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Molecular Aspects of Medicine Apr 2024Massively parallel sequencing technologies have long been used in both basic research and clinical routine. The recent introduction of digital sequencing has made... (Review)
Review
Massively parallel sequencing technologies have long been used in both basic research and clinical routine. The recent introduction of digital sequencing has made previously challenging applications possible by significantly improving sensitivity and specificity to now allow detection of rare sequence variants, even at single molecule level. Digital sequencing utilizes unique molecular identifiers (UMIs) to minimize sequencing-induced errors and quantification biases. Here, we discuss the principles of UMIs and how they are used in digital sequencing. We outline the properties of different UMI types and the consequences of various UMI approaches in relation to experimental protocols and bioinformatics. Finally, we describe how digital sequencing can be applied in specific research fields, focusing on cancer management where it can be used in screening of asymptomatic individuals, diagnosis, treatment prediction, prognostication, monitoring treatment efficacy and early detection of treatment resistance as well as relapse.
Topics: Humans; High-Throughput Nucleotide Sequencing; Computational Biology; Sensitivity and Specificity
PubMed: 38367531
DOI: 10.1016/j.mam.2024.101253 -
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 -
British Journal of Haematology Jan 2020Myelodysplastic syndromes (MDS) are clonal haematological disorders characterized by haematopoietic cell dysplasia, peripheral blood cytopenias, and a predisposition for... (Review)
Review
Myelodysplastic syndromes (MDS) are clonal haematological disorders characterized by haematopoietic cell dysplasia, peripheral blood cytopenias, and a predisposition for developing acute myeloid leukaemia (AML). Cytogenetics have historically been important in diagnosis and prognosis in MDS, but the growing accessibility of next generation sequencing (NGS) has led to growing research in the roles of molecular genetic variation on clinical decision-making in these disorders. Multiple genes have been previously studied and found to be associated with specific outcomes or disease types within MDS and knowledge of mutations in these genes provides insight into previously defined MDS subtypes. Knowledge of these mutations also informs development of novel therapies in the treatment of MDS. The precise role of NGS in the diagnosis, prognosis and monitoring of MDS remains unclear but the improvements in NGS technology and accessibility affords clinicians an additional practice tool to provide the best care for patients.
Topics: High-Throughput Nucleotide Sequencing; Humans; Myelodysplastic Syndromes; Prognosis
PubMed: 31571207
DOI: 10.1111/bjh.16212 -
Frontiers in Public Health 2022
Topics: High-Throughput Nucleotide Sequencing
PubMed: 35757642
DOI: 10.3389/fpubh.2022.945595 -
International Journal of Molecular... Aug 2020The early 1970s marked two breakthroughs in the field of biology: (i) The development of nucleotide sequencing technology; and, (ii) the discovery of the viroids. The... (Review)
Review
The early 1970s marked two breakthroughs in the field of biology: (i) The development of nucleotide sequencing technology; and, (ii) the discovery of the viroids. The first DNA sequences were obtained by two-dimensional chromatography which was later replaced by sequencing using electrophoresis technique. The subsequent development of fluorescence-based sequencing method which made DNA sequencing not only easier, but many orders of magnitude faster. The knowledge of DNA sequences has become an indispensable tool for both basic and applied research. It has shed light biology of viroids, the highly structured, circular, single-stranded non-coding RNA molecules that infect numerous economically important plants. Our understanding of viroid molecular biology and biochemistry has been intimately associated with the evolution of nucleic acid sequencing technologies. With the development of the next-generation sequence method, viroid research exponentially progressed, notably in the areas of the molecular mechanisms of viroids and viroid diseases, viroid pathogenesis, viroid quasi-species, viroid adaptability, and viroid-host interactions, to name a few examples. In this review, the progress in the understanding of viroid biology in conjunction with the improvements in nucleotide sequencing technology is summarized. The future of viroid research with respect to the use of third-generation sequencing technology is also briefly envisaged.
Topics: Biomedical Research; High-Throughput Nucleotide Sequencing; Host-Pathogen Interactions; Models, Genetic; Nucleic Acids; Plant Diseases; Plant Viruses; RNA, Viral; Viroids; Virus Replication
PubMed: 32752288
DOI: 10.3390/ijms21155532 -
Emerging Topics in Life Sciences Dec 2023Tandem repeat DNA sequences constitute a significant proportion of the human genome. While previously considered to be functionally inert, these sequences are now... (Review)
Review
Tandem repeat DNA sequences constitute a significant proportion of the human genome. While previously considered to be functionally inert, these sequences are now broadly accepted as important contributors to genetic diversity. However, the polymorphic nature of these sequences can lead to expansion beyond a gene-specific threshold, causing disease. More than 50 pathogenic repeat expansions have been identified to date, many of which have been discovered in the last decade as a result of advances in sequencing technologies and associated bioinformatic tools. Commonly utilised diagnostic platforms including Sanger sequencing, capillary array electrophoresis, and Southern blot are generally low throughput and are often unable to accurately determine repeat size, composition, and epigenetic signature, which are important when characterising repeat expansions. The rapid advances in bioinformatic tools designed specifically to interrogate short-read sequencing and the development of long-read single molecule sequencing is enabling a new generation of high throughput testing for repeat expansion disorders. In this review, we discuss some of the challenges surrounding the identification and characterisation of disease-causing repeat expansions and the technological advances that are poised to translate the promise of genomic medicine to individuals and families affected by these disorders.
Topics: Humans; Tandem Repeat Sequences; Sequence Analysis, DNA; Computational Biology; High-Throughput Nucleotide Sequencing
PubMed: 37888797
DOI: 10.1042/ETLS20230019 -
The Journal of Molecular Diagnostics :... Sep 2019An enduring goal of personalized medicine in cancer is the ability to identify patients who are likely to respond to specific therapies. Our growing understanding of the... (Review)
Review
An enduring goal of personalized medicine in cancer is the ability to identify patients who are likely to respond to specific therapies. Our growing understanding of the biology and molecular signatures of individual tumor types has facilitated the identification of predictive biomarkers and has led to an increasing number of diagnostic tests to be performed, often as serial and distinct assays on limited tumor specimens. The biomarker diagnostics field has been revolutionized by next-generation sequencing (NGS), which provides a comprehensive overview of the genomic profile of a tumor. Many preanalytic variables can influence the accuracy and reliability of NGS results. Standardization of preanalytic variables is, however, complicated by the plethora of specimen acquisition and processing methods. Variables across the tissue journey, including specimen acquisition, specimen fixation, and sectioning, as well as postfixation processing, such as nucleic acid extraction, library preparation, and choice of sequencing methods, are critical for the reliability of NGS analysis; thus, standardization would be beneficial. In this article, each step in the tissue journey is outlined, with specific focus on preanalytic variables that can influence NGS results. Practical considerations for standardization of these variables are provided to facilitate accurate, reliable, and reproducible NGS-based molecular characterization of tumors, ultimately informing diagnosis and guiding treatment.
Topics: Gene Library; High-Throughput Nucleotide Sequencing; Humans; Neoplasms; Precision Medicine; Sequence Analysis, DNA; Specimen Handling
PubMed: 31251989
DOI: 10.1016/j.jmoldx.2019.05.004 -
Nucleic Acids Research Dec 2023We presented an experimental method called FLOUR-seq, which combines BD Rhapsody and nanopore sequencing to detect the RNA lifecycle (including nascent, mature, and...
We presented an experimental method called FLOUR-seq, which combines BD Rhapsody and nanopore sequencing to detect the RNA lifecycle (including nascent, mature, and degrading RNAs) in cells. Additionally, we updated our HIT-scISOseq V2 to discover a more accurate RNA lifecycle using 10x Chromium and Pacbio sequencing. Most importantly, to explore how single-cell full-length RNA sequencing technologies could help improve the RNA velocity approach, we introduced a new algorithm called 'Region Velocity' to more accurately configure cellular RNA velocity. We applied this algorithm to study spermiogenesis and compared the performance of FLOUR-seq with Pacbio-based HIT-scISOseq V2. Our findings demonstrated that 'Region Velocity' is more suitable for analyzing single-cell full-length RNA data than traditional RNA velocity approaches. These novel methods could be useful for researchers looking to discover full-length RNAs in single cells and comprehensively monitor RNA lifecycle in cells.
Topics: Algorithms; High-Throughput Nucleotide Sequencing; Nanopore Sequencing; Sequence Analysis, RNA; Single-Cell Analysis
PubMed: 37941145
DOI: 10.1093/nar/gkad969 -
PLoS Computational Biology Sep 2022The development of increasingly efficient and cost-effective high throughput DNA sequencing techniques has enhanced the possibility of studying complex microbial... (Review)
Review
The development of increasingly efficient and cost-effective high throughput DNA sequencing techniques has enhanced the possibility of studying complex microbial systems. Recently, researchers have shown great interest in studying the microorganisms that characterise different ecological niches. Differential abundance analysis aims to find the differences in the abundance of each taxa between two classes of subjects or samples, assigning a significance value to each comparison. Several bioinformatic methods have been specifically developed, taking into account the challenges of microbiome data, such as sparsity, the different sequencing depth constraint between samples and compositionality. Differential abundance analysis has led to important conclusions in different fields, from health to the environment. However, the lack of a known biological truth makes it difficult to validate the results obtained. In this work we exploit metaSPARSim, a microbial sequencing count data simulator, to simulate data with differential abundance features between experimental groups. We perform a complete comparison of recently developed and established methods on a common benchmark with great effort to the reliability of both the simulated scenarios and the evaluation metrics. The performance overview includes the investigation of numerous scenarios, studying the effect on methods' results on the main covariates such as sample size, percentage of differentially abundant features, sequencing depth, feature variability, normalisation approach and ecological niches. Mainly, we find that methods show a good control of the type I error and, generally, also of the false discovery rate at high sample size, while recall seem to depend on the dataset and sample size.
Topics: Benchmarking; Computational Biology; High-Throughput Nucleotide Sequencing; Microbiota; Reproducibility of Results
PubMed: 36074761
DOI: 10.1371/journal.pcbi.1010467 -
Cold Spring Harbor Perspectives in... Sep 2019The application of next-generation sequencing (NGS) technology to the study of cancer genomes has been transformational. Not only has this technology revealed the... (Review)
Review
The application of next-generation sequencing (NGS) technology to the study of cancer genomes has been transformational. Not only has this technology revealed the genetic and epigenetic underpinnings of disease onset and progression, but also has redefined our clinical diagnosis and treatment paradigms. This rapid translation from discovery to clinical platform has occurred in the context of new pharmaceutical paradigms, enabling the use of NGS for the diagnosis and definition of therapeutic vulnerabilities of cancer. This review explores this transformation and identifies cutting-edge applications of NGS that will result in its additional utility in cancer care.
Topics: Genetic Predisposition to Disease; Genome, Human; High-Throughput Nucleotide Sequencing; Humans; Neoplasms; Sequence Analysis, DNA
PubMed: 30397020
DOI: 10.1101/cshperspect.a036269