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Molecular Biology Reports Aug 2014Next generation sequencing (NGS) is perhaps one of the most exciting advances in the field of life sciences and biomedical research in the last decade. With the... (Review)
Review
Next generation sequencing (NGS) is perhaps one of the most exciting advances in the field of life sciences and biomedical research in the last decade. With the availability of massive parallel sequencing, human DNA blueprint can be decoded to explore the hidden information with reduced time and cost. This technology has been used to understand the genetic aspects of various diseases including cardiomyopathies. Mutations for different cardiomyopathies have been identified and cataloging mutations on phenotypic basis are underway and are expected to lead to new discoveries that may translate to novel diagnostic, prognostic and therapeutic targets. With ease in handling NGS, cost effectiveness and fast data output, NGS is now considered as a diagnostic tool for cardiomyopathy by providing targeted gene sequencing. In addition to the number of genetic variants that are identified in cardiomyopathies, there is a need of quicker and easy way to screen multiple genes associated with the disease. In this review, an attempt has been made to explain the NGS technology, methods and applications in cardiomyopathies and their perspective in clinical practice and challenges which are to be addressed.
Topics: Cardiomyopathies; Genomics; High-Throughput Nucleotide Sequencing; Humans; Phenotype; Precision Medicine
PubMed: 24908287
DOI: 10.1007/s11033-014-3418-9 -
The Veterinary Clinics of North... Mar 2023High-throughput sequencing (HTS) techniques have revolutionized the way we understand microbial communities in both research and clinical settings and are bringing new... (Review)
Review
High-throughput sequencing (HTS) techniques have revolutionized the way we understand microbial communities in both research and clinical settings and are bringing new insights into what constitutes a healthy ocular surface (and a diseased one). As more diagnostic laboratories incorporate HTS into their technique repertoire, practitioners can expect this technology to become increasingly accessible for clinical practice, potentially becoming the new standard. However, particularly regarding ophthalmic microbiota, considerable research remains to render HTS accessible and applicable.
Topics: Animals; Bacteria; Microbiota; High-Throughput Nucleotide Sequencing
PubMed: 36813387
DOI: 10.1016/j.cvsm.2022.10.004 -
Cell Sep 2013Genomics is a relatively new scientific discipline, having DNA sequencing as its core technology. As technology has improved the cost and scale of genome... (Review)
Review
Genomics is a relatively new scientific discipline, having DNA sequencing as its core technology. As technology has improved the cost and scale of genome characterization over sequencing's 40-year history, the scope of inquiry has commensurately broadened. Massively parallel sequencing has proven revolutionary, shifting the paradigm of genomics to address biological questions at a genome-wide scale. Sequencing now empowers clinical diagnostics and other aspects of medical care, including disease risk, therapeutic identification, and prenatal testing. This Review explores the current state of genomics in the massively parallel sequencing era.
Topics: Animals; Disease; Genetic Variation; Genomics; High-Throughput Nucleotide Sequencing; History, 20th Century; History, 21st Century; Humans; Mutation; Sequence Analysis, DNA
PubMed: 24074859
DOI: 10.1016/j.cell.2013.09.006 -
International Journal of Legal Medicine Jul 2020In the last decade, next-generation sequencing (NGS) technology, alternatively massive parallel sequencing (MPS), was applied to all fields of biological research. Its... (Review)
Review
In the last decade, next-generation sequencing (NGS) technology, alternatively massive parallel sequencing (MPS), was applied to all fields of biological research. Its introduction to the field of forensics was slower, mainly due to lack of accredited sequencers, kits, and relatively higher sequencing error rates as compared with standardized Sanger sequencing. Currently, a majority of the problematic issues have been solved, which is proven by the body of reports in the literature. Here, we discuss the utility of NGS sequencing in forensics, emphasizing the advantages, issues, the technical aspects of the experiments, commercial solutions, and the potentially interesting applications of MPS.
Topics: Forensic Sciences; High-Throughput Nucleotide Sequencing; Sequence Analysis, DNA
PubMed: 32451905
DOI: 10.1007/s00414-020-02294-0 -
PloS One 2013Next generation sequencing of ribosomal DNA is increasingly used to assess the diversity and structure of microbial communities. Here we test the ability of 454...
Next generation sequencing of ribosomal DNA is increasingly used to assess the diversity and structure of microbial communities. Here we test the ability of 454 pyrosequencing to detect the number of species present, and assess the relative abundance in terms of cell numbers and biomass of protists in the phylum Haptophyta. We used a mock community consisting of equal number of cells of 11 haptophyte species and compared targeting DNA and RNA/cDNA, and two different V4 SSU rDNA haptophyte-biased primer pairs. Further, we tested four different bioinformatic filtering methods to reduce errors in the resulting sequence dataset. With sequencing depth of 11000-20000 reads and targeting cDNA with Haptophyta specific primers Hap454 we detected all 11 species. A rarefaction analysis of expected number of species recovered as a function of sampling depth suggested that minimum 1400 reads were required here to recover all species in the mock community. Relative read abundance did not correlate to relative cell numbers. Although the species represented with the largest biomass was also proportionally most abundant among the reads, there was generally a weak correlation between proportional read abundance and proportional biomass of the different species, both with DNA and cDNA as template. The 454 sequencing generated considerable spurious diversity, and more with cDNA than DNA as template. With initial filtering based only on match with barcode and primer we observed 100-fold more operational taxonomic units (OTUs) at 99% similarity than the number of species present in the mock community. Filtering based on quality scores, or denoising with PyroNoise resulted in ten times more OTU99% than the number of species. Denoising with AmpliconNoise reduced the number of OTU99% to match the number of species present in the mock community. Based on our analyses, we propose a strategy to more accurately depict haptophyte diversity using 454 pyrosequencing.
Topics: Biodiversity; DNA, Complementary; DNA, Ribosomal; Haptophyta; High-Throughput Nucleotide Sequencing; Metagenome; Phylogeny
PubMed: 24069303
DOI: 10.1371/journal.pone.0074371 -
Human Immunology Dec 2015
Topics: HLA Antigens; High-Throughput Nucleotide Sequencing; Histocompatibility Testing; Humans
PubMed: 26455298
DOI: 10.1016/j.humimm.2015.10.003 -
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 -
European Journal of Medical Genetics Dec 2023Rare diseases encompass a diverse group of genetic disorders that affect a small proportion of the population. Identifying the underlying genetic causes of these... (Review)
Review
Rare diseases encompass a diverse group of genetic disorders that affect a small proportion of the population. Identifying the underlying genetic causes of these conditions presents significant challenges due to their genetic heterogeneity and complexity. Conventional short-read sequencing (SRS) techniques have been widely used in diagnosing and investigating of rare diseases, with limitations due to the nature of short-read lengths. In recent years, long read sequencing (LRS) technologies have emerged as a valuable tool in overcoming these limitations. This minireview provides a concise overview of the applications of LRS in rare disease research and diagnosis, including the identification of disease-causing tandem repeat expansions, structural variations, and comprehensive analysis of pathogenic variants with LRS.
Topics: Humans; Rare Diseases; High-Throughput Nucleotide Sequencing; Sequence Analysis, DNA
PubMed: 38832911
DOI: 10.1016/j.ejmg.2023.104871 -
Expert Review of Molecular Diagnostics May 2017The advent of next-generation sequencing (NGS) has introduced an exciting new era in biomedical research. NGS forms the foundation of current genetic testing approaches,... (Review)
Review
The advent of next-generation sequencing (NGS) has introduced an exciting new era in biomedical research. NGS forms the foundation of current genetic testing approaches, including targeted gene panel testing, as well as more comprehensive whole-exome and whole-genome sequencing. Together, these approaches promise to provide critical insights into the understanding of health and disease. However, with NGS testing come many ethical questions and concerns, particularly when testing involves children. These concerns are especially relevant for children with cancer, where the testing of tumor and germline tissues is increasingly being incorporated into clinical care. Areas covered: In this manuscript, we explore the key ethical considerations related to conducting germline NGS testing in pediatric oncology, focusing on the four main principles of beneficence, non-maleficence, autonomy and justice. Expert commentary: The ethical issues surrounding germline NGS testing are complex and result in part from our limited understanding of the medical relevance of many of the results obtained and poor knowledge of the impacts of testing, both beneficial and detrimental, on patients and their families. In this article we discuss the risks and benefits of germline NGS testing and the arguments for and against such testing in children with cancer.
Topics: Adolescent; Child; Child, Preschool; Ethics; Female; Germ-Line Mutation; High-Throughput Nucleotide Sequencing; Humans; Infant; Male; Neoplasms
PubMed: 28399664
DOI: 10.1080/14737159.2017.1316665 -
British Journal of Cancer Feb 2017In the context of solid tumours, the evolution of cancer therapies to more targeted and nuanced approaches has led to the impetus for personalised medicine. The targets... (Review)
Review
In the context of solid tumours, the evolution of cancer therapies to more targeted and nuanced approaches has led to the impetus for personalised medicine. The targets for these therapies are largely based on the driving genetic mutations of the tumours. To track these multiple driving mutations the use of next generation sequencing (NGS) coupled with a morphomolecular approach to tumours, has the potential to deliver on the promises of personalised medicine. A review of NGS and its application in a universal healthcare (UHC) setting is undertaken as the technology has a wide appeal and utility in diagnostic, clinical trial and research paradigms. Furthermore, we suggest that these can be accommodated with a unified integromic approach. Challenges remain in bringing NGS to routine clinical use and these include validation, handling of the large amounts of information flow and production of a clinically useful report. These challenges are particularly acute in the setting of UHC where tests are not reimbursed and there are finite resources available. It is our opinion that the challenges faced in applying NGS in a UHC setting are surmountable and we outline our approach for its routine application in diagnostic, clinical trial and research paradigms.
Topics: Clinical Trials as Topic; High-Throughput Nucleotide Sequencing; Humans; Insurance, Health; Mutation; Neoplasms; Precision Medicine; Sequence Analysis, DNA
PubMed: 28103613
DOI: 10.1038/bjc.2016.452