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PLoS Computational Biology Mar 2020Life scientists are increasingly turning to high-throughput sequencing technologies in their research programs, owing to the enormous potential of these methods. In a...
Life scientists are increasingly turning to high-throughput sequencing technologies in their research programs, owing to the enormous potential of these methods. In a parallel manner, the number of core facilities that provide bioinformatics support are also increasing. Notably, the generation of complex large datasets has necessitated the development of bioinformatics support core facilities that aid laboratory scientists with cost-effective and efficient data management, analysis, and interpretation. In this article, we address the challenges-related to communication, good laboratory practice, and data handling-that may be encountered in core support facilities when providing bioinformatics support, drawing on our own experiences working as support bioinformaticians on multidisciplinary research projects. Most importantly, the article proposes a list of guidelines that outline how these challenges can be preemptively avoided and effectively managed to increase the value of outputs to the end user, covering the entire research project lifecycle, including experimental design, data analysis, and management (i.e., sharing and storage). In addition, we highlight the importance of clear and transparent communication, comprehensive preparation, appropriate handling of samples and data using monitoring systems, and the employment of appropriate tools and standard operating procedures to provide effective bioinformatics support.
Topics: Biomedical Research; Communication; Computational Biology; High-Throughput Nucleotide Sequencing; Humans; Research Design
PubMed: 32214318
DOI: 10.1371/journal.pcbi.1007531 -
Methods (San Diego, Calif.) May 2019A large number of catalytic RNAs, or ribozymes, have been identified in the genomes of various organisms and viruses. Ribozymes are involved in biological processes such... (Review)
Review
A large number of catalytic RNAs, or ribozymes, have been identified in the genomes of various organisms and viruses. Ribozymes are involved in biological processes such as regulation of gene expression and viral replication, but biological roles of many ribozymes still remain unknown. Ribozymes have also inspired researchers to engineer synthetic ribozymes that function as sensors or gene switches. To gain deeper understanding of the sequence-function relationship of ribozymes and to efficiently engineer synthetic ribozymes, a large number of ribozyme variants need to be examined which was limited to hundreds of sequences by Sanger sequencing. The advent of high-throughput sequencing technologies, however, has allowed us to sequence millions of ribozyme sequences at low cost. This review focuses on the recent applications of high-throughput sequencing to both characterize and engineer ribozymes, to highlight how the large-scale sequence data can advance ribozyme research and engineering.
Topics: Animals; Genetic Engineering; High-Throughput Nucleotide Sequencing; Humans; Mutation; RNA, Catalytic
PubMed: 30738128
DOI: 10.1016/j.ymeth.2019.02.001 -
Genomics Dec 2014We chronicle and dissect the history of the field of Experimental Microbial Evolution, beginning with work by Monod. Early research was largely carried out by... (Review)
Review
We chronicle and dissect the history of the field of Experimental Microbial Evolution, beginning with work by Monod. Early research was largely carried out by microbiologists and biochemists, who used experimental evolutionary change as a tool to understand structure-function relationships. These studies attracted the interest of evolutionary biologists who recognized the power of the approach to address issues such as the tempo of adaptive change, the costs and benefits of sex, parallelism, and the role which contingency plays in the evolutionary process. In the 1980s and 1990s, an ever-expanding body of microbial, physiological and biochemical data, together with new technologies for manipulating microbial genomes, allowed such questions to be addressed in ever-increasing detail. Since then, technological advances leading to low-cost, high-throughput DNA sequencing have made it possible for these and other fundamental questions in evolutionary biology to be addressed at the molecular level.
Topics: Databases, Genetic; Directed Molecular Evolution; Evolution, Molecular; Genome, Microbial; High-Throughput Nucleotide Sequencing; History, 20th Century; History, 21st Century
PubMed: 25315137
DOI: 10.1016/j.ygeno.2014.10.004 -
Biosensors & Bioelectronics Feb 2019To explore genome mutation meaningfully, it is in urgent need to develop an automated and inexpensive platform for DNA mutation analysis. Digital microfluidics is a...
To explore genome mutation meaningfully, it is in urgent need to develop an automated and inexpensive platform for DNA mutation analysis. Digital microfluidics is a powerful platform for a broad range of applications due to the advantages of high automatization and low reagent consumption. Pyrosequencing enables DNA sequencing based on non-electrophoresis bioluminescence, which is suitable for rapid and sensitive analysis of short sequences. Herein, we describe a palmtop sequencing platform for automatic, real-time and portable analysis of DNA mutations, which is based on the pyrosequencing principle and implemented by digital microfluidics. The portable system can sequence a DNA template with up to 53 bp with 100% accuracy within 2 h. Mutation in the KRAS gene can be detected within 30 min with a LOD as low as 5% mutant level. Portable and accurate gender identification was further demonstrated by sequencing a short amelogenin fragment. With the advantages of portability, ease of use, high accuracy, and low cost, the palmtop sequencing platform shows great potential for portable genetic testing in a variety of circumstances.
Topics: Biosensing Techniques; DNA; DNA Mutational Analysis; High-Throughput Nucleotide Sequencing; Humans; Luminescent Measurements; Microfluidics; Mutation
PubMed: 30497021
DOI: 10.1016/j.bios.2018.09.092 -
Protein & Cell Jun 2010As one of the key technologies in biomedical research, DNA sequencing has not only improved its productivity with an exponential growth rate but also been applied to new... (Review)
Review
As one of the key technologies in biomedical research, DNA sequencing has not only improved its productivity with an exponential growth rate but also been applied to new areas of application over the past few years. This is largely due to the advent of newer generations of sequencing platforms, offering ever-faster and cheaper ways to analyze sequences. In our previous review, we looked into technical characteristics of the next-generation sequencers and provided prospective insights into their future development. In this article, we present a brief overview of the advantages and shortcomings of key commercially available platforms with a focus on their suitability for a broad range of applications.
Topics: Animals; DNA-Binding Proteins; Epigenomics; Gene Expression Profiling; Genomics; High-Throughput Nucleotide Sequencing; Humans; Nanostructures; RNA, Small Untranslated
PubMed: 21204006
DOI: 10.1007/s13238-010-0065-3 -
Anti-cancer Drugs Jan 2017Mutation detection in tumors started with classical cytogenetics as the method of choice more than 50 years ago. Karyotyping proved to be sensitive enough to detect... (Review)
Review
Mutation detection in tumors started with classical cytogenetics as the method of choice more than 50 years ago. Karyotyping proved to be sensitive enough to detect deletions or duplications of large chromosome segments, and translocations. Over time, new techniques were developed to detect mutations that are much smaller in scope. The availability of Sanger sequencing and the invention of the PCR improved the discriminatory power of mutation detection to just one base change in the genomic DNA sequence. Techniques derived from PCR (allele-specific PCR, qPCR) and improved or modified sequencing methods (capillary electrophoresis, pyrosequencing) considerably increased the efficiency and sample throughput of mutation detection assays. With the advent of massive parallel sequencing [also called next-generation sequencing (NGS)] in the past decade, a major shift to even higher sample throughput and a significant decrease in cost per sequenced base occurred. The application of the new technology provided a whole slew of novel biomarkers and potential therapy targets to improve diagnosis and treatment. It even led to changes in cancer classification as new information on the mutation profile of tumors became available that characterizes some disease entities better than morphology. NGS, which usually interrogates multiple genes at once and is a prime example of a multianalyte assay, started to replace older single analyte assays focused on analysis of one target, one gene. However, the transition to these extremely complex NGS-based assays is associated with multiple challenges. There are issues with adequate tissue source of nucleic acids, sequencing library preparation, bioinformatics, government regulations and oversight, reimbursement, and electronic medical records that need to be resolved to successfully implement the new technology in a clinical laboratory.
Topics: Alleles; DNA Mutational Analysis; High-Throughput Nucleotide Sequencing; Humans; Mutation; Neoplasms; Polymerase Chain Reaction
PubMed: 27575332
DOI: 10.1097/CAD.0000000000000427 -
Human Heredity 2011
Topics: Genetic Linkage; High-Throughput Nucleotide Sequencing; Humans
PubMed: 22189464
DOI: 10.1159/000334421 -
JAMA Neurology Jun 2013The availability of high-throughput genome sequencing technologies is expected to revolutionize our understanding of not only hereditary neurological diseases but also... (Review)
Review
The availability of high-throughput genome sequencing technologies is expected to revolutionize our understanding of not only hereditary neurological diseases but also sporadic neurological diseases. The molecular bases of sporadic diseases, particularly those of sporadic neurodegenerative diseases, largely remain unknown. As potential molecular bases, various mechanisms can be considered, which include those underlying apparently sporadic neurological diseases with low-penetrant mutations in the gene for hereditary diseases, sporadic diseases with de novo mutations, and sporadic diseases with variations in disease-susceptible genes. With unprecedentedly robust power, high-throughput genome sequencing technologies will enable us to explore all of these possibilities. These new technologies will soon be applied in clinical practice. It will be a new era of datacentric clinical practice.
Topics: Animals; Genetic Predisposition to Disease; Genomics; High-Throughput Nucleotide Sequencing; Humans; Mutation; Nervous System Diseases
PubMed: 23571861
DOI: 10.1001/jamaneurol.2013.734 -
Methods in Molecular Biology (Clifton,... 2018With the rapid evolution of genomics technologies over the past decade, whole genome sequencing (WGS) has become an increasingly accessible tool in biomedical research.... (Review)
Review
With the rapid evolution of genomics technologies over the past decade, whole genome sequencing (WGS) has become an increasingly accessible tool in biomedical research. WGS applications include analysis of genomic DNA from single individuals, multiple related family members, and tumor/normal samples from the same patient in the context of oncology. A number of different modalities are available for performing WGS; this chapter focuses on wet lab library construction procedures for complex short insert WGS libraries using the KAPA Hyper Prep Kit (Kapa Biosystems), and includes a discussion of appropriate quality control measures for sequencing on the Illumina HiSeq2000 platform. Additional modifications to the protocol for long insert WGS library construction, to assess structural alterations and copy number changes, are also described.
Topics: Animals; Gene Library; Genome, Human; High-Throughput Nucleotide Sequencing; Humans
PubMed: 29423797
DOI: 10.1007/978-1-4939-7471-9_8 -
Drug Discovery Today Apr 2017The progress of next-generation sequencing has a major impact on medical and genomic research. This high-throughput technology can now produce billions of short DNA or... (Review)
Review
The progress of next-generation sequencing has a major impact on medical and genomic research. This high-throughput technology can now produce billions of short DNA or RNA fragments in excess of a few terabytes of data in a single run. This leads to massive datasets used by a wide range of applications including personalized cancer treatment and precision medicine. In addition to the hugely increased throughput, the cost of using high-throughput technologies has been dramatically decreasing. A low sequencing cost of around US$1000 per genome has now rendered large population-scale projects feasible. However, to make effective use of the produced data, the design of big data algorithms and their efficient implementation on modern high performance computing systems is required.
Topics: Algorithms; Computing Methodologies; Databases, Genetic; Genome; Genomics; High-Throughput Nucleotide Sequencing; Humans; Sequence Analysis, DNA
PubMed: 28163155
DOI: 10.1016/j.drudis.2017.01.014