DNA methylation between the two life cycle stages is indicative that it could have a role in regulatory processes during development and, dDNMT2 may have a role as a co-factor for the hitherto undiscovered DNA methyltransferase in D. melanogaster.

scholarly journals Extraction and high-throughput sequencing of oak heartwood DNA: Assessing the feasibility of genome-wide DNA methylation profiling

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0254971
Author(s):  
Federico Rossi ◽  
Alessandro Crnjar ◽  
Federico Comitani ◽  
Rodrigo Feliciano ◽  
Leonie Jahn ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Tree Ring ◽  
High Throughput ◽  
Environmental Conditions ◽  
High Throughput Sequencing ◽  
Wood Formation ◽  
Whole Genome ◽  
Library Preparation ◽  
Bisulfite Treatment ◽  
Standing Trees

Tree ring features are affected by environmental factors and therefore are the basis for dendrochronological studies to reconstruct past environmental conditions. Oak wood often provides the data for these studies because of the durability of oak heartwood and hence the availability of samples spanning long time periods of the distant past. Wood formation is regulated in part by epigenetic mechanisms such as DNA methylation. Studies of the methylation state of DNA preserved in oak heartwood thus could identify epigenetic tree ring features informing on past environmental conditions. In this study, we aimed to establish protocols for the extraction of DNA, the high-throughput sequencing of whole-genome DNA libraries (WGS) and the profiling of DNA methylation by whole-genome bisulfite sequencing (WGBS) for oak (Quercus robur) heartwood drill cores taken from the trunks of living standing trees spanning the AD 1776-2014 time period. Heartwood contains little DNA, and large amounts of phenolic compounds known to hinder the preparation of high-throughput sequencing libraries. Whole-genome and DNA methylome library preparation and sequencing consistently failed for oak heartwood samples more than 100 and 50 years of age, respectively. DNA fragmentation increased with sample age and was exacerbated by the additional bisulfite treatment step during methylome library preparation. Relative coverage of the non-repetitive portion of the oak genome was sparse. These results suggest that quantitative methylome studies of oak hardwood will likely be limited to relatively recent samples and will require a high sequencing depth to achieve sufficient genome coverage.

scholarly journals Behavior Individuality: A Focus on Drosophila melanogaster

2021 ◽  
Vol 12 ◽  
Author(s):  
Rubén Mollá-Albaladejo ◽  
Juan A. Sánchez-Alcañiz
Keyword(s):  
Gene Expression ◽  
Drosophila Melanogaster ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Great Effort ◽  
Behavioral Differences ◽  
Adult Behavior ◽  
Large Numbers ◽  
Environmental Variations ◽  
And Function

Among individuals, behavioral differences result from the well-known interplay of nature and nurture. Minute differences in the genetic code can lead to differential gene expression and function, dramatically affecting developmental processes and adult behavior. Environmental factors, epigenetic modifications, and gene expression and function are responsible for generating stochastic behaviors. In the last decade, the advent of high-throughput sequencing has facilitated studying the genetic basis of behavior and individuality. We can now study the genomes of multiple individuals and infer which genetic variations might be responsible for the observed behavior. In addition, the development of high-throughput behavioral paradigms, where multiple isogenic animals can be analyzed in various environmental conditions, has again facilitated the study of the influence of genetic and environmental variations in animal personality. Mainly, Drosophila melanogaster has been the focus of a great effort to understand how inter-individual behavioral differences emerge. The possibility of using large numbers of animals, isogenic populations, and the possibility of modifying neuronal function has made it an ideal model to search for the origins of individuality. In the present review, we will focus on the recent findings that try to shed light on the emergence of individuality with a particular interest in D. melanogaster.

scholarly journals A high-throughput small molecule screen identifies synergism between DNA methylation and Aurora kinase pathways for X reactivation

2016 ◽  
Vol 113 (50) ◽  
pp. 14366-14371 ◽  
Author(s):  
Derek Lessing ◽  
Thomas O. Dial ◽  
Chunyao Wei ◽  
Bernhard Payer ◽  
Lieselot L. G. Carrette ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Small Molecules ◽  
High Throughput ◽  
Dosage Compensation ◽  
Dna Methyltransferase ◽  
Aurora Kinase ◽  
Functional Genes ◽  
X Chromosomes ◽  
Gfp Reporter ◽  
Small Molecule Screen

X-chromosome inactivation is a mechanism of dosage compensation in which one of the two X chromosomes in female mammals is transcriptionally silenced. Once established, silencing of the inactive X (Xi) is robust and difficult to reverse pharmacologically. However, the Xi is a reservoir of >1,000 functional genes that could be potentially tapped to treat X-linked disease. To identify compounds that could reactivate the Xi, here we screened ∼367,000 small molecules in an automated high-content screen using an Xi-linked GFP reporter in mouse fibroblasts. Given the robust nature of silencing, we sensitized the screen by “priming” cells with the DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine (5azadC). Compounds that elicited GFP activity include VX680, MLN8237, and 5azadC, which are known to target the Aurora kinase and DNA methylation pathways. We demonstrate that the combinations of VX680 and 5azadC, as well as MLN8237 and 5azadC, synergistically up-regulate genes on the Xi. Thus, our work identifies a synergism between the DNA methylation and Aurora kinase pathways as being one of interest for possible pharmacological reactivation of the Xi.

scholarly journals Developmental regulation of edited CYb and COIII mitochondrial mRNAs is achieved by distinct mechanisms in Trypanosoma brucei

2020 ◽  
Vol 48 (15) ◽  
pp. 8704-8723
Author(s):  
Joseph T Smith Jr. ◽  
Eva Doleželová ◽  
Brianna Tylec ◽  
Jonathan E Bard ◽  
Runpu Chen ◽  
...  
Keyword(s):  
Life Cycle ◽  
Trypanosoma Brucei ◽  
High Throughput Sequencing ◽  
Environmental Changes ◽  
Developmental Regulation ◽  
Complex Life Cycle ◽  
Mrna Levels ◽  
Developmentally Regulated ◽  
Life Cycle Stages

Abstract Trypanosoma brucei is a parasitic protozoan that undergoes a complex life cycle involving insect and mammalian hosts that present dramatically different nutritional environments. Mitochondrial metabolism and gene expression are highly regulated to accommodate these environmental changes, including regulation of mRNAs that require extensive uridine insertion/deletion (U-indel) editing for their maturation. Here, we use high throughput sequencing and a method for promoting life cycle changes in vitro to assess the mechanisms and timing of developmentally regulated edited mRNA expression. We show that edited CYb mRNA is downregulated in mammalian bloodstream forms (BSF) at the level of editing initiation and/or edited mRNA stability. In contrast, edited COIII mRNAs are depleted in BSF by inhibition of editing progression. We identify cell line-specific differences in the mechanisms abrogating COIII mRNA editing, including the possible utilization of terminator gRNAs that preclude the 3′ to 5′ progression of editing. By examining the developmental timing of altered mitochondrial mRNA levels, we also reveal transcript-specific developmental checkpoints in epimastigote (EMF), metacyclic (MCF), and BSF. These studies represent the first analysis of the mechanisms governing edited mRNA levels during T. brucei development and the first to interrogate U-indel editing in EMF and MCF life cycle stages.

scholarly journals Population Genomic Inferences from Sparse High-Throughput Sequencing of Two Populations of Drosophila melanogaster

2009 ◽  
Vol 1 ◽  
pp. 449-465 ◽  
Author(s):  
Timothy B. Sackton ◽  
Rob J. Kulathinal ◽  
Casey M. Bergman ◽  
Aaron R. Quinlan ◽  
Erik B. Dopman ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Population Genomic ◽  
Two Populations

scholarly journals Targeted DNA Methylation Analysis by High-Throughput Sequencing in Hepatic Carcinoma Tissue and Adjacent Normal Tissues

2020 ◽  
Author(s):  
Guangmou Zhang ◽  
Huigen Feng ◽  
Zhiqing Yuan
Keyword(s):  
Dna Methylation ◽  
Liver Cancer ◽  
Clinical Diagnosis ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Methylation Analysis ◽  
Hepatic Carcinoma ◽  
Sequencing Technology ◽  
The Relationship ◽  
Dna Methylation Analysis

Abstract Background: The relationship between epigenetic abnormalities and tumorigenesi has been investigated in the past decade and made major advances, particularly the abnormal expression of small RNAs, DNA methylation, and histone modification in cancer. In many tumor-related studies, the regulatory changes in DNA methylation during cancer development and the development of resistance to anticancer drugs have show that DNA methylation can be used as a biomarker for cancer diagnosis and concomitant diagnosis, but there is a lack of clinically useful biomarkers associated with hepatic carcinoma. Using high-throughput sequencing technology, appropriate testing and validation can be carried out in large samples. The relationship between DNA methylation and tumor development can be explored, contributing to clinical diagnosis and personalized treatment of hepatic carcinoma. Methods: In this study, we implemented and evaluated the effectiveness of high-throughput sequencing for DNA methylation analysis in hepatic carcinoma. For the relationship between DNA methylation and gene expression, Pearson correlation analysis was used to evaluate the correlation. Twenty-five isolated genomic regions were amplified by PCR using bisulfite-transformed liver cancer tissue (Ca) and paracancer tissue (T) as template DNA. PCR final product sequence information was obtained by sequence analysis using Illumina Hiseq/Miseq platform. Results: The average depth of coverage across all amplicons was 30,548 for T and 29,346 for Ca, with a maximum of 3,675 at the ARID1A amplicon and a minimum of 65 at the PTEN amplicon. Methylation spectra were obtained for each genomic locus of the two groups of samples, and the results showed that methylation was significantly different at the X target loci and slightly different at the Y target loci. Cluster analysis showed that all T tissues were clustered in one group (except tissues T2 and T3), while Ca tissues were clustered on the other side. The results showed that DNA methylation at the loci may be closely related to liver cancer, providing references for the research and development of biomarkers in clinical diagnosis. Conclusions: The study demonstrates that high-throughput sequencing technology is a powerful and cost-effective method for methylation analysis of target DNA in cancer tissues.

scholarly journals Gut Bacterial Diversity in Different Life Cycle Stages of Adelphocoris suturalis (Hemiptera: Miridae)

2021 ◽  
Vol 12 ◽  
Author(s):  
Hui Xue ◽  
Xiangzhen Zhu ◽  
Li Wang ◽  
Kaixin Zhang ◽  
Dongyang Li ◽  
...  
Keyword(s):  
Life Cycle ◽  
Relative Abundance ◽  
High Throughput Sequencing ◽  
Developmental Stages ◽  
Control Strategies ◽  
Symbiotic Bacteria ◽  
Microbial Composition ◽  
Agricultural Industry ◽  
Life Cycle Stages ◽  
Major Pest

Bacteria and insects have a mutually beneficial symbiotic relationship. Bacteria participate in several physiological processes such as reproduction, metabolism, and detoxification of the host. Adelphocoris suturalis is considered a pest by the agricultural industry and is now a major pest in cotton, posing a serious threat to agricultural production. As with many insects, various microbes live inside A. suturalis. However, the microbial composition and diversity of its life cycle have not been well-studied. To identify the species and community structure of symbiotic bacteria in A. suturalis, we used the HiSeq platform to perform high-throughput sequencing of the V3–V4 region in the 16S rRNA of symbiotic bacteria found in A. suturalis throughout its life stages. Our results demonstrated that younger nymphs (1st and 2nd instar nymphs) have higher species richness. Proteobacteria (87.06%) and Firmicutes (9.43%) were the dominant phyla of A. suturalis. At the genus level, Erwinia (28.98%), Staphylococcus (5.69%), and Acinetobacter (4.54%) were the dominant bacteria. We found that the relative abundance of Erwinia was very stable during the whole developmental stage. On the contrary, the relative abundance of Staphylococcus, Acinetobacter, Pseudomonas, and Corynebacterium showed significant dynamic changes at different developmental stages. Functional prediction of symbiotic bacteria mainly focuses on metabolic pathways. Our findings document symbiotic bacteria across the life cycle of A. suturalis, as well as differences in both the composition and richness in nymph and adult symbiotic bacteria. Our analysis of the bacteria in A. suturalis provides important information for the development of novel biological control strategies.

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