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This is an introductory text and laboratory manual to be used primarily in undergraduate courses. It is also useful for graduate students and research scientists who require an introduction to the theory and methods of nanopore sequencing. The book has clear explanations of the principles of this emerging technology, together with instructional material written by experts that describes how to use a MinION nanopore instrument for sequencing in research or the classroom.At Harvard University the book serves as a textbook and lab manual for a university laboratory course designed to intensify the intellectual experience of incoming undergraduates while exploring biology as a field of concentration. Nanopore sequencing is an ideal topic as a path to encourage students about the range of courses they will take in Biology by pre-emptively addressing the complaint about having to take a course in Physics or Maths while majoring in Biology. The book addresses this complaint by concretely demonstrating the range of topics — from electricity to biochemistry, protein structure, molecular engineering, and informatics — that a student will have to master in subsequent courses if he or she is to become a scientist who truly understands what his or her biology instrument is measuring when investigating biological phenomena.
This volume provides comprehensive dry and wet experiments, methods, and applications on nanopore sequencing. Chapters guide readers through bioinformatic procedures, genome sequencing, analysis of repetitive regions, structural variations, rapid and on-site microbial identification, epidemiology, and transcriptome analysis. Written in the format of the highly successful Methods in Molecular Biology series, each chapter includes an introduction to the topic, lists necessary materials and methods, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols. Authoritative and cutting-edge, Nanopore Sequencing: Methods and Protocols aims to be comprehensive guide for researchers.
DNA contains the code of life, forming the molecular basis for all of life's diversity. The past several decades have witnessed remarkable progress in our ability to read and understand life's code through DNA sequencing. While fast and cheap DNA sequencing technologies are revolutionizing both science and healthcare, a new generation of technologies capable of single-molecule sequencing1 promise to further revolutionize the field of DNA sequencing by addressing many of limitations of the previous methods. Nanopore DNA sequencing is one such emerging single-molecule sequencing technology, capable of long reads and direct detection of epigenetically-relevant modified bases. The basic nanopore sequencing devices consists of two wells filled with a conductive electrolyte solution separated by an impermeable membrane containing a single nanometer-size hole, or nanopore. A voltage applied across the membrane drives an ionic current through the nanopore. DNA is negatively charged in solution and so will by drawn through the pore by the voltage, blocking some of the ionic current. As the different nucleotides along the DNA block the ionic current to different extents, the series of current fluctuations in the recorded time series can be used to decode the sequence of the DNA molecule moving through the pore. DNA motion through the pore is controlled using a DNA-processing motor enzyme, which steps the DNA through in discrete steps slow enough to allow resolution of the sequence-dependent fluctuations in the ionic current. Commercial nanopore sequencing devices have recently become available, making good on the decades-long promise of this technology. However, despite considerable early success and fanfare accompanying these first nanopore sequencers, technology development is not complete. Particularly, the single-read de novo sequencing accuracy must be improved for this technology to reach its full potential2. In order to fully realize its promise, we must both improve the accuracy of nanopore sequencing and devise better methods of handling error-prone sequencing data. In this dissertation, I discuss my work in the Gundlach nanopore lab at the University of Washington towards the goals of improved nanopore sequencing accuracy and improved application of existing error-prone sequencing data. In chapter 1, I introduce the broad field of DNA sequencing. I cover the history of scientific interest in DNA and DNA sequencing and provide motivation for DNA sequencing as a worthwhile pursuit both for its scientific and medical merits. I also discuss previous and existing DNA sequencing technologies, as well as the limitations of these technologies that motivate the development of new methods such as nanopore sequencing. In chapter 2 I describe and introduce nanopore sequencing. I summarize the development of nanopore sequencing technology, how various challenges were overcome, and how currently available nanopore sequencing devices work, setting the stage for understanding the primary error modes limiting the sequencing accuracy of this technologies. In chapter 3, in I present my work on improving nanopore sequencing accuracy using a new method of DNA control for enzyme-actuated nanopore DNA sequencing. This new method, in which we use a time-varying voltage to control DNA motion through the pore in addition to a DNA-processing enzyme, is able to mitigate two of the primary error modes in nanopore sequencing and dramatically improve sequencing accuracy. I discuss the motivation behind this new method, outline how we were able to realize nanopore sequencing using this method, and demonstrate the improved sequencing accuracy it affords. In chapter 4, I shift the discussion over to my work on improving the application of nanopore sequencing data. Specifically, I introduce a method of aligning nanopore data that enables highly sensitive and specific sequence alignment and species identification even for low accuracy reads. I go over the motivation for this method, and present our findings of its improved performance over alternative methods. Finally, I conclude in chapter 5 where I discuss the implications of the demonstrated advances in the accuracy and application of nanopore sequencing, as well as look out towards further progress that can be made in both arenas.
Nanopores are nanometer scale holes formed naturally by proteins or cells, and can be used for a variety of applications, including sequencing DNA and detecting anthrax. They can be integrated into artificially constructed encapsulated cells of silicon wafers while allowing small molecules like oxygen, glucose and insulin to pass, while keeping out large system molecules. "Nanopores: Sensing and Fundamental Biological Interactions" examines the emerging research directions surrounding nanopores such as genome sequencing and early disease detection using biomarker identification. Covering the applications of nanopores in genetics, proteomics, drug discovery, early disease detection and detection of emerging environmental threats, it is a must-have book for biomedicalengineers and research scientists.
The goal of this book is to introduce the biological and technical aspects of next generation sequencing methods, as well as algorithms to assemble these sequences into whole genomes. The book is organized into two parts; part 1 introduces NGS methods and part 2 reviews assembly algorithms and gives a good insight to these methods for readers new to the field. Gathering information, about sequencing and assembly methods together, helps both biologists and computer scientists to get a clear idea about the field. Chapters will include information about new sequencing technologies such as ChIp-seq, ChIp-chip, and De Novo sequence assembly. ​
Genomics has transformed the biological sciences. From epidemiology and medicine to evolution and forensics, the ability to determine an organism's complete genetic makeup has changed the way science is done and the questions that can be asked of it. Its most celebrated achievement was the Human Genome Project, a technologically challenging endeavor that took thousands of scientists around the world 13 years and over 3 billion US dollars to complete. In this Very Short Introduction John Archibald explores the science of genomics and its rapidly expanding toolbox. Sequencing a human genome now takes only a few days and costs as little as $1,000. The genomes of simple bacteria and viruses can be sequenced in a matter of hours on a device that fits in the palm of your hand. The resulting sequences can be used to better understand our biology in health and disease and to 'personalize' medicine. Archibald shows how the field of genomics is on the cusp of another quantum leap; the implications for science and society are profound. ABOUT THE SERIES: The Very Short Introductions series from Oxford University Press contains hundreds of titles in almost every subject area. These pocket-sized books are the perfect way to get ahead in a new subject quickly. Our expert authors combine facts, analysis, perspective, new ideas, and enthusiasm to make interesting and challenging topics highly readable.
The introduction of Next Generation Sequencing (NGS) technologies resulted in a major transformation in the way scientists extract genetic information from biological systems, revealing limitless insight about the genome, transcriptome and epigenome of any species. However, with NGS, came its own challenges that require continuous development in the sequencing technologies and bioinformatics analysis of the resultant raw data and assembly of the full length genome and transcriptome. Such developments lead to outstanding improvements of the performance and coverage of sequencing and improved quality for the assembled sequences, nevertheless, challenges such as sequencing errors, expensive processing and memory usage for assembly and sequencer specific errors remains major challenges in the field. This book aims to provide brief overviews the NGS field with special focus on the challenges facing the NGS field, including information on different experimental platforms, assembly algorithms and software tools, assembly error correction approaches and the correlated challenges.
This introduction to the methodology of DNA sequencing should be useful to those embarking on DNA sequencing for the first time. DNA sequencing is a very widely used technique, which has been automated to a certain degree.
Genetic information plays an increasingly important role in life sciences and medicine today. The ultimate goal of my Ph.D. project was to develop a nanopore-based DNA sequencing device that could drastically reduce the cost of acquiring genetic information. Chapter 1 is an overview of DNA sequencing methods, including the Sanger dideoxy-termination method and many emerging "next-generation" approaches. The potential of the nanopore DNA sequencing concept, as well as the requirements that need to be met for turning it into reality, are discussed at the end. Chapter 2 starts with an introduction to the biochemical and biophysical features of the most widely used protein ([alpha]HL) in the nanopore DNA sequencing field. It is followed by describing proof-of-principle experiments that suggest the feasibility of the nanopore DNA sequencing concept. More specifically, the pseudorotaxane design demonstrated that single-nucleotide variations in an intact ssDNA can be recognized, and the rotaxane design demonstrated that the natural DNA polymerase motor function can be employed to effect oligonucleotide translocation in unidirectional single-nucleotide steps. Chapter 3 begins with the discussion of a previously overlooked configuration dynamics associated with the [alpha]HL*DNA-PEG phos rotaxane. Such a dynamic feature of the rotaxane allows us to control and monitor oligonucleotide strand translocation in real-time. While DNA polymerase remains integral to most DNA sequencing methods, we came full circle by presenting preliminary data showing that the high spatial and temporal resolution of this rotaxane system provide the means for mechanistic studies of the DNA polymerase at the single-molecule level. Chapter 4 touches upon the importance of read-length in genome-scale sequencing, and the potential impact of the nanopore DNA sequencing method in this area of research.