-
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 -
Microbiology Spectrum Jan 2019In the decade and a half since the introduction of next-generation sequencing (NGS), the technical feasibility, cost, and overall utility of sequencing have changed... (Review)
Review
In the decade and a half since the introduction of next-generation sequencing (NGS), the technical feasibility, cost, and overall utility of sequencing have changed dramatically, including applications for infectious disease epidemiology. Massively parallel sequencing technologies have decreased the cost of sequencing by more than 6 orders or magnitude over this time, with a corresponding increase in data generation and complexity. This review provides an overview of the basic principles, chemistry, and operational mechanics of current sequencing technologies, including both conventional Sanger and NGS approaches. As the generation of large amounts of sequence data becomes increasingly routine, the role of bioinformatics in data analysis and reporting becomes all the more critical, and the successful deployment of NGS in public health settings requires careful consideration of changing information technology, bioinformatics, workforce, and regulatory requirements. While there remain important challenges to the sustainable implementation of NGS in public health, in terms of both laboratory and bioinformatics capacity, the impact of these technologies on infectious disease surveillance and outbreak investigations has been nothing short of revolutionary. Understanding the important role that NGS plays in modern public health laboratory practice is critical, as is the need to ensure appropriate workforce, infrastructure, facilities, and funding consideration for routine NGS applications, future innovation, and rapidly scaling NGS-based infectious disease surveillance and outbreak response activities. *This article is part of a curated collection.
Topics: Computational Biology; DNA; Data Analysis; Gene Library; High-Throughput Nucleotide Sequencing; Humans; Sequence Analysis, DNA
PubMed: 30737915
DOI: 10.1128/microbiolspec.AME-0005-2018 -
Methods in Molecular Biology (Clifton,... 2018Next-generation sequencing (NGS) enables the analysis of both microRNA expression and sequence, allowing for elucidation of a comprehensive landscape of miRNAs in a...
Next-generation sequencing (NGS) enables the analysis of both microRNA expression and sequence, allowing for elucidation of a comprehensive landscape of miRNAs in a given tissue and sample type. NGS analysis requires high-quality RNA extraction and preparation of microRNA libraries. In this chapter, we describe the methods used for RNA extraction from tissue specimens, serum, cytological slides, and formalin-fixed paraffin-embedded samples. Although the described library preparation and sequencing approaches are based on Illumina NextSeq 500 sequencing technology, the presented principles shall be compatible with other commercially available sequencing platforms.
Topics: Animals; Gene Library; High-Throughput Nucleotide Sequencing; Humans; MicroRNAs
PubMed: 29959676
DOI: 10.1007/978-1-4939-8624-8_8 -
Nature Methods Feb 2021
Topics: Algorithms; Computational Biology; Genome, Human; High-Throughput Nucleotide Sequencing; Humans; Sequence Analysis, DNA
PubMed: 33526887
DOI: 10.1038/s41592-021-01057-y -
Yi Chuan = Hereditas Mar 2015Last decade witnessed the explosive development of the third-generation sequencing strategy, including single-molecule real-time sequencing (SMRT), true single-molecule... (Review)
Review
Last decade witnessed the explosive development of the third-generation sequencing strategy, including single-molecule real-time sequencing (SMRT), true single-molecule sequencing (tSMSTM) and the single-molecule nanopore DNA sequencing. In this review, we summarize the principle, performance and application of the SMRT sequencing technology. Compared with the traditional Sanger method and the next-generation sequencing (NGS) technologies, the SMRT approach has several advantages, including long read length, high speed, PCR-free and the capability of direct detection of epigenetic modifications. However, the disadvantage of its low accuracy, most of which resulted from insertions and deletions, is also notable. So, the raw sequence data need to be corrected before assembly. Up to now, the SMRT is a good fit for applications in the de novo genomic sequencing and the high-quality assemblies of small genomes. In the future, it is expected to play an important role in epigenetics, transcriptomic sequencing, and assemblies of large genomes.
Topics: Animals; Bacteria; High-Throughput Nucleotide Sequencing; Humans; Software; Viruses
PubMed: 25787000
DOI: 10.16288/j.yczz.14-323 -
Mutagenesis Sep 2014Demand for new technologies that deliver fast, inexpensive and accurate genome information has never been greater. This challenge has catalysed the rapid development of... (Review)
Review
Demand for new technologies that deliver fast, inexpensive and accurate genome information has never been greater. This challenge has catalysed the rapid development of advances in next-generation sequencing (NGS). The generation of large volumes of sequence data and the speed of data acquisition are the primary advantages over previous, more standard methods. In 2013, the Food and Drug Administration granted marketing authorisation for the first high-throughput NG sequencer, Illumina's MiSeqDx, which allowed the development and use of a large number of new genome-based tests. Here, we present a review of template preparation, nucleic acid sequencing and imaging, genome assembly and alignment approaches as well as recent advances in current and near-term commercially available NGS instruments. We also outline the broad range of applications for NGS technologies and provide guidelines for platform selection to best address biological questions of interest. DNA sequencing has revolutionised biological and medical research, and is poised to have a similar impact on the practice of medicine. This tool is but one of an increasing arsenal of developing tools that enhance our capabilities to identify, quantify and functionally characterise the components of biological networks that keep us healthy or make us sick. Despite advances in other 'omic' technologies, DNA sequencing and analysis, in many respects, have played the leading role to date. The new technologies provide a bridge between genotype and phenotype, both in man and model organisms, and have revolutionised how risk of developing a complex human disease may be assessed. The generation of large DNA sequence data sets is producing a wealth of medically relevant information on a large number of individuals and populations that will potentially form the basis of truly individualised medical care in the future.
Topics: Computational Biology; Genetics, Medical; Genome, Human; Genotype; High-Throughput Nucleotide Sequencing; Humans; Phenotype; Sequence Analysis, DNA
PubMed: 25150023
DOI: 10.1093/mutage/geu031 -
Methods in Molecular Biology (Clifton,... 2019Drop-Seq is a low-cost, high-throughput platform to profile thousands of cells by encapsualting them into individual droplets. Uniquely barcoded mRNA capture...
Drop-Seq is a low-cost, high-throughput platform to profile thousands of cells by encapsualting them into individual droplets. Uniquely barcoded mRNA capture microparticles and cells are coconfined through a microfluidic device within the droplets where they undergo cell lysis and RNA hybridiztion. After breaking the droplets and pooling the hybridized particles, reverse transcription, PCR, and sequencing in single reactions allow to generate data from thousands of single-cell transcriptomes while maintaining information on the cellular origin of each transcript.
Topics: Animals; Equipment Design; Gene Expression Profiling; Gene Library; High-Throughput Nucleotide Sequencing; Humans; Lab-On-A-Chip Devices; Single-Cell Analysis; Transcriptome
PubMed: 31028633
DOI: 10.1007/978-1-4939-9240-9_6 -
Methods in Molecular Biology (Clifton,... 2022RNA-Seq is now a routinely employed assay to measure gene expression. As the technique matured over the last decade, so have dedicated analytic tools. In this chapter,... (Review)
Review
RNA-Seq is now a routinely employed assay to measure gene expression. As the technique matured over the last decade, so have dedicated analytic tools. In this chapter, we first describe the mainstream as well as the most up-to-date protocols and their implications on downstream analysis. We then detail the steps entailing RNA-Seq analysis in three main stages: (i) preprocessing and data preparation, (ii) upstream processing, and (iii) high-level analyses. We review the most recent and relevant tools as one workflow following a stepwise order. The chapter further encompasses in-depth features of these tools. Details of the required code are made available throughout the chapter, as well as of the underlying statistics. We illustrate these steps with analysis of publicly available RNA-Seq data.
Topics: Gene Expression; Gene Expression Profiling; High-Throughput Nucleotide Sequencing; RNA-Seq; Sequence Analysis, RNA; Software
PubMed: 35737247
DOI: 10.1007/978-1-0716-2376-3_20 -
Methods in Molecular Biology (Clifton,... 2019microRNAs are evolutionarily conserved, endogenously produced, noncoding RNAs (ncRNAs) of approximately 19-24 nucleotides (nts) in length known to exhibit gene silencing... (Review)
Review
microRNAs are evolutionarily conserved, endogenously produced, noncoding RNAs (ncRNAs) of approximately 19-24 nucleotides (nts) in length known to exhibit gene silencing of complementary target sequence. Their deregulated expression is reported in various disease conditions and thus has therapeutic implications. In the last decade, various computational resources are published in this field. In this chapter, we have reviewed bioinformatics resources, i.e., miRNA-centered databases, algorithms, and tools to predict miRNA targets. First section has enlisted more than 75 databases, which mainly covers information regarding miRNA registries, targets, disease associations, differential expression, interactions with other noncoding RNAs, and all-in-one resources. In the algorithms section, we have compiled about 140 algorithms from eight subcategories, viz. for the prediction of precursor (pre-) and mature miRNAs. These algorithms are developed on various sequence, structure, and thermodynamic based features incorporated into different machine learning techniques (MLTs). In addition, computational identification of miRNAs from high-throughput next generation sequencing (NGS) data and their variants, viz. isomiRs, differential expression, miR-SNPs, and functional annotation, are discussed. Prediction and analysis of miRNAs and their associated targets are also evaluated under miR-targets section providing knowledge regarding novel miRNA targets and complex host-pathogen interactions. In conclusion, we have provided comprehensive review of in silico resources published in miRNA research to help scientific community be updated and choose the appropriate tool according to their needs.
Topics: Computational Biology; Databases, Genetic; Gene Regulatory Networks; Gene Silencing; High-Throughput Nucleotide Sequencing; Host-Pathogen Interactions; Humans; Machine Learning; MicroRNAs; Polymorphism, Single Nucleotide; Sequence Analysis, RNA
PubMed: 30635896
DOI: 10.1007/978-1-4939-8982-9_9 -
ACS Synthetic Biology Apr 2024Conventional biological experiments often focus on in vitro assays because of the inherent limitations when handling multiple variables in vivo, including... (Review)
Review
Conventional biological experiments often focus on in vitro assays because of the inherent limitations when handling multiple variables in vivo, including labor-intensive and time-consuming procedures. Often only a subset of samples demonstrating significant efficacy in the in vitro assays can be evaluated in vivo. Nonetheless, because of the low correlation between the in vitro and in vivo tests, evaluation of the variables under examination in vivo and not solely in vitro is critical. An emerging approach to achieve high-throughput in vivo tests involves using a barcode system consisting of various nucleotide combinations. Unique barcodes for each variant enable the simultaneous testing of multiple entities, eliminating the need for separate individual tests. Subsequently, to identify crucial parameters, samples were collected and analyzed using barcode sequencing. This review explores the development of barcode design and its applications, including the evaluation of nucleic acid delivery systems and the optimization of gene expression in vivo.
Topics: Nucleic Acids; Technology; High-Throughput Nucleotide Sequencing
PubMed: 38526308
DOI: 10.1021/acssynbio.3c00602