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Nature Reviews. Genetics Jul 2024
Topics: Humans; Tandem Repeat Sequences; High-Throughput Nucleotide Sequencing; Sequence Analysis, DNA
PubMed: 38898331
DOI: 10.1038/s41576-024-00751-9 -
Journal of Vascular and Interventional... Aug 2023The discovery of increasing numbers of actionable molecular and gene targets for cancer treatment has driven the demand for tissue sampling for next-generation... (Review)
Review
The discovery of increasing numbers of actionable molecular and gene targets for cancer treatment has driven the demand for tissue sampling for next-generation sequencing (NGS). Requirements for sequencing can be very specific, and inadequate sampling leads to delays in management and decision making. It is important that interventional radiologists are aware of NGS technologies and their common applications and be cognizant of the factors that contribute to successful sample sequencing. This review summarizes the fundamentals of cancer tissue collection and processing for NGS. It elaborates on sequencing technologies and their applications with the aim of providing readers with a working understanding that can enhance their clinical practice. It then describes imaging, tumor, biopsy, and sample collection factors that improve the chances of NGS success. Finally, it discusses future practice, highlighting the problem of undersampling in both clinical and research settings and the opportunities within interventional radiology to address this.
Topics: Humans; Neoplasms; Biopsy; High-Throughput Nucleotide Sequencing
PubMed: 36977432
DOI: 10.1016/j.jvir.2023.03.012 -
Cell Biology International Sep 2020The recent development of next-generation sequencing technologies has offered valuable insights into individual cells. This technology is centered on the... (Review)
Review
The recent development of next-generation sequencing technologies has offered valuable insights into individual cells. This technology is centered on the characterization of single cells for epigenomics, genomics, and transcriptomics. Ever since the first report appeared in 2009, the single-cell RNA-sequencing saga started to explore deeper into the mechanics intrigued within a single cell. microRNA (miRNA) has been increasingly recognized as an essential molecule triggering an additional layer for gene regulation. Therefore, single-cell sequencing of miRNAs is crucial to explore the logical riddles surrounding the epigenomics, genomics, and transcriptomics of an individual cell. Scientists from the Vienna Biocenter Campus have lately performed single-cell sequencing of miRNAs in the fly, Drosophila, and nematode, Caenorhabditis elegans. In this review, we present the latest scientific explorations supported by all-inclusive data on this novel subject matter of next-generation sequencing.
Topics: Animals; Computational Biology; Epigenomics; Gene Expression Profiling; Genomics; High-Throughput Nucleotide Sequencing; Humans; MicroRNAs; Sequence Analysis, RNA; Single-Cell Analysis
PubMed: 32379363
DOI: 10.1002/cbin.11376 -
Methods in Molecular Biology (Clifton,... 2022DEL technology is dependent on the generation and analysis of large amounts of DNA sequence data. In this chapter, we describe a method to sequence DEL libraries that...
DEL technology is dependent on the generation and analysis of large amounts of DNA sequence data. In this chapter, we describe a method to sequence DEL libraries that uses a customized preparative PCR protocol, along with standard steps for purification, analysis, and sequencing of the amplified library DNA on an Illumina sequencing platform. Compared with standard Illumina sequencing library preparation protocols, in which a PCR reaction is followed by end repair, adenylation, ligation of Illumina adapters, and a second preparative PCR reaction, the customized operations described here provide significantly improved process efficiency and sequencing quality.
Topics: DNA; Gene Library; High-Throughput Nucleotide Sequencing; Polymerase Chain Reaction; Sequence Analysis, DNA
PubMed: 36083556
DOI: 10.1007/978-1-0716-2545-3_21 -
Methods in Molecular Biology (Clifton,... 2023As single-cell RNA sequencing experiments continue to advance scientific discoveries across biological disciplines, an increasing number of analysis tools and workflows...
As single-cell RNA sequencing experiments continue to advance scientific discoveries across biological disciplines, an increasing number of analysis tools and workflows for analyzing the data have been developed. In this chapter, we describe a standard workflow and elaborate on relevant data analysis tools for analyzing single-cell RNA sequencing data. We provide recommendations for the appropriate use of commonly used methods, with code examples and analysis interpretations.
Topics: Gene Expression Profiling; Sequence Analysis, RNA; High-Throughput Nucleotide Sequencing; Single-Cell Gene Expression Analysis; Workflow; Single-Cell Analysis; Software
PubMed: 36929075
DOI: 10.1007/978-1-0716-2986-4_6 -
International Journal of Molecular... Aug 2021Aptamers feature a number of advantages, compared to antibodies. However, their application has been limited so far, mainly because of the complex selection process.... (Review)
Review
Aptamers feature a number of advantages, compared to antibodies. However, their application has been limited so far, mainly because of the complex selection process. 'High-throughput sequencing fluorescent ligand interaction profiling' (HiTS-FLIP) significantly increases the selection efficiency and is consequently a very powerful and versatile technology for the selection of high-performance aptamers. It is the first experiment to allow the direct and quantitative measurement of the affinity and specificity of millions of aptamers simultaneously by harnessing the potential of optical next-generation sequencing platforms to perform fluorescence-based binding assays on the clusters displayed on the flow cells and determining their sequence and position in regular high-throughput sequencing. Many variants of the experiment have been developed that allow automation and in situ conversion of DNA clusters into base-modified DNA, RNA, peptides, and even proteins. In addition, the information from mutational assays, performed with HiTS-FLIP, provides deep insights into the relationship between the sequence, structure, and function of aptamers. This enables a detailed understanding of the sequence-specific rules that determine affinity, and thus, supports the evolution of aptamers. Current variants of the HiTS-FLIP experiment and its application in the field of aptamer selection, characterisation, and optimisation are presented in this review.
Topics: Aptamers, Nucleotide; Automation, Laboratory; High-Throughput Nucleotide Sequencing; Mutagenesis; Optical Devices; Sequence Analysis, DNA
PubMed: 34502110
DOI: 10.3390/ijms22179202 -
Methods in Molecular Biology (Clifton,... 2023Specific length amplified fragment sequencing (SLAF-seq) technology is a simplified genome sequencing technology based on next-generation sequencing. SLAF-seq technology...
Specific length amplified fragment sequencing (SLAF-seq) technology is a simplified genome sequencing technology based on next-generation sequencing. SLAF-seq technology has several distinguishing characteristics: 1. Deep sequencing to ensure accuracy of genotyping; 2. Effectively reduce sequencing costs; 3. Pre-designed simplified representation scheme to optimize marker efficiency; 4. Doubled barcode system for large populations. The advantages and technical process of SLAF-seq are described briefly with summarized results for the application of SLAF-seq in development of molecular markers, construction of high-density genetic map and gene mapping in ornamental plants. Finally, the difficulties and prospects of this method are discussed in application.
Topics: Quantitative Trait Loci; Sequence Analysis, DNA; Chromosome Mapping; High-Throughput Nucleotide Sequencing; Polymorphism, Single Nucleotide
PubMed: 36781641
DOI: 10.1007/978-1-0716-3024-2_11 -
Methods in Molecular Biology (Clifton,... 2023Chromatin immunoprecipitation sequencing (ChIP-seq) has been widely performed to identify protein binding information along the genome. The sequencing protocol is quite...
Chromatin immunoprecipitation sequencing (ChIP-seq) has been widely performed to identify protein binding information along the genome. The sequencing protocol is quite flexible and mature to measure different types of protein binding as long as sequencing parameters are properly tailored to accommodate protein features. Two distinct types of protein binding are point-source-like binding by transcription factors and diffused-distribution binding by histone modifications. Consequently, statistical approaches have been proposed to address ChIP-seq-related questions according to different protein features. In this chapter, we briefly summarize statistical principles, approaches, and tools that are widely implemented in modeling ChIP-seq data, from raw data quality control to final result reporting. We discuss the key solutions in addressing eight routine questions in ChIP-seq applications. We also include discussion on approaches fitting unique data features in different ChIP-seq types. We hope this chapter will serve as a brief guide, especially for ChIP-seq beginners, to provide them with a high-level overview to understand and design processing plans for their ChIP-seq experiments.
Topics: Chromatin Immunoprecipitation Sequencing; Chromatin Immunoprecipitation; Transcription Factors; Genome; Protein Binding; Sequence Analysis, DNA; High-Throughput Nucleotide Sequencing
PubMed: 36929078
DOI: 10.1007/978-1-0716-2986-4_9 -
Journal of Visualized Experiments : JoVE Jul 2022The proper use of statistical modeling in NGS data analysis requires an advanced level of expertise. There has recently been a growing consensus on using generalized...
The proper use of statistical modeling in NGS data analysis requires an advanced level of expertise. There has recently been a growing consensus on using generalized linear models for differential analysis of RNA-Seq data and the advantage of mixture models to perform co-expression analysis. To offer a managed setting to use these modeling approaches, we developed DiCoExpress that provides a standardized R pipeline to perform an RNA-Seq analysis. Without any particular knowledge in statistics or R programming, beginners can perform a complete RNA-Seq analysis from quality controls to co-expression through differential analysis based on contrasts inside a generalized linear model. An enrichment analysis is proposed both on the lists of differentially expressed genes, and the co-expressed gene clusters. This video tutorial is conceived as a step-by-step protocol to help users take full advantage of DiCoExpress and its potential in empowering the biological interpretation of an RNA-Seq experiment.
Topics: Gene Expression Profiling; High-Throughput Nucleotide Sequencing; RNA-Seq; Sequence Analysis, RNA; Software; Exome Sequencing
PubMed: 35969075
DOI: 10.3791/62566 -
Briefings in Bioinformatics Jul 2022The study of the Human Virome remains challenging nowadays. Viral metagenomics, through high-throughput sequencing data, is the best choice for virus discovery. The...
MOTIVATION
The study of the Human Virome remains challenging nowadays. Viral metagenomics, through high-throughput sequencing data, is the best choice for virus discovery. The metagenomics approach is culture-independent and sequence-independent, helping search for either known or novel viruses. Though it is estimated that more than 40% of the viruses found in metagenomics analysis are not recognizable, we decided to analyze several tools to identify and discover viruses in RNA-seq samples.
RESULTS
We have analyzed eight Virus Tools for the identification of viruses in RNA-seq data. These tools were compared using a synthetic dataset of 30 viruses and a real one. Our analysis shows that no tool succeeds in recognizing all the viruses in the datasets. So we can conclude that each of these tools has pros and cons, and their choice depends on the application domain.
AVAILABILITY
Synthetic data used through the review and raw results of their analysis can be found at https://zenodo.org/record/6426147. FASTQ files of real data can be found in GEO (https://www.ncbi.nlm.nih.gov/gds) or ENA (https://www.ebi.ac.uk/ena/browser/home). Raw results of their analysis can be downloaded from https://zenodo.org/record/6425917.
Topics: High-Throughput Nucleotide Sequencing; Humans; Metagenomics; Viruses
PubMed: 35753694
DOI: 10.1093/bib/bbac235