Identification of Genomic Loci Responsible for the Formation of Nuclear Domains Using Lampbrush Chromosomes

In the cell nuclei, various types of nuclear domains assemble as a result of transcriptional activity at specific chromosomal loci. Giant transcriptionally active lampbrush chromosomes, which form in oocyte nuclei of amphibians and birds enable the mapping of genomic sequences with high resolution and the visualization of individual transcription units. This makes avian and amphibian oocyte nuclei an advantageous model for studying locus-specific nuclear domains. We developed two strategies for identification and comprehensive analysis of the genomic loci involved in nuclear domain formation on lampbrush chromosomes. The first approach was based on the sequential FISH-mapping of BAC clones containing genomic DNA fragments with a known chromosomal position close to the locus of a nuclear domain. The second approach involved mechanical microdissection of the chromosomal region adjacent to the nuclear domain followed by the generation of FISH-probes and DNA sequencing. Furthermore, deciphering the DNA sequences from the dissected material by high throughput sequencing technologies and their mapping to the reference genome helps to identify the genomic region responsible for the formation of the nuclear domain. For those nuclear domains structured by nascent transcripts, identification of genomic loci of their formation is a crucial step in the identification of scaffold RNAs.

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CRAFT: Compact genome Representation towards large-scale Alignment-Free daTabase

10.1101/2020.07.10.196741 ◽

2020 ◽

Author(s):

Yang Young Lu ◽

Jiaxing Bai ◽

Yiwen Wang ◽

Ying Wang ◽

Fengzhu Sun

Keyword(s):

Dna Sequences ◽

Sequence Comparison ◽

Large Scale ◽

High Throughput Sequencing ◽

Sequence Data ◽

Practical Interest ◽

Supplementary Information ◽

Computationally Efficient ◽

Sequencing Technologies ◽

Alignment Free

AbstractMotivationRapid developments in sequencing technologies have boosted generating high volumes of sequence data. To archive and analyze those data, one primary step is sequence comparison. Alignment-free sequence comparison based on k-mer frequencies offers a computationally efficient solution, yet in practice, the k-mer frequency vectors for large k of practical interest lead to excessive memory and storage consumption.ResultsWe report CRAFT, a general genomic/metagenomic search engine to learn compact representations of sequences and perform fast comparison between DNA sequences. Specifically, given genome or high throughput sequencing (HTS) data as input, CRAFT maps the data into a much smaller embedding space and locates the best matching genome in the archived massive sequence repositories. With 102 – 104-fold reduction of storage space, CRAFT performs fast query for gigabytes of data within seconds or minutes, achieving comparable performance as six state-of-the-art alignment-free measures.AvailabilityCRAFT offers a user-friendly graphical user interface with one-click installation on Windows and Linux operating systems, freely available at https://github.com/jiaxingbai/[email protected]; [email protected] informationSupplementary data are available at Bioinformatics online.

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In Silico Estimation of the Abundance and Phylogenetic Significance of the Composite Oct4-Sox2 Binding Motifs within a Wide Range of Species

Data ◽

10.3390/data5040111 ◽

2020 ◽

Vol 5 (4) ◽

pp. 111

Author(s):

Arman Kulyyassov ◽

Ruslan Kalendar

Keyword(s):

High Throughput ◽

Dna Sequences ◽

High Throughput Sequencing ◽

Regulatory Elements ◽

Scoring Method ◽

Sequencing Technologies ◽

Wide Range ◽

Multiple Species ◽

Eukaryotic Organisms ◽

The Impact

High-throughput sequencing technologies have greatly accelerated the progress of genomics, transcriptomics, and metagenomics. Currently, a large amount of genomic data from various organisms is being generated, the volume of which is increasing every year. Therefore, the development of methods that allow the rapid search and analysis of DNA sequences is urgent. Here, we present a novel motif-based high-throughput sequence scoring method that generates genome information. We found and identified Utf1-like, Fgf4-like, and Hoxb1-like motifs, which are cis-regulatory elements for the pluripotency transcription factors Sox2 and Oct4 within the genomes of different eukaryotic organisms. The genome-wide analysis of these motifs was performed to understand the impact of their diversification on mammalian genome evolution. Utf1-like, Fgf4-like, and Hoxb1-like motif diversity was evaluated across genomes from multiple species.

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Reassortment of Genome Segments Creates Stable Lineages Among Strains of Orchid Fleck Virus Infecting Citrus in Mexico

Phytopathology ◽

10.1094/phyto-07-19-0253-fi ◽

2020 ◽

Vol 110 (1) ◽

pp. 106-120 ◽

Cited By ~ 1

Author(s):

Avijit Roy ◽

Andrew L. Stone ◽

Gabriel Otero-Colina ◽

Gang Wei ◽

Ronald H. Brlansky ◽

...

Keyword(s):

High Throughput Sequencing ◽

Sensu Stricto ◽

Genome Segment ◽

Rt Pcr ◽

Sequence Comparisons ◽

Orchid Fleck Virus ◽

Reverse Transcription Pcr ◽

Sequencing Technologies ◽

Negative Sense

The genus Dichorhavirus contains viruses with bipartite, negative-sense, single-stranded RNA genomes that are transmitted by flat mites to hosts that include orchids, coffee, the genus Clerodendrum, and citrus. A dichorhavirus infecting citrus in Mexico is classified as a citrus strain of orchid fleck virus (OFV-Cit). We previously used RNA sequencing technologies on OFV-Cit samples from Mexico to develop an OFV-Cit–specific reverse transcription PCR (RT-PCR) assay. During assay validation, OFV-Cit–specific RT-PCR failed to produce an amplicon from some samples with clear symptoms of OFV-Cit. Characterization of this virus revealed that dichorhavirus-like particles were found in the nucleus. High-throughput sequencing of small RNAs from these citrus plants revealed a novel citrus strain of OFV, OFV-Cit2. Sequence comparisons with known orchid and citrus strains of OFV showed variation in the protein products encoded by genome segment 1 (RNA1). Strains of OFV clustered together based on host of origin, whether orchid or citrus, and were clearly separated from other dichorhaviruses described from infected citrus in Brazil. The variation in RNA1 between the original (now OFV-Cit1) and the new (OFV-Cit2) strain was not observed with genome segment 2 (RNA2), but instead, a common RNA2 molecule was shared among strains of OFV-Cit1 and -Cit2, a situation strikingly similar to OFV infecting orchids. We also collected mites at the affected groves, identified them as Brevipalpus californicus sensu stricto, and confirmed that they were infected by OFV-Cit1 or with both OFV-Cit1 and -Cit2. OFV-Cit1 and -Cit2 have coexisted at the same site in Toliman, Queretaro, Mexico since 2012. OFV strain-specific diagnostic tests were developed.

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Application of Oxford Nanopore Technology to Plant Virus Detection

Viruses ◽

10.3390/v13081424 ◽

2021 ◽

Vol 13 (8) ◽

pp. 1424

Author(s):

Lia W. Liefting ◽

David W. Waite ◽

Jeremy R. Thompson

Keyword(s):

Plant Virus ◽

High Throughput Sequencing ◽

Virus Detection ◽

Diagnostic Methods ◽

Plant Virus Detection ◽

Sequencing Technologies ◽

Oxford Nanopore ◽

Virus Diagnostics ◽

Post Entry ◽

Read Accuracy

The adoption of Oxford Nanopore Technologies (ONT) sequencing as a tool in plant virology has been relatively slow despite its promise in more recent years to yield large quantities of long nucleotide sequences in real time without the need for prior amplification. The portability of the MinION and Flongle platforms combined with lowering costs and continued improvements in read accuracy make ONT an attractive method for both low- and high-scale virus diagnostics. Here, we provide a detailed step-by-step protocol using the ONT Flongle platform that we have developed for the routine application on a range of symptomatic post-entry quarantine and domestic surveillance plant samples. The aim of this methods paper is to highlight ONT’s feasibility as a valuable component to the diagnostician’s toolkit and to hopefully stimulate other laboratories towards the eventual goal of integrating high-throughput sequencing technologies as validated plant virus diagnostic methods in their own right.

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Assessing genotyping errors in mammalian museum study skins using high-throughput genotyping-by-sequencing

Conservation Genetics Resources ◽

10.1007/s12686-021-01213-8 ◽

2021 ◽

Author(s):

Stella C. Yuan ◽

Eric Malekos ◽

Melissa T. R. Hawkins

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Massively Parallel Sequencing ◽

Massively Parallel ◽

Museum Specimens ◽

Museum Specimen ◽

Genotyping Errors ◽

Allelic Dropout ◽

Parallel Sequencing ◽

Sequencing Technologies

AbstractThe use of museum specimens held in natural history repositories for population and conservation genetic research is increasing in tandem with the use of massively parallel sequencing technologies. Short Tandem Repeats (STRs), or microsatellite loci, are commonly used genetic markers in wildlife and population genetic studies. However, they traditionally suffered from a host of issues including length homoplasy, high costs, low throughput, and difficulties in reproducibility across laboratories. Massively parallel sequencing technologies can address these problems, but the incorporation of museum specimen derived DNA suffers from significant fragmentation and exogenous DNA contamination. Combatting these issues requires extra measures of stringency in the lab and during data analysis, yet there have not been any high-throughput sequencing studies evaluating microsatellite allelic dropout from museum specimen extracted DNA. In this study, we evaluate genotyping errors derived from mammalian museum skin DNA extracts for previously characterized microsatellites across PCR replicates utilizing high-throughput sequencing. We found it useful to classify samples based on DNA concentration, which determined the rate by which genotypes were accurately recovered. Longer microsatellites performed worse in all museum specimens. Allelic dropout rates across loci were dependent on sample quantity, with high concentration museum specimens performing as well and recovering quality metrics nearly as high as the frozen tissue sample. Based on our results, we provide a set of best practices for quality assurance and incorporation of reliable genotypes from museum specimens.

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Variability of the Sheep Lung Microbiota

Applied and Environmental Microbiology ◽

10.1128/aem.00540-16 ◽

2016 ◽

Vol 82 (11) ◽

pp. 3225-3238 ◽

Cited By ~ 27

Author(s):

Laura Glendinning ◽

Steven Wright ◽

Jolinda Pollock ◽

Peter Tennant ◽

David Collie ◽

...

Keyword(s):

Spatial Variability ◽

Dna Sequences ◽

Bacterial Communities ◽

16S Rrna Genes ◽

Rrna Genes ◽

Large Animal ◽

Local Factors ◽

Adult Sheep ◽

Sequencing Technologies ◽

Host Influence

ABSTRACTSequencing technologies have recently facilitated the characterization of bacterial communities present in lungs during health and disease. However, there is currently a dearth of information concerning the variability of such data in health both between and within subjects. This study seeks to examine such variability using healthy adult sheep as our model system. Protected specimen brush samples were collected from three spatially disparate segmental bronchi of six adult sheep (age, 20 months) on three occasions (day 0, 1 month, and 3 months). To further explore the spatial variability of the microbiotas, more-extensive brushing samples (n= 16) and a throat swab were taken from a separate sheep. The V2 and V3 hypervariable regions of the bacterial 16S rRNA genes were amplified and sequenced via Illumina MiSeq. DNA sequences were analyzed using the mothur software package. Quantitative PCR was performed to quantify total bacterial DNA. Some sheep lungs contained dramatically different bacterial communities at different sampling sites, whereas in others, airway microbiotas appeared similar across the lung. In our spatial variability study, we observed clustering related to the depth within the lung from which samples were taken. Lung depth refers to increasing distance from the glottis, progressing in a caudal direction. We conclude that both host influence and local factors have impacts on the composition of the sheep lung microbiota.IMPORTANCEUntil recently, it was assumed that the lungs were a sterile environment which was colonized by microbes only during disease. However, recent studies using sequencing technologies have found that there is a small population of bacteria which exists in the lung during health, referred to as the “lung microbiota.” In this study, we characterize the variability of the lung microbiotas of healthy sheep. Sheep not only are economically important animals but also are often used as large animal models of human respiratory disease. We conclude that, while host influence does play a role in dictating the types of microbes which colonize the airways, it is clear that local factors also play an important role in this regard. Understanding the nature and influence of these factors will be key to understanding the variability in, and functional relevance of, the lung microbiota.

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Multi-omics approach to precision medicine for immune-mediated diseases

Inflammation and Regeneration ◽

10.1186/s41232-021-00173-8 ◽

2021 ◽

Vol 41 (1) ◽

Author(s):

Mineto Ota ◽

Keishi Fujio

Keyword(s):

Treatment Response ◽

High Throughput Sequencing ◽

Disease Risk ◽

Clinical Information ◽

Clinical Settings ◽

Social Significance ◽

Sequencing Technologies ◽

Immune Mediated ◽

Recent Innovation ◽

Future Direction

AbstractRecent innovation in high-throughput sequencing technologies has drastically empowered the scientific research. Consequently, now, it is possible to capture comprehensive profiles of samples at multiple levels including genome, epigenome, and transcriptome at a time. Applying these kinds of rich information to clinical settings is of great social significance. For some traits such as cardiovascular diseases, attempts to apply omics datasets in clinical practice for the prediction of the disease risk have already shown promising results, although still under way for immune-mediated diseases. Multiple studies have tried to predict treatment response in immune-mediated diseases using genomic, transcriptomic, or clinical information, showing various possible indicators. For better prediction of treatment response or disease outcome in immune-mediated diseases, combining multi-layer information together may increase the power. In addition, in order to efficiently pick up meaningful information from the massive data, high-quality annotation of genomic functions is also crucial. In this review, we discuss the achievement so far and the future direction of multi-omics approach to immune-mediated diseases.

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Periphyton diversity in two different Antarctic lakes assessed using metabarcoding

Antarctic Science ◽

10.1017/s0954102021000316 ◽

2021 ◽

pp. 1-9

Author(s):

Paulo E.A.S. Câmara ◽

Láuren M.D. De Souza ◽

Otávio Henrique Bezerra Pinto ◽

Peter Convey ◽

Eduardo T. Amorim ◽

...

Keyword(s):

Dna Sequences ◽

High Throughput Sequencing ◽

Shannon Index ◽

South Shetland Islands ◽

King George Island ◽

Deception Island ◽

Maritime Antarctic ◽

Shetland Islands ◽

Antarctic Lakes ◽

Significant Difference

Abstract Antarctic lakes have generally simple periphyton communities when compared with those of lower latitudes. To date, assessment of microbial diversity in Antarctica has relied heavily on traditional direct observation and cultivation methods. In this study, sterilized cotton baits were left submerged for two years in two lakes on King George Island and Deception Island, South Shetland Islands (Maritime Antarctic), followed by assessment of diversity by metabarcoding using high-throughput sequencing. DNA sequences of 44 taxa belonging to four kingdoms and seven phyla were found. Thirty-six taxa were detected in Hennequin Lake on King George Island and 20 taxa were detected in Soto Lake on Deception Island. However, no significant difference in species composition was detected between the two assemblages (Shannon index). Our data suggest that metabarcoding provides a suitable method for the assessment of periphyton biodiversity in oligotrophic Antarctic lakes.

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Profiling DNA Methylation Based on Next-Generation Sequencing Approaches: New Insights and Clinical Applications

Genes ◽

10.3390/genes9090429 ◽

2018 ◽

Vol 9 (9) ◽

pp. 429 ◽

Cited By ~ 38

Author(s):

Daniela Barros-Silva ◽

C. Marques ◽

Rui Henrique ◽

Carmen Jerónimo

Keyword(s):

Dna Methylation ◽

Next Generation Sequencing ◽

High Throughput Sequencing ◽

Epigenetic Modification ◽

Response To Therapy ◽

Next Generation ◽

Sequencing Technologies ◽

Prognosis And Prediction ◽

Novel Biomarkers ◽

Generation Sequencing

DNA methylation is an epigenetic modification that plays a pivotal role in regulating gene expression and, consequently, influences a wide variety of biological processes and diseases. The advances in next-generation sequencing technologies allow for genome-wide profiling of methyl marks both at a single-nucleotide and at a single-cell resolution. These profiling approaches vary in many aspects, such as DNA input, resolution, coverage, and bioinformatics analysis. Thus, the selection of the most feasible method according with the project’s purpose requires in-depth knowledge of those techniques. Currently, high-throughput sequencing techniques are intensively used in epigenomics profiling, which ultimately aims to find novel biomarkers for detection, diagnosis prognosis, and prediction of response to therapy, as well as to discover new targets for personalized treatments. Here, we present, in brief, a portrayal of next-generation sequencing methodologies’ evolution for profiling DNA methylation, highlighting its potential for translational medicine and presenting significant findings in several diseases.

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Genome-Wide Identification of 5-Methylcytosine Sites in Bacterial Genomes By High-Throughput Sequencing of MspJI Restriction Fragments

10.1101/2021.02.10.430591 ◽

2021 ◽

Author(s):

Brian P. Anton ◽

Alexey Fomenkov ◽

Victoria Wu ◽

Richard J. Roberts

Keyword(s):

Single Molecule ◽

Dna Sequences ◽

High Throughput Sequencing ◽

Cost Effective ◽

Restriction Enzymes ◽

Specific Sequence ◽

Genome Wide ◽

Cost Effective Alternative ◽

Simple Column ◽

Sequencing Platforms

ABSTRACTSingle-molecule Real-Time (SMRT) sequencing can easily identify sites of N6-methyladenine and N4-methylcytosine within DNA sequences, but similar identification of 5-methylcytosine sites is not as straightforward. In prokaryotic DNA, methylation typically occurs within specific sequence contexts, or motifs, that are a property of the methyltransferases that “write” these epigenetic marks. We present here a straightforward, cost-effective alternative to both SMRT and bisulfite sequencing for the determination of prokaryotic 5-methylcytosine methylation motifs. The method, called MFRE-Seq, relies on excision and isolation of fully methylated fragments of predictable size using MspJI-Family Restriction Enzymes (MFREs), which depend on the presence of 5-methylcytosine for cleavage. We demonstrate that MFRE-Seq is compatible with both Illumina and Ion Torrent sequencing platforms and requires only a digestion step and simple column purification of size-selected digest fragments prior to standard library preparation procedures. We applied MFRE-Seq to numerous bacterial and archaeal genomic DNA preparations and successfully confirmed known motifs and identified novel ones. This method should be a useful complement to existing methodologies for studying prokaryotic methylomes and characterizing the contributing methyltransferases.

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