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Nature Biotechnology Nov 2021Rapid advances in nanopore technologies for sequencing single long DNA and RNA molecules have led to substantial improvements in accuracy, read length and throughput.... (Review)
Review
Rapid advances in nanopore technologies for sequencing single long DNA and RNA molecules have led to substantial improvements in accuracy, read length and throughput. These breakthroughs have required extensive development of experimental and bioinformatics methods to fully exploit nanopore long reads for investigations of genomes, transcriptomes, epigenomes and epitranscriptomes. Nanopore sequencing is being applied in genome assembly, full-length transcript detection and base modification detection and in more specialized areas, such as rapid clinical diagnoses and outbreak surveillance. Many opportunities remain for improving data quality and analytical approaches through the development of new nanopores, base-calling methods and experimental protocols tailored to particular applications.
Topics: Computational Biology; High-Throughput Nucleotide Sequencing; Nanopore Sequencing; Nanopores; Sequence Analysis, DNA; Technology
PubMed: 34750572
DOI: 10.1038/s41587-021-01108-x -
Cold Spring Harbor Perspectives in... Nov 2019Although DNA and RNA sequencing has a history spanning five decades, large-scale massively parallel sequencing, or next-generation sequencing (NGS), has only been... (Review)
Review
Although DNA and RNA sequencing has a history spanning five decades, large-scale massively parallel sequencing, or next-generation sequencing (NGS), has only been commercially available for about 10 years. Nonetheless, the meteoric increase in sequencing throughput with NGS has dramatically changed our understanding of our genome and ourselves. Sequencing the first human genome as a haploid reference took nearly 10 years but now a full diploid human genome sequence can be accomplished in just a few days. NGS has also reduced the cost of generating sequence data and a plethora of sequence-based methods for probing a genome have emerged using NGS as the readout and have been applied to many species. NGS methods have also entered the medical realm and will see an increasing use in diagnosis and treatment. NGS has largely been driven by short-read generation (150 bp) but new platforms have emerged and are now capable of generating long multikilobase reads. These latter platforms enable reference-independent genome assemblies and long-range haplotype generation. Rapid DNA and RNA sequencing is now mainstream and will continue to have an increasing impact on biology and medicine.
Topics: Computational Biology; Genome, Human; Genomics; High-Throughput Nucleotide Sequencing; Humans; Sequence Analysis, DNA; Sequence Analysis, RNA
PubMed: 30478097
DOI: 10.1101/cshperspect.a036798 -
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 -
Briefings in Bioinformatics Nov 2019Differential gene expression (DGE) analysis is one of the most common applications of RNA-sequencing (RNA-seq) data. This process allows for the elucidation of... (Review)
Review
Differential gene expression (DGE) analysis is one of the most common applications of RNA-sequencing (RNA-seq) data. This process allows for the elucidation of differentially expressed genes across two or more conditions and is widely used in many applications of RNA-seq data analysis. Interpretation of the DGE results can be nonintuitive and time consuming due to the variety of formats based on the tool of choice and the numerous pieces of information provided in these results files. Here we reviewed DGE results analysis from a functional point of view for various visualizations. We also provide an R/Bioconductor package, Visualization of Differential Gene Expression Results using R, which generates information-rich visualizations for the interpretation of DGE results from three widely used tools, Cuffdiff, DESeq2 and edgeR. The implemented functions are also tested on five real-world data sets, consisting of one human, one Malus domestica and three Vitis riparia data sets.
Topics: Computational Biology; Gene Expression; High-Throughput Nucleotide Sequencing; Sequence Analysis, RNA
PubMed: 30099484
DOI: 10.1093/bib/bby067 -
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 -
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 -
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 -
BioTechniques Jul 2020Emulsion PCR (ePCR) is an important technique that permits amplification of DNA molecules in physically separated picoliter-volume water-in-oil droplets, and thus avoids...
Emulsion PCR (ePCR) is an important technique that permits amplification of DNA molecules in physically separated picoliter-volume water-in-oil droplets, and thus avoids formation of unproductive chimeras and other artifacts between similar DNA sequences. However, the recovery of ePCR products involves repeated extraction with hazardous organic solvents followed by purification using silica-based columns, making the overall process cumbersome. In this benchmark, we have described a quick ePCR extraction protocol for the purification of ePCR products, which directly employs silica-based DNA purification columns; products purified using this method have been found to be compatible with gene cloning and next-generation sequencing applications. The method described here makes ePCR easy, safe and within the reach of every laboratory.
Topics: DNA; Emulsions; High-Throughput Nucleotide Sequencing; Polymerase Chain Reaction; Silicon Dioxide
PubMed: 32338528
DOI: 10.2144/btn-2019-0161 -
International Journal of Molecular... Feb 2023As an emerging sequencing technology, single-cell RNA sequencing (scRNA-Seq) has become a powerful tool for describing cell subpopulation classification and cell... (Review)
Review
As an emerging sequencing technology, single-cell RNA sequencing (scRNA-Seq) has become a powerful tool for describing cell subpopulation classification and cell heterogeneity by achieving high-throughput and multidimensional analysis of individual cells and circumventing the shortcomings of traditional sequencing for detecting the average transcript level of cell populations. It has been applied to life science and medicine research fields such as tracking dynamic cell differentiation, revealing sensitive effector cells, and key molecular events of diseases. This review focuses on the recent technological innovations in scRNA-Seq, highlighting the latest research results with scRNA-Seq as the core technology in frontier research areas such as embryology, histology, oncology, and immunology. In addition, this review outlines the prospects for its innovative application in traditional Chinese medicine (TCM) research and discusses the key issues currently being addressed by scRNA-Seq and its great potential for exploring disease diagnostic targets and uncovering drug therapeutic targets in combination with multiomics technologies.
Topics: Single-Cell Analysis; Sequence Analysis, RNA; High-Throughput Nucleotide Sequencing; Multiomics; Technology; Gene Expression Profiling
PubMed: 36769267
DOI: 10.3390/ijms24032943 -
Journal of Clinical Microbiology Dec 2019Metagenomic sequencing for infectious disease diagnostics is an important tool that holds promise for use in the clinical laboratory. Challenges for implementation so... (Review)
Review
Metagenomic sequencing for infectious disease diagnostics is an important tool that holds promise for use in the clinical laboratory. Challenges for implementation so far include high cost, the length of time to results, and the need for technical and bioinformatics expertise. However, the recent technological innovation of nanopore sequencing from Oxford Nanopore Technologies (ONT) has the potential to address these challenges. ONT sequencing is an attractive platform for clinical laboratories to adopt due to its low cost, rapid turnaround time, and user-friendly bioinformatics pipelines. However, this method still faces the problem of base-calling accuracy compared to other platforms. This review highlights the general challenges of pathogen detection in clinical specimens by metagenomic sequencing, the advantages and disadvantages of the ONT platform, and how research to date supports the potential future use of nanopore sequencing in infectious disease diagnostics.
Topics: Clinical Laboratory Services; Clinical Laboratory Techniques; Communicable Diseases; High-Throughput Nucleotide Sequencing; Humans; Nanopore Sequencing
PubMed: 31619531
DOI: 10.1128/JCM.01315-19