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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 -
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 -
Trends in Genetics : TIG Feb 2015Science is defined in part by an honest exposition of the uncertainties that arise in measurements and propagate through calculations and inferences, so that the... (Review)
Review
Science is defined in part by an honest exposition of the uncertainties that arise in measurements and propagate through calculations and inferences, so that the reliabilities of its conclusions are made apparent. The recent rapid development of high-throughput DNA sequencing technologies has dramatically increased the number of measurements made at the biochemical and molecular level. These data come from many different DNA-sequencing technologies, each with their own platform-specific errors and biases, which vary widely. Several statistical studies have tried to measure error rates for basic determinations, but there are no general schemes to project these uncertainties so as to assess the surety of the conclusions drawn about genetic, epigenetic, and more general biological questions. We review here the state of uncertainty quantification in DNA sequencing applications, describe sources of error, and propose methods that can be used for accounting and propagating these errors and their uncertainties through subsequent calculations.
Topics: Base Sequence; High-Throughput Nucleotide Sequencing; Humans; Models, Statistical; Sequence Analysis, DNA; Uncertainty
PubMed: 25579994
DOI: 10.1016/j.tig.2014.12.002 -
Seminars in Diagnostic Pathology Sep 2019From a technical perspective, specimen identity determination in surgical pathology over the last several decades has primarily focused on analysis of repetitive DNA... (Review)
Review
From a technical perspective, specimen identity determination in surgical pathology over the last several decades has primarily focused on analysis of repetitive DNA sequences, specifically microsatellite repeats. However, a number of techniques have recently been developed that have similar, if not greater, utility in surgical pathology, most notably analysis of single nucleotide polymorphism (SNPs) and gene panels by next generation sequencing (NGS). For cases with an extremely limited sample or a degraded sample, sequence analysis of mitochondrial DNA continues to be the method of choice. From a diagnostic perspective, interest in identity determination in surgical pathology is usually centered on resolving issues of specimen provenance due to specimen labeling/accessioning deficiencies and possible contamination, but is also frequently performed in cases for which the patient's clinical course following definitive therapy is remarkably atypical, in cases of an unexpected diagnosis, and by patient request for "peace of mind". However, the methods used for identity determination have a much broader range of applications in surgical pathology beyond tissue provenance analysis. The methods can be used to provide ancillary information for cases in which the histomorphology is not definitively diagnostic, as for example for tumors that have a virtually identical microscopic appearance but for which the differential diagnosis includes synchronous/metachronous tumors versus a metastasis, and for the diagnosis of hydropic early gestations versus hydatidiform molar pregnancies. The methods also have utility in several other clinical settings, for example to rule out a donor-transmitted malignancy in a transplant recipient, to monitor bone marrow transplant engraftment, and to evaluate natural chimerism.
Topics: High-Throughput Nucleotide Sequencing; Humans; Pathology, Surgical
PubMed: 31196743
DOI: 10.1053/j.semdp.2019.06.001 -
Molekuliarnaia Biologiia 2023This review describes the application of oligonucleotides, which are mainly obtained using DNA synthesizers of a new generation (microarray DNA synthesizers), for the... (Review)
Review
This review describes the application of oligonucleotides, which are mainly obtained using DNA synthesizers of a new generation (microarray DNA synthesizers), for the enrichment of target genomic fragments. The methods of molecular hybridization, polymerase chain reaction, and CRISPR-Cas9 system for this purpose are considered. Examples of the practical use of the developed methods for research and diagnostic purposes are given.
Topics: CRISPR-Cas Systems; DNA; Polymerase Chain Reaction; Genome; High-Throughput Nucleotide Sequencing
PubMed: 37326047
DOI: No ID Found -
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 -
Methods in Molecular Biology (Clifton,... 2021The advent of deep sequencing technologies has greatly improved the study of complex eukaryotic genomes and transcriptomes, allowing the investigation of...
The advent of deep sequencing technologies has greatly improved the study of complex eukaryotic genomes and transcriptomes, allowing the investigation of posttranscriptional molecular mechanisms as alternative splicing and RNA editing at unprecedented throughput and resolution. The most prevalent type of RNA editing in higher eukaryotes is the deamination of adenosine to inosine (A-to-I) in double-stranded RNAs. Depending on the RNA type or the RNA region involved, A-to-I RNA editing contributes to the transcriptome and proteome diversity.Hereafter, we present an easy and reproducible computational protocol for the identification of candidate RNA editing sites in humans using deep transcriptome (RNA-Seq) and genome (DNA-Seq) sequencing.
Topics: Animals; Computational Biology; Computers; DNA; High-Throughput Nucleotide Sequencing; Humans; RNA; RNA Editing; Sequence Analysis, RNA; Software; Transcriptome
PubMed: 32729082
DOI: 10.1007/978-1-0716-0787-9_12 -
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 -
Circulation Research Sep 2015
Topics: Cost-Benefit Analysis; Heart Diseases; High-Throughput Nucleotide Sequencing; Humans
PubMed: 26358106
DOI: 10.1161/CIRCRESAHA.115.307344 -
Methods in Molecular Biology (Clifton,... 2021A-to-I RNA editing in humans plays a relevant role since it can influence gene expression and increase proteome diversity. In addition, its deregulation has been linked...
A-to-I RNA editing in humans plays a relevant role since it can influence gene expression and increase proteome diversity. In addition, its deregulation has been linked to a variety of human diseases, including neurological disorders and cancer.In the last decade, massive transcriptome sequencing through the RNAseq technology has dramatically improved the investigation of RNA editing at single nucleotide resolution. Nowadays, different bioinformatics resources to discover and/or collect A-to-I events have been released. Hereafter, we initially provide an overview of the state-of-the-art RNA editing databases and, then, we focus on REDIportal, the largest collection of A-to-I events with more than 4.5 million sites from 2660 humans GTEx samples.
Topics: Animals; Computational Biology; Databases, Genetic; Genome, Human; Genomics; High-Throughput Nucleotide Sequencing; Humans; Internet; RNA Editing; Sequence Analysis, RNA; Software; Transcriptome; User-Computer Interface
PubMed: 33835458
DOI: 10.1007/978-1-0716-1307-8_25