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Human Immunology Nov 2021Since the days of Sanger sequencing, next-generation sequencing technologies have significantly evolved to provide increased data output, efficiencies, and applications.... (Review)
Review
Since the days of Sanger sequencing, next-generation sequencing technologies have significantly evolved to provide increased data output, efficiencies, and applications. These next generations of technologies can be categorized based on read length. This review provides an overview of these technologies as two paradigms: short-read, or "second-generation," technologies, and long-read, or "third-generation," technologies. Herein, short-read sequencing approaches are represented by the most prevalent technologies, Illumina and Ion Torrent, and long-read sequencing approaches are represented by Pacific Biosciences and Oxford Nanopore technologies. All technologies are reviewed along with reported advantages and disadvantages. Until recently, short-read sequencing was thought to provide high accuracy limited by read-length, while long-read technologies afforded much longer read-lengths at the expense of accuracy. Emerging developments for third-generation technologies hold promise for the next wave of sequencing evolution, with the co-existence of longer read lengths and high accuracy.
Topics: DNA Probes, HLA; Genotyping Techniques; HLA Antigens; High-Throughput Nucleotide Sequencing; Humans; Sequence Analysis, DNA
PubMed: 33745759
DOI: 10.1016/j.humimm.2021.02.012 -
Methods in Molecular Biology (Clifton,... 2021Thanks to innovative sample-preparation and sequencing technologies, gene expression in individual cells can now be measured for thousands of cells in a single...
Thanks to innovative sample-preparation and sequencing technologies, gene expression in individual cells can now be measured for thousands of cells in a single experiment. Since its introduction, single-cell RNA sequencing (scRNA-seq) approaches have revolutionized the genomics field as they created unprecedented opportunities for resolving cell heterogeneity by exploring gene expression profiles at a single-cell resolution. However, the rapidly evolving field of scRNA-seq invoked the emergence of various analytics approaches aimed to maximize the full potential of this novel strategy. Unlike population-based RNA sequencing approaches, scRNA seq necessitates comprehensive computational tools to address high data complexity and keep up with the emerging single-cell associated challenges. Despite the vast number of analytical methods, a universal standardization is lacking. While this reflects the fields' immaturity, it may also encumber a newcomer to blend in.In this review, we aim to bridge over the abovementioned hurdle and propose four ready-to-use pipelines for scRNA-seq analysis easily accessible by a newcomer, that could fit various biological data types. Here we provide an overview of the currently available single-cell technologies for cell isolation and library preparation and a step by step guide that covers the entire canonical analytic workflow to analyse scRNA-seq data including read mapping, quality controls, gene expression quantification, normalization, feature selection, dimensionality reduction, and cell clustering useful for trajectory inference and differential expression. Such workflow guidelines will escort novices as well as expert users in the analysis of complex scRNA-seq datasets, thus further expanding the research potential of single-cell approaches in basic science, and envisaging its future implementation as best practice in the field.
Topics: Algorithms; Animals; Cluster Analysis; Gene Expression Profiling; Genomics; High-Throughput Nucleotide Sequencing; Humans; Quality Control; Sequence Analysis, RNA; Single-Cell Analysis; Software; Transcriptome
PubMed: 33835452
DOI: 10.1007/978-1-0716-1307-8_19 -
Cold Spring Harbor Perspectives in... Jul 2019More than a decade ago, the term "next-generation" sequencing was coined to describe what was, at the time, revolutionary new methods to sequence RNA and DNA at a faster... (Review)
Review
More than a decade ago, the term "next-generation" sequencing was coined to describe what was, at the time, revolutionary new methods to sequence RNA and DNA at a faster pace and cheaper cost than could be performed by standard bench-top protocols. Since then, the field of DNA sequencing has evolved at a rapid pace, with new breakthroughs allowing capacity to exponentially increase and cost to dramatically decrease. As genome-scale sequencing has become routine, a paradigm shift is occurring in genomics, which uses the power of high-throughput, rapid sequencing power with large-scale studies. These new approaches to genetic discovery will provide direct impact to fields such as personalized medicine, evolution, and biodiversity. This work reviews recent technology advances and methods in next-generation sequencing and highlights current large-scale sequencing efforts driving the evolution of the genomics space.
Topics: Genome, Human; Genomics; High-Throughput Nucleotide Sequencing; Humans; Sequence Analysis, DNA
PubMed: 30323017
DOI: 10.1101/cshperspect.a025791 -
Journal of Veterinary Diagnostic... Nov 2020Genetic sequencing, or DNA sequencing, using the Sanger technique has become widely used in the veterinary diagnostic community. This technology plays a role in...
Genetic sequencing, or DNA sequencing, using the Sanger technique has become widely used in the veterinary diagnostic community. This technology plays a role in verification of PCR results and is used to provide the genetic sequence data needed for phylogenetic analysis, epidemiologic studies, and forensic investigations. The Laboratory Technology Committee of the American Association of Veterinary Laboratory Diagnosticians has prepared guidelines for sample preparation, submission to sequencing facilities or instrumentation, quality assessment of nucleic acid sequence data performed, and for generating basic sequencing data and phylogenetic analysis for diagnostic applications. This guidance is aimed at assisting laboratories in providing consistent, high-quality, and reliable sequence data when using Sanger-based genetic sequencing as a component of their laboratory services.
Topics: Animal Diseases; Animals; Base Sequence; High-Throughput Nucleotide Sequencing; Humans; Laboratories; Phylogeny; Polymerase Chain Reaction; Sequence Analysis, DNA
PubMed: 32070230
DOI: 10.1177/1040638720905833 -
Methods in Molecular Biology (Clifton,... 2022With the ability to obtain several millions of reads per sample, high-throughput RNA sequencing (RNA-Seq) enables investigation of any transcriptome at a fine...
With the ability to obtain several millions of reads per sample, high-throughput RNA sequencing (RNA-Seq) enables investigation of any transcriptome at a fine resolution. Not just the messenger RNA (mRNA), but a wide variety of different RNA populations (e.g., total RNA, microRNA, long ncRNA, pre-mRNA) can also be investigated using RNA-Seq. While facilitating accurate quantification of gene expression, RNA-Seq offers the opportunity to estimate abundance of isoforms and find novel transcripts and allele-specific transcripts. In this chapter, we describe a protocol to construct an RNA-Seq library for sequencing on Illumina NGS platforms and a computational pipeline to perform RNA-Seq data analysis. The protocols described in this chapter can be applied to the analysis of differential gene expression in control versus 17β-estradiol treatment of in vivo or in vitro systems.
Topics: Data Analysis; High-Throughput Nucleotide Sequencing; RNA-Seq; Transcriptome
PubMed: 35119677
DOI: 10.1007/978-1-0716-1920-9_22 -
Methods in Molecular Biology (Clifton,... 2021Advances in next generation sequencing (NGS) technologies resulted in a broad array of large-scale gene expression studies and an unprecedented volume of whole messenger... (Review)
Review
Advances in next generation sequencing (NGS) technologies resulted in a broad array of large-scale gene expression studies and an unprecedented volume of whole messenger RNA (mRNA) sequencing data, or the transcriptome (also known as RNA sequencing, or RNA-seq). These include the Genotype Tissue Expression project (GTEx) and The Cancer Genome Atlas (TCGA), among others. Here we cover some of the commonly used datasets, provide an overview on how to begin the analysis pipeline, and how to explore and interpret the data provided by these publicly available resources.
Topics: Databases, Genetic; High-Throughput Nucleotide Sequencing; Humans; RNA, Messenger; RNA-Seq; Sequence Analysis, RNA; Transcriptome
PubMed: 33606253
DOI: 10.1007/978-1-0716-1103-6_4 -
Biomolecules Jul 2021Recent developments have revolutionized the study of biomolecules. Among them are molecular markers, amplification and sequencing of nucleic acids. The latter is... (Review)
Review
Recent developments have revolutionized the study of biomolecules. Among them are molecular markers, amplification and sequencing of nucleic acids. The latter is classified into three generations. The first allows to sequence small DNA fragments. The second one increases throughput, reducing turnaround and pricing, and is therefore more convenient to sequence full genomes and transcriptomes. The third generation is currently pushing technology to its limits, being able to sequence single molecules, without previous amplification, which was previously impossible. Besides, this represents a new revolution, allowing researchers to directly sequence RNA without previous retrotranscription. These technologies are having a significant impact on different areas, such as medicine, agronomy, ecology and biotechnology. Additionally, the study of biomolecules is revealing interesting evolutionary information. That includes deciphering what makes us human, including phenomena like non-coding RNA expansion. All this is redefining the concept of gene and transcript. Basic analyses and applications are now facilitated with new genome editing tools, such as CRISPR. All these developments, in general, and nucleic-acid sequencing, in particular, are opening a new exciting era of biomolecule analyses and applications, including personalized medicine, and diagnosis and prevention of diseases for humans and other animals.
Topics: Animals; Base Sequence; DNA; Genome; Genomics; High-Throughput Nucleotide Sequencing; History, 20th Century; History, 21st Century; Humans; RNA, Messenger; Sequence Analysis, DNA; Sequence Analysis, RNA; Whole Genome Sequencing
PubMed: 34439777
DOI: 10.3390/biom11081111 -
Methods in Molecular Biology (Clifton,... 2021A complete RNA-Seq analysis involves the use of several different tools, with substantial software and computational requirements. The Galaxy platform simplifies the...
A complete RNA-Seq analysis involves the use of several different tools, with substantial software and computational requirements. The Galaxy platform simplifies the execution of such bioinformatics analyses by embedding the needed tools in its web interface, while also providing reproducibility. Here, we describe how to perform a reference-based RNA-Seq analysis using Galaxy, from data upload to visualization and functional enrichment analysis of differentially expressed genes.
Topics: Animals; Computational Biology; Data Analysis; Datasets as Topic; Gene Expression Profiling; High-Throughput Nucleotide Sequencing; Humans; RNA-Seq; Reproducibility of Results; Sequence Analysis, RNA; Software; Exome Sequencing
PubMed: 33835453
DOI: 10.1007/978-1-0716-1307-8_20 -
Clinics in Laboratory Medicine Sep 2019Next-generation sequencing (NGS) applications have been transitioning from research tools to diagnostic methods and are becoming more commonplace in clinical... (Review)
Review
Next-generation sequencing (NGS) applications have been transitioning from research tools to diagnostic methods and are becoming more commonplace in clinical microbiology laboratories. These applications include (1) whole-genome sequencing, (2) targeted next-generation sequencing methods, and (3) metagenomic next-generation sequencing. The introduction of these methods into the clinical microbiology laboratory has led to the theoretic question of "Will NGS-based methods supplant traditional methods for strain typing, identification, and antimicrobial susceptibility prediction?" The authors address this question and discuss where we are at now with clinical NGS applications for infectious diseases, what does the future hold, and at what cost?
Topics: Bacteria; Bacterial Infections; High-Throughput Nucleotide Sequencing; Humans; Microbiological Techniques
PubMed: 31383265
DOI: 10.1016/j.cll.2019.05.003 -
Briefings in Bioinformatics May 2022With recent advances in high-throughput next-generation sequencing, it is possible to describe the regulation and expression of genes at multiple levels. An assay for... (Review)
Review
With recent advances in high-throughput next-generation sequencing, it is possible to describe the regulation and expression of genes at multiple levels. An assay for transposase-accessible chromatin using sequencing (ATAC-seq), which uses Tn5 transposase to sequence protein-free binding regions of the genome, can be combined with chromatin immunoprecipitation coupled with deep sequencing (ChIP-seq) and ribonucleic acid sequencing (RNA-seq) to provide a detailed description of gene expression. Here, we reviewed the literature on ATAC-seq and described the characteristics of ATAC-seq publications. We then briefly introduced the principles of RNA-seq, ChIP-seq and ATAC-seq, focusing on the main features of the techniques. We built a phylogenetic tree from species that had been previously studied by using ATAC-seq. Studies of Mus musculus and Homo sapiens account for approximately 90% of the total ATAC-seq data, while other species are still in the process of accumulating data. We summarized the findings from human diseases and other species, illustrating the cutting-edge discoveries and the role of multi-omics data analysis in current research. Moreover, we collected and compared ATAC-seq analysis pipelines, which allowed biological researchers who lack programming skills to better analyze and explore ATAC-seq data. Through this review, it is clear that multi-omics analysis and single-cell sequencing technology will become the mainstream approach in future research.
Topics: Animals; Bibliometrics; Chromatin Immunoprecipitation Sequencing; Gene Expression; High-Throughput Nucleotide Sequencing; Humans; Mice; Phylogeny; RNA; Sequence Analysis, DNA
PubMed: 35255493
DOI: 10.1093/bib/bbac061