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Pathobiology : Journal of... 2017Interrogation of tissue informs on patient management through delivery of a diagnosis together with associated clinically relevant data. The diagnostic pathologist will... (Review)
Review
Interrogation of tissue informs on patient management through delivery of a diagnosis together with associated clinically relevant data. The diagnostic pathologist will usually evaluate the morphological appearances of a tissue sample and, occasionally, the pattern of expression of a limited number of biomarkers. Recent developments in sequencing technology mean that DNA and RNA from tissue samples can now be interrogated in great detail. These new technologies, collectively known as next-generation sequencing (NGS), generate huge amounts of data which can be used to support patient management. In order to maximize the utility of tissue interrogation, the molecular data need to be interpreted and integrated with the morphological data. However, in order to interpret the molecular data, the pathologist must understand the utility and the limitations of NGS data. In this review, the principles behind NGS technologies are described. In addition, the caveats in the interpretation of the data are discussed, and a scheme is presented to "classify" the types of data which are generated. Finally, a glossary of new terminology is included to help pathologists become familiar with the lexicon of NGS-derived molecular data.
Topics: Gene Library; High-Throughput Nucleotide Sequencing; Histological Techniques; Humans; Pathology, Molecular; Sequence Analysis, DNA; Statistics as Topic
PubMed: 29131018
DOI: 10.1159/000480089 -
Annual Review of Microbiology Sep 2020Shotgun metagenomic sequencing has revolutionized our ability to detect and characterize the diversity and function of complex microbial communities. In this review, we... (Review)
Review
Shotgun metagenomic sequencing has revolutionized our ability to detect and characterize the diversity and function of complex microbial communities. In this review, we highlight the benefits of using metagenomics as well as the breadth of conclusions that can be made using currently available analytical tools, such as greater resolution of species and strains across phyla and functional content, while highlighting challenges of metagenomic data analysis. Major challenges remain in annotating function, given the dearth of functional databases for environmental bacteria compared to model organisms, and the technical difficulties of metagenome assembly and phasing in heterogeneous environmental samples. In the future, improvements and innovation in technology and methodology will lead to lowered costs. Data integration using multiple technological platforms will lead to a better understanding of how to harness metagenomes. Subsequently, we will be able not only to characterize complex microbiomes but also to manipulate communities to achieve prosperous outcomes for health, agriculture, and environmental sustainability.
Topics: Bacteria; Computational Biology; High-Throughput Nucleotide Sequencing; Metagenome; Metagenomics; Microbiota
PubMed: 32603623
DOI: 10.1146/annurev-micro-012520-072314 -
Nature Reviews. Genetics Jul 2015Research involving ancient DNA (aDNA) has experienced a true technological revolution in recent years through advances in the recovery of aDNA and, particularly, through... (Review)
Review
Research involving ancient DNA (aDNA) has experienced a true technological revolution in recent years through advances in the recovery of aDNA and, particularly, through applications of high-throughput sequencing. Formerly restricted to the analysis of only limited amounts of genetic information, aDNA studies have now progressed to whole-genome sequencing for an increasing number of ancient individuals and extinct species, as well as to epigenomic characterization. Such advances have enabled the sequencing of specimens of up to 1 million years old, which, owing to their extensive DNA damage and contamination, were previously not amenable to genetic analyses. In this Review, we discuss these varied technical challenges and solutions for sequencing ancient genomes and epigenomes.
Topics: Animals; DNA; Epigenomics; Genomics; High-Throughput Nucleotide Sequencing; History, Ancient; Humans
PubMed: 26055157
DOI: 10.1038/nrg3935 -
FEMS Microbiology Letters Aug 2018Industrial biotechnology develops and applies microorganisms for the production of bioproducts and enzymes with applications ranging from food and feed ingredients and... (Review)
Review
Industrial biotechnology develops and applies microorganisms for the production of bioproducts and enzymes with applications ranging from food and feed ingredients and processing to bio-based chemicals, biofuels and pharmaceutical products. Next generation DNA sequencing technologies play an increasingly important role in improving and accelerating microbial strain development for existing and novel bio-products via screening, gene and pathway discovery, metabolic engineering and additional optimization and understanding of large-scale manufacturing. In this mini-review, we describe novel DNA sequencing and analysis technologies with a focus on applications to industrial strain development, enzyme discovery and microbial community analysis.
Topics: Bacteria; High-Throughput Nucleotide Sequencing; Industrial Microbiology
PubMed: 30010862
DOI: 10.1093/femsle/fny103 -
Methods in Molecular Biology (Clifton,... 2019As in any endeavor, the strategy applied to a genome project can mean the difference between success and failure. This is especially important when limited funding often... (Review)
Review
As in any endeavor, the strategy applied to a genome project can mean the difference between success and failure. This is especially important when limited funding often means only a single approach may be tried at a given time. Although the advance of all areas of genomics and transcriptomics in recent years has led to an embarrassment of riches, methods in the field have not quite reached the turn-key production status for all species, despite being closer than ever. Here I contrast and compare the technical approaches to genome projects in the hope of enabling strategy choices with higher probabilities of success. Finally, I review the new technologies that are not yet widely distributed which are revolutionizing the future of genomics.
Topics: Animals; Arthropods; Genome, Insect; Genomics; High-Throughput Nucleotide Sequencing; Sequence Analysis, DNA
PubMed: 30414106
DOI: 10.1007/978-1-4939-8775-7_1 -
Methods in Enzymology 2019Aptamers are small, functional nucleic acids that bind a variety of targets, often with high specificity and affinity. Genomic aptamers constitute the ligand-binding... (Review)
Review
Aptamers are small, functional nucleic acids that bind a variety of targets, often with high specificity and affinity. Genomic aptamers constitute the ligand-binding domains of riboswitches, whereas synthetic aptamers find applications as diagnostic and therapeutic tools, and as ligand-binding domains of regulatory RNAs in synthetic biology. Discovery and characterization of aptamers has been limited by a lack of high-throughput approaches that uncover the target-binding domains and the biochemical properties of individual sequences. With the advent of high-throughput sequencing, large-scale analysis of in vitro selected populations of aptamers (and catalytic nucleic acids, such as ribozymes and DNAzmes) became possible. In recent years the development of new experimental approaches and software tools has led to significant streamlining of the selection-pool analysis. This article provides an overview of post-selection data analysis and describes high-throughput methods that facilitate rapid discovery and biochemical characterization of aptamers.
Topics: Animals; Aptamers, Nucleotide; High-Throughput Nucleotide Sequencing; Humans; Nucleic Acids; SELEX Aptamer Technique; Software
PubMed: 31128787
DOI: 10.1016/bs.mie.2019.02.009 -
The FEBS Journal Apr 2019During the past decade, the number of novel technologies to interrogate biological systems at the single-cell level has skyrocketed. Numerous approaches for measuring... (Review)
Review
During the past decade, the number of novel technologies to interrogate biological systems at the single-cell level has skyrocketed. Numerous approaches for measuring the proteome, genome, transcriptome and epigenome at the single-cell level have been pioneered, using a variety of technologies. All these methods have one thing in common: they generate large and high-dimensional datasets that require advanced computational modelling tools to highlight and interpret interesting patterns in these data, potentially leading to novel biological insights and hypotheses. In this work, we provide an overview of the computational approaches used to interpret various types of single-cell data in an automated and unbiased way.
Topics: Cluster Analysis; Computational Biology; Flow Cytometry; High-Throughput Nucleotide Sequencing; Quality Control; Single-Cell Analysis; Workflow
PubMed: 30058136
DOI: 10.1111/febs.14613 -
Methods in Molecular Biology (Clifton,... 2019Noncoding RNAs (ncRNAs) have received much attention due to their central role in gene expression and translational regulation as well as due to their involvement in... (Review)
Review
Noncoding RNAs (ncRNAs) have received much attention due to their central role in gene expression and translational regulation as well as due to their involvement in several biological processes and disease development. Small noncoding RNAs (sncRNAs), such as microRNAs and piwiRNAs, have been thoroughly investigated and functionally characterized. Long noncoding RNAs (lncRNAs), known to play an important role in chromatin-interacting transcription regulation, posttranscriptional regulation, cell-to-cell signaling, and protein regulation, are also being investigated to further elucidate their functional roles.Next-generation sequencing (NGS) technologies have greatly aided in characterizing the ncRNAome. Moreover, the coupling of NGS technology together with bioinformatics tools has been essential to the genome-wide detection of RNA modifications in ncRNAs. RNA editing, a common human co-transcriptional and posttranscriptional modification, is a dynamic biological phenomenon able to alter the sequence and the structure of primary transcripts (both coding and noncoding RNAs) during the maturation process, consequently influencing the biogenesis, as well as the function, of ncRNAs. In particular, the dysregulation of the RNA editing machineries have been associated with the onset of human diseases.In this chapter we discuss the potential functions of ncRNA editing and describe the knowledge base and bioinformatics resources available to investigate such phenomenon.
Topics: Animals; Computational Biology; Gene Expression Regulation; High-Throughput Nucleotide Sequencing; Humans; Mice; RNA Editing; RNA, Untranslated; Sequence Analysis, RNA
PubMed: 30635893
DOI: 10.1007/978-1-4939-8982-9_6 -
International Journal of Legal Medicine Jan 2022Since methylation analysis has become an important tool in forensic genetics, the reliability and credibility of the method must be ensured. After a successful...
Since methylation analysis has become an important tool in forensic genetics, the reliability and credibility of the method must be ensured. After a successful validation and establishment of several pyrosequencing assays using a PyroMark® Q48 Autoprep instrument (Qiagen, Hilden, Germany), we decided to expand the method further purchasing a second instrument. But after initializing this second instrument side by side with the first, the majority of analyses failed (97 samples of 133 samples (73%)). The number of error messages increased rapidly and the average RFU values decreased. After purchasing two anti-vibration weighing tables for the PyroMark® instruments and repeating the analyses under the same conditions and with identical samples the results improved considerably, 115 samples of 130 samples (88%) showed successful and reproducible results. These findings demonstrate the impact of vibrations and percussions on PyroMark® Q48 Autoprep performance and the reliability of methylation analyses.
Topics: CpG Islands; DNA Methylation; High-Throughput Nucleotide Sequencing; Humans; Reproducibility of Results; Sequence Analysis, DNA; Vibration
PubMed: 34637025
DOI: 10.1007/s00414-021-02716-7 -
Annual Review of Genomics and Human... Aug 2016The term next-generation sequencing is almost a decade old, but it remains the colloquial way to describe highly parallel or high-output sequencing methods that produce... (Review)
Review
The term next-generation sequencing is almost a decade old, but it remains the colloquial way to describe highly parallel or high-output sequencing methods that produce data at or beyond the genome scale. Since the introduction of these technologies, the number of applications and methods that leverage the power of genome-scale sequencing has increased at an exponential pace. This review highlights recent concepts, technologies, and methods from next-generation sequencing to illustrate the breadth and depth of the applications and research areas that are driving progress in genomics.
Topics: Genome, Human; Genomics; High-Throughput Nucleotide Sequencing; Humans; Sequence Analysis, DNA
PubMed: 27362342
DOI: 10.1146/annurev-genom-083115-022413