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Expert Review of Molecular Diagnostics Sep 2016The last two decades have witnessed revolutionary changes in clinical diagnostics, fueled by the Human Genome Project and advances in high throughput, Next Generation... (Review)
Review
INTRODUCTION
The last two decades have witnessed revolutionary changes in clinical diagnostics, fueled by the Human Genome Project and advances in high throughput, Next Generation Sequencing (NGS). We review the current state of sequencing-based pediatric diagnostics, associated challenges, and future prospects.
AREAS COVERED
We present an overview of genetic disease in children, review the technical aspects of Next Generation Sequencing and the strategies to make molecular diagnoses for children with genetic disease. We discuss the challenges of genomic sequencing including incomplete current knowledge of variants, lack of data about certain genomic regions, mosaicism, and the presence of regions with high homology. Expert commentary: NGS has been a transformative technology and the gap between the research and clinical communities has never been so narrow. Therapeutic interventions are emerging based on genomic findings and the applications of NGS are progressing to prenatal genetics, epigenomics and transcriptomics.
Topics: Adolescent; Child; Child, Preschool; Female; Genetic Diseases, Inborn; Genome, Human; Genomics; High-Throughput Nucleotide Sequencing; Humans; Infant; Infant, Newborn; Male; Mosaicism
PubMed: 27388938
DOI: 10.1080/14737159.2016.1209411 -
Omics : a Journal of Integrative Biology Feb 2014High-throughput sequencing technologies, also known as next-generation sequencing (NGS) technologies, have revolutionized the way that genomic research is advancing. In... (Review)
Review
High-throughput sequencing technologies, also known as next-generation sequencing (NGS) technologies, have revolutionized the way that genomic research is advancing. In addition to the static genome, these state-of-art technologies have been recently exploited to analyze the dynamic transcriptome, and the resulting technology is termed RNA sequencing (RNA-seq). RNA-seq is free from many limitations of other transcriptomic approaches, such as microarray and tag-based sequencing method. Although RNA-seq has only been available for a short time, studies using this method have completely changed our perspective of the breadth and depth of eukaryotic transcriptomes. In terms of the transcriptomics of teleost fishes, both model and non-model species have benefited from the RNA-seq approach and have undergone tremendous advances in the past several years. RNA-seq has helped not only in mapping and annotating fish transcriptome but also in our understanding of many biological processes in fish, such as development, adaptive evolution, host immune response, and stress response. In this review, we first provide an overview of each step of RNA-seq from library construction to the bioinformatic analysis of the data. We then summarize and discuss the recent biological insights obtained from the RNA-seq studies in a variety of fish species.
Topics: Animals; Fishes; Gene Expression Profiling; Gene Library; Genome; High-Throughput Nucleotide Sequencing; Molecular Sequence Annotation; Polymorphism, Single Nucleotide; RNA; Sequence Analysis, RNA; Transcriptome
PubMed: 24380445
DOI: 10.1089/omi.2013.0110 -
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 -
Genetics in Medicine : Official Journal... Sep 2015
Topics: Genomics; High-Throughput Nucleotide Sequencing; Humans; Molecular Diagnostic Techniques
PubMed: 26331192
DOI: 10.1038/gim.2015.114 -
Clinical Microbiology and Infection :... Jan 2013Recent advances in nucleic acid sequencing technologies, referred to as 'next-generation' sequencing (NGS), have produced a true revolution and opened new perspectives... (Review)
Review
Recent advances in nucleic acid sequencing technologies, referred to as 'next-generation' sequencing (NGS), have produced a true revolution and opened new perspectives for research and diagnostic applications, owing to the high speed and throughput of data generation. So far, NGS has been applied to metagenomics-based strategies for the discovery of novel viruses and the characterization of viral communities. Additional applications include whole viral genome sequencing, detection of viral genome variability, and the study of viral dynamics. These applications are particularly suitable for viruses such as human immunodeficiency virus, hepatitis B virus, and hepatitis C virus, whose error-prone replication machinery, combined with the high replication rate, results, in each infected individual, in the formation of many genetically related viral variants referred to as quasi-species. The viral quasi-species, in turn, represents the substrate for the selective pressure exerted by the immune system or by antiviral drugs. With traditional approaches, it is difficult to detect and quantify minority genomes present in viral quasi-species that, in fact, may have biological and clinical relevance. NGS provides, for each patient, a dataset of clonal sequences that is some order of magnitude higher than those obtained with conventional approaches. Hence, NGS is an extremely powerful tool with which to investigate previously inaccessible aspects of viral dynamics, such as the contribution of different viral reservoirs to replicating virus in the course of the natural history of the infection, co-receptor usage in minority viral populations harboured by different cell lineages, the dynamics of development of drug resistance, and the re-emergence of hidden genomes after treatment interruptions. The diagnostic application of NGS is just around the corner.
Topics: High-Throughput Nucleotide Sequencing; Humans; Metagenome; Molecular Diagnostic Techniques; Virology
PubMed: 23279287
DOI: 10.1111/1469-0691.12056 -
Trends in Molecular Medicine Jun 2017Single-cell RNA sequencing (scRNA-seq) is an exciting new technology allowing the analysis of transcriptomes from individual cells, and is ideally suited to address the... (Review)
Review
Single-cell RNA sequencing (scRNA-seq) is an exciting new technology allowing the analysis of transcriptomes from individual cells, and is ideally suited to address the inherent complexity and dynamics of the central nervous system. scRNA-seq has already been applied to the study of molecular taxonomy of the brain. These works have paved the way to expanding our understanding of the nervous system and provide insights into cellular susceptibilities and molecular mechanisms in neurological and neurodegenerative diseases. We discuss recent progress and challenges in applying this technology to advance our understanding of the brain. We advocate the application of scRNA-seq in the discovery of targets and biomarkers as a new approach in developing novel therapeutics for the treatment of neurodegenerative diseases.
Topics: Animals; Biomarkers; High-Throughput Nucleotide Sequencing; Humans; Neurodegenerative Diseases; Sequence Analysis, RNA
PubMed: 28501348
DOI: 10.1016/j.molmed.2017.04.006 -
Frontiers in Cellular and Infection... 2022We hypothesized that targeted NGS sequencing might have an advantage over Sanger sequencing, especially in polymicrobial infections. The study included 55 specimens from...
We hypothesized that targeted NGS sequencing might have an advantage over Sanger sequencing, especially in polymicrobial infections. The study included 55 specimens from 51 patients. We compared targeted NGS to Sanger sequencing in clinical samples submitted for Sanger sequencing. The overall concordance rate was 58% (32/55) for NGS vs. Sanger. NGS identified 9 polymicrobial and 2 monomicrobial infections among 19 Sanger-negative samples and 8 polymicrobial infections in 11 samples where a 16S gene was identified by gel electrophoresis, but could not be mapped to an identified pathogen by Sanger. We estimated that NGS could have contributed to patient management in 6/18 evaluated patients and thus has an advantage over Sanger sequencing in certain polymicrobial infections.
Topics: Humans; High-Throughput Nucleotide Sequencing; Coinfection; Mutation
PubMed: 36339334
DOI: 10.3389/fcimb.2022.955481 -
Blood Nov 2013The development of novel technologies for high-throughput DNA sequencing is having a major impact on our ability to measure and define normal and pathologic variation in... (Review)
Review
The development of novel technologies for high-throughput DNA sequencing is having a major impact on our ability to measure and define normal and pathologic variation in humans. This review discusses advances in DNA sequencing that have been applied to benign hematologic disorders, including those affecting the red blood cell, the neutrophil, and other white blood cell lineages. Relevant examples of how these approaches have been used for disease diagnosis, gene discovery, and studying complex traits are provided. High-throughput DNA sequencing technology holds significant promise for impacting clinical care. This includes development of improved disease detection and diagnosis, better understanding of disease progression and stratification of risk of disease-specific complications, and development of improved therapeutic strategies, particularly patient-specific pharmacogenomics-based therapy, with monitoring of therapy by genomic biomarkers.
Topics: Exome; Genetic Association Studies; Genome-Wide Association Study; Hematologic Diseases; Hematology; High-Throughput Nucleotide Sequencing; Humans
PubMed: 24021670
DOI: 10.1182/blood-2013-07-460337 -
The Journal of Molecular Diagnostics :... Feb 2020Sample tracking and identity are essential when processing multiple samples in parallel. Sequencing applications often involve high sample numbers, and the data are...
Sample tracking and identity are essential when processing multiple samples in parallel. Sequencing applications often involve high sample numbers, and the data are frequently used in a clinical setting. As such, a simple and accurate intrinsic sample tracking process through a sequencing pipeline is essential. Various solutions have been implemented to verify sample identity, including variant detection at the start and end of the pipeline using arrays or genotyping, bioinformatic comparisons, and optical barcoding of samples. None of these approaches are optimal. To establish a more effective approach using genetic barcoding, we developed a panel of unique DNA sequences cloned into a common vector. A unique DNA sequence is added to the sample when it is first received and can be detected by PCR and/or sequencing at any stage of the process. The control sequences are approximately 200 bases long with low identity to any sequence in the National Center for Biotechnology Information nonredundant database (<30 bases) and contain no long homopolymer (>7) stretches. When a spiked next-generation sequencing library is sequenced, sequence reads derived from this control sequence are generated along with the standard sequencing run and are used to confirm sample identity and determine cross-contamination levels. This approach is used in our targeted clinical diagnostic whole-genome and RNA-sequencing pipelines and is an inexpensive, flexible, and platform-agnostic solution.
Topics: Computational Biology; DNA Contamination; Databases, Nucleic Acid; Gene Library; High-Throughput Nucleotide Sequencing; Humans; Reference Standards; Reproducibility of Results; Sequence Analysis, DNA
PubMed: 31837431
DOI: 10.1016/j.jmoldx.2019.10.011 -
Annual Review of Biomedical Data Science Aug 2023The human microbiome is complex, variable from person to person, essential for health, and related to both the risk for disease and the efficacy of our treatments. There... (Review)
Review
The human microbiome is complex, variable from person to person, essential for health, and related to both the risk for disease and the efficacy of our treatments. There are robust techniques to describe microbiota with high-throughput sequencing, and there are hundreds of thousands of already-sequenced specimens in public archives. The promise remains to use the microbiome both as a prognostic factor and as a target for precision medicine. However, when used as an input in biomedical data science modeling, the microbiome presents unique challenges. Here, we review the most common techniques used to describe microbial communities, explore these unique challenges, and discuss the more successful approaches for biomedical data scientists seeking to use the microbiome as an input in their studies.
Topics: Humans; Microbiota; Precision Medicine; High-Throughput Nucleotide Sequencing
PubMed: 37159872
DOI: 10.1146/annurev-biodatasci-020722-043017