DNA Sequencing Sensors: An Overview
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paper_title: Nucleotide sequence of bacteriophage phi X174 DNA.

paper_content:

A DNA sequence for the genome of bacteriophage phi X174 of approximately 5,375 nucleotides has been determined using the rapid and simple 'plus and minus' method. The sequence identifies many of the features responsible for the production of the proteins of the nine known genes of the organism, including initiation and termination sites for the proteins and RNAs. Two pairs of genes are coded by the same region of DNA using different reading frames.
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paper_title: Next-Generation Sequencing Platforms

paper_content:

Automated DNA sequencing instruments embody an elegant interplay among chemistry, engineering, software, and molecular biology and have built upon Sanger's founding discovery of dideoxynucleotide sequencing to perform once-unfathomable tasks. Combined with innovative physical mapping approaches that helped to establish long-range relationships between cloned stretches of genomic DNA, fluorescent DNA sequencers produced reference genome sequences for model organisms and for the reference human genome. New types of sequencing instruments that permit amazing acceleration of data-collection rates for DNA sequencing have been developed. The ability to generate genome-scale data sets is now transforming the nature of biological inquiry. Here, I provide an historical perspective of the field, focusing on the fundamental developments that predated the advent of next-generation sequencing instruments and providing information about how these instruments work, their application to biological research, and the newes...
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paper_title: Initial sequencing and analysis of the human genome.

paper_content:

The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.
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paper_title: Direct detection of nucleic acid hybridization on the surface of a charge coupled device

paper_content:

A method is described for the detection of DNA hybrids formed on a solid support, based upon the pairing of oligonucleotide chemistry and the technologies of electronic microdevice design. Surface matrices have been created in which oligonucleotide probes are covalently linked to a thin SiO2 film. 32P labeled target nucleic acid is then hybridized to this probe matrix under conditions of high stringency. The salient feature of the method is that to achieve the highest possible collection efficiency, the hybridization matrix is placed directly on the surface of a charge coupled device (CCD), which is used to detect 32P decay from hybridized target molecules (1, Eggers, M.D., Hogan, M.E., Reich, R.K., Lamture, J.B., Beattie, K.L., Hollis, M.A., Ehrilich, D.J., Kosicki, B.B., Shumaker, J.M., Varma, R.S., Burke, B.E., Murphy, A., and Rathman, D.D., (1993), Advances in DNA Sequencing Technology, Proc. SPIE, 1891, 13-26). Two implementations of the technology have been employed. The first involves direct attachment of the matrix to the surface of a CCD. The second involves attachment of the matrix to a disposible SiO2 coated chip, which is then placed face to face upon the CCD surface. As can be predicted from this favorable collection geometry and the known characteristics of a CCD, it is found that as measured by the time required to obtain equivalent signal to noise ratios, 32P detection speed by the direct CCD approach is at least 10 fold greater than can be obtained with a commercial gas phase array detector, and at least 100 fold greater than when X-ray film is used for 32P detection. Thus, it is shown that excellent quality hybridization signals can be obtained from a standard hybridization reaction, after only 1 second of CCD data acquisition.
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paper_title: Sequencing the Genome, Fast

paper_content:

The biotechnology company Celera has generated controversy by reporting that it will sequence the human genome in 3 years with the aid of 230 new high-throughput DNA sequencing machines, well before the efforts of the Human Genome Project are slated to be competed. Now J. C. Mullikin and A. A. McMurray report their evaluation of this sequencer, the ABI 3700 from Perkin-Elmer.
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paper_title: Studies on the chemical nature of the substance inducing transformation of pneumococcal types. Inductions of transformation by a desoxyribonucleic acid fraction isolated from pneumococcus type III

paper_content:

1. From Type III pneumococci a biologically active fraction has been isolated in highly purified form which in exceedingly minute amounts is capable under appropriate cultural conditions of inducing the transformation of unencapsulated R variants of Pneumococcus Type II into fully encapsulated cells of the same specific type as that of the heat-killed microorganisms from which the inducing material was recovered. 2. Methods for the isolation and purification of the active transforming material are described. 3. The data obtained by chemical, enzymatic, and serological analyses together with the results of preliminary studies by electrophoresis, ultracentrifugation, and ultraviolet spectroscopy indicate that, within the limits of the methods, the active fraction contains no demonstrable protein, unbound lipid, or serologically reactive polysaccharide and consists principally, if not solely, of a highly polymerized, viscous form of desoxyribonucleic acid. 4. Evidence is presented that the chemically induced alterations in cellular structure and function are predictable, type-specific, and transmissible in series. The various hypotheses that have been advanced concerning the nature of these changes are reviewed.
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paper_title: High-Throughput Sequencing Technologies

paper_content:

The human genome sequence has profoundly altered our understanding of biology, human diversity, and disease. The path from the first draft sequence to our nascent era of personal genomes and genomic medicine has been made possible only because of the extraordinary advancements in DNA sequencing technologies over the past 10 years. Here, we discuss commonly used high-throughput sequencing platforms, the growing array of sequencing assays developed around them, as well as the challenges facing current sequencing platforms and their clinical application.
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paper_title: High-throughput DNA sequencing – concepts and limitations

paper_content:

Recent advances in DNA sequencing have revolutionized the field of genomics, making it possible for even single research groups to generate large amounts of sequence data very rapidly and at a substantially lower cost. These high-throughput sequencing technologies make deep transcriptome sequencing and transcript quantification, whole genome sequencing and resequencing available to many more researchers and projects. However, while the cost and time have been greatly reduced, the error profiles and limitations of the new platforms differ significantly from those of previous sequencing technologies. The selection of an appropriate sequencing platform for particular types of experiments is an important consideration, and requires a detailed understanding of the technologies available; including sources of error, error rate, as well as the speed and cost of sequencing. We review the relevant concepts and compare the issues raised by the current high-throughput DNA sequencing technologies. We analyze how future developments may overcome these limitations and what challenges remain.
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paper_title: Sequencing technologies — the next generation

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Demand has never been greater for revolutionary technologies that deliver fast, inexpensive and accurate genome information. This challenge has catalysed the development of next-generation sequencing (NGS) technologies. The inexpensive production of large volumes of sequence data is the primary advantage over conventional methods. Here, I present a technical review of template preparation, sequencing and imaging, genome alignment and assembly approaches, and recent advances in current and near-term commercially available NGS instruments. I also outline the broad range of applications for NGS technologies, in addition to providing guidelines for platform selection to address biological questions of interest.
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paper_title: Coming of age: ten years of next-generation sequencing technologies

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Advances in DNA sequencing technologies have led to vast increases in the diversity of sequencing-based applications and in the amount of data generated. This Review discusses the current state-of-the-art technologies in both short-read and long-read DNA sequencing, their underlying mechanisms, relative strengths and limitations, and emerging applications.
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paper_title: Advanced sequencing technologies and their wider impact in microbiology

paper_content:

SUMMARY In the past 10 years, microbiology has undergone a revolution that has been ::: driven by access to cheap high-throughput DNA sequencing. It was not long ago ::: that the cloning and sequencing of a target gene could take months or years, ::: whereas now this entire process has been replaced by a 10 min Internet search ::: of a public genome database. There has been no single innovation that has ::: initiated this rapid technological change; in fact, the core chemistry of DNA ::: sequencing is the same as it was 30 years ago. Instead, progress has been ::: driven by large sequencing centers that have incrementally industrialized the ::: Sanger sequencing method. A side effect of this industrialization is that ::: large-scale sequencing has moved out of small research labs, and the vast ::: majority of sequence data is now generated by large genome centers. Recently, ::: there have been advances in technology that will enable high-throughput genome ::: sequencing to be established in research labs using bench-top instrumentation. ::: These new technologies are already being used to explore the vast microbial ::: diversity in the natural environment and the untapped genetic variation that ::: can occur in bacterial species. It is expected that these powerful new methods ::: will open up new questions to genomic investigation and will also allow ::: high-throughput sequencing to be more than just a discovery exercise but also ::: a routine assay for hypothesis testing. While this review will concentrate on ::: microorganisms, many of the important arguments about the need to measure and ::: understand variation at the species, population and ecosystem level will hold ::: true for many other biological systems.
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paper_title: Multiwavelength fluorescence detection for DNA sequencing using capillary electrophoresis

paper_content:

Abstract ::: Multiwavelength detection of laser induced fluorescence for dideoxynucleotide DNA sequencing with four different fluorophores and separation by capillary gel electrophoresis is described. A cryogenically cooled, low readout noise, 2-dimensional charge-coupled device is used as a detector for the on-line, on-column recording of emission spectra. The detection system has no moving parts and provides wavelength selectivity on a single detector device. The detection limit of fluorescently labeled oligonucleotides meets the high sensitivity requirements for capillary DNA sequencing largely due to the efficient operation of the CCD detector with a 94% duty cycle. Using the condition number as a selectivity criterion, multiwavelength detection provides better analytical selectivity than detection with four bandpass filters. Monte Carlo studies and analytical estimates show that base assignment errors are reduced with peak identification based on entire emission spectra. High-speed separation of sequencing samples and the treatment of the 2-dimensional electropherogram data is presented. Comparing the DNA sequence of a sample separated by slab gel electrophoresis with sequence from capillary gel electrophoresis and multiwavelength detection we find no significant difference in the amount of error attributable to the instrumentation.
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paper_title: Ten years of next-generation sequencing technology.

paper_content:

Ten years ago next-generation sequencing (NGS) technologies appeared on the market. During the past decade, tremendous progress has been made in terms of speed, read length, and throughput, along with a sharp reduction in per-base cost. Together, these advances democratized NGS and paved the way for the development of a large number of novel NGS applications in basic science as well as in translational research areas such as clinical diagnostics, agrigenomics, and forensic science. Here we provide an overview of the evolution of NGS and discuss the most significant improvements in sequencing technologies and library preparation protocols. We also explore the current landscape of NGS applications and provide a perspective for future developments.
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paper_title: PYROSEQUENCING- PRINCIPLES AND APPLICATIONS

paper_content:

Pyrosequencing is the first alternative to the conventional Sanger method for de novo DNA sequencing. Pyrosequencing is a DNA sequencing technology based on the sequencingbysynthesis principle. It employs a series of four enzymes to accurately detect nucleic acid sequences during the synthesis. Pyrosequencing has the potential advantages of accuracy, flexibility, parallel processing, and can be easily automated. Furthermore, the technique dispenses with the need for labeled primers, labeled nucleotides, and gelelectrophoresis. Pyrosequencing has opened up npossibilities for performing sequencebased DNA analysis. The method has been proven highly suitable for single nucleotide polymorphism analysis and sequencing of short stretches of DNA. Pyrosequencing has been successful for both confirmatory sequencing and de novo sequencing . By increasing the read length to higher scores and by shortening the sequence reaction time per base calling, pyrosequencing may take over many broad areas of DNA sequencing applications as the trend is directed to analysis of fewer amounts of specimens and larges cale settings, with higher throughput and lower cost. This article considers key features regarding different aspects of pyrosequencing technology, including the general principles, enzyme properties, sequencing modes, instrumentation, limitations, potential and future applications.
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paper_title: Long-read pyrosequencing using pure 2'-deoxyadenosine-5'-O'-(1-thiotriphosphate) Sp-isomer.

paper_content:

Pyrosequencing, a nonelectrophoretic DNA sequencing method that uses a luciferase-based enzymatic system to monitor DNA synthesis in real time, has so far been limited to sequencing of short stretches of DNA. To increase the signal-to-noise ratio in pyrosequencing the natural dATP was replaced by dATPalphaS (M. Ronaghi et al., 1996, Anal. Biochem. 242, 84-89). The applied dATPalphaS was a mixture of two isomers (Sp and Rp). We show here that by the introduction of pure 2'-deoxyadenosine-5'-O'-(1-thiotriphosphate) Sp-isomer in pyrosequencing substantial longer reads could be obtained. The pure Sp-isomer allowed lower nucleotide concentration to be used and improved the possibility to read through poly(T) regions. In general, a doubling of the read length could be obtained by the use of pure Sp-isomer. Pyrosequencing data for 50 to 100 bases could be generated on different types of template. The longer read will enable numerous new applications, such as identification and typing of medically important microorganisms as well as resequencing of DNA fragments for mutation screening and clone checking.
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paper_title: Ten years of next-generation sequencing technology.

paper_content:

Ten years ago next-generation sequencing (NGS) technologies appeared on the market. During the past decade, tremendous progress has been made in terms of speed, read length, and throughput, along with a sharp reduction in per-base cost. Together, these advances democratized NGS and paved the way for the development of a large number of novel NGS applications in basic science as well as in translational research areas such as clinical diagnostics, agrigenomics, and forensic science. Here we provide an overview of the evolution of NGS and discuss the most significant improvements in sequencing technologies and library preparation protocols. We also explore the current landscape of NGS applications and provide a perspective for future developments.
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paper_title: Correction of sequencing errors in a mixed set of reads

paper_content:

Motivation: High-throughput sequencing technologies produce large sets of short reads that may contain errors. These sequencing errors make de novo assembly challenging. Error correction aims to reduce the error rate prior assembly. Many de novo sequencing projects use reads from several sequencing technologies to get the benefits of all used technologies and to alleviate their shortcomings. However, combining such a mixed set of reads is problematic as many tools are specific to one sequencing platform. The SOLiD sequencing platform is especially problematic in this regard because of the two base color coding of the reads. Therefore, new tools for working with mixed read sets are needed. ::: ::: Results: We present an error correction tool for correcting substitutions, insertions and deletions in a mixed set of reads produced by various sequencing platforms. We first develop a method for correcting reads from any sequencing technology producing base space reads such as the SOLEXA/Illumina and Roche/454 Life Sciences sequencing platforms. We then further refine the algorithm to correct the color space reads from the Applied Biosystems SOLiD sequencing platform together with normal base space reads. Our new tool is based on the SHREC program that is aimed at correcting SOLEXA/Illumina reads. Our experiments show that we can detect errors with 99% sensitivity and >98% specificity if the combined sequencing coverage of the sets is at least 12. We also show that the error rate of the reads is greatly reduced. ::: ::: Availability: The JAVA source code is freely available at http://www.cs.helsinki.fi/u/lmsalmel/hybrid-shrec/ ::: ::: Contact: leena.salmela@cs.helsinki.fi
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paper_title: Toward the Single-Hour High-Quality Genome

paper_content:

Today, resequencing of a human genome can be performed in approximately a week using a single instrument. Thanks to a steady logarithmic rate of increase in performance for DNA sequencing platforms over the past seven years, DNA sequencing is one of the fastest developing technology fields. As the process becomes faster, it opens up possibilities within health care, diagnostics, and entirely new fields of research. Immediate genetic characterization of contagious outbreaks has been exemplified, and with such applications for the direct benefit of human health, expectations of future sensitive, rapid, high-throughput, and cost-effective technologies are steadily growing. Simultaneously, some of the limitations of a rapidly growing field have become apparent, and questions regarding the quality of some of the data deposited into databases have been raised. A human genome sequenced in only an hour is likely to become a reality in the future, but its definition may not be as certain.
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paper_title: A high-resolution , nucleosome position map of C . elegans reveals a lack of universal sequence-dictated positioning

paper_content:

Using the massively parallel technique of sequencing by oligonucleotide ligation and detection (SOLiD; Applied Biosystems), we have assessed the in vivo positions of more than 44 million putative nucleosome cores in the multicellular genetic model organism Caenorhabditis elegans. These analyses provide a global view of the chromatin architecture of a multicellular animal at extremely high density and resolution. While we observe some degree of reproducible positioning throughout the genome in our mixed stage population of animals, we note that the major chromatin feature in the worm is a diversity of allowed nucleosome positions at the vast majority of individual loci. While absolute positioning of nucleosomes can vary substantially, relative positioning of nucleosomes (in a repeated array structure likely to be maintained at least in part by steric constraints) appears to be a significant property of chromatin structure. The high density of nucleosomal reads enabled a substantial extension of previous analysis describing the usage of individual oligonucleotide sequences along the span of the nucleosome core and linker. We release this data set, via the UCSC Genome Browser, as a resource for the high-resolution analysis of chromatin conformation and DNA accessibility at individual loci within the C. elegans genome.
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paper_title: SHRiMP: Accurate Mapping of Short Color-space Reads

paper_content:

The development of Next Generation Sequencing technologies, capable of sequencing hundreds of millions of short reads (25–70 bp each) in a single run, is opening the door to population genomic studies of non-model species. In this paper we present SHRiMP - the SHort Read Mapping Package: a set of algorithms and methods to map short reads to a genome, even in the presence of a large amount of polymorphism. Our method is based upon a fast read mapping technique, separate thorough alignment methods for regular letter-space as well as AB SOLiD (color-space) reads, and a statistical model for false positive hits. We use SHRiMP to map reads from a newly sequenced Ciona savignyi individual to the reference genome. We demonstrate that SHRiMP can accurately map reads to this highly polymorphic genome, while confirming high heterozygosity of C. savignyi in this second individual. SHRiMP is freely available at http://compbio.cs.toronto.edu/shrimp.
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paper_title: Nucleotide sequence of bacteriophage phi X174 DNA.

paper_content:

A DNA sequence for the genome of bacteriophage phi X174 of approximately 5,375 nucleotides has been determined using the rapid and simple 'plus and minus' method. The sequence identifies many of the features responsible for the production of the proteins of the nine known genes of the organism, including initiation and termination sites for the proteins and RNAs. Two pairs of genes are coded by the same region of DNA using different reading frames.
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paper_title: Total internal reflection fluorescence microscopy in cell biology.

paper_content:

Key events in cellular trafficking occur at the cell surface, and it is desirable to visualize these events without interference from other regions deeper within. This review describes a microscopy technique based on total internal reflection fluorescence which is well suited for optical sectioning at cell-substrate regions with an unusually thin region of fluorescence excitation. The technique has many other applications as well, most notably for studying biochemical kinetics and single biomolecule dynamics at surfaces. A brief summary of these applications is provided, followed by presentations of the physical basis for the technique and the various ways to implement total internal reflection fluorescence in a standard fluorescence microscope.
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paper_title: Total internal reflection STED microscopy

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Stimulated emission depletion (STED) microscopy achieves diffraction-unlimited resolution in far-field fluorescence microscopy well below 100 nm. As common for (single-lens) far-field microscopy techniques, the lateral resolution is better than the axial sectioning capabilities. Here we present the first implementation of total internal reflection (TIR) illumination into STED microscopy which limits fluorophore excitation to ~70 nm in the vicinity of the cover slip while simultaneously providing ~50 nm lateral resolution. We demonstrate the performance of this new microscope technique with fluorescent bead test samples as well as immuno-stained microtubules. Total internal reflection STED microscopy provides superior axial sectioning capabilities with the potential to reduce photo-bleaching and photo-damage in live cell imaging.
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paper_title: Applications of New Sequencing Technologies for Transcriptome Analysis

paper_content:

Transcriptome analysis has been a key area of biological inquiry for decades. Over the years, research in the field has progressed from candidate gene-based detection of RNAs using Northern blotting to high-throughput expression profiling driven by the advent of microarrays. Next-generation sequencing technologies have revolutionized transcriptomics by providing opportunities for multidimensional examinations of cellular transcriptomes in which high-throughput expression data are obtained at a single-base resolution.
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paper_title: Accurate whole human genome sequencing using reversible terminator chemistry

paper_content:

DNA sequence information underpins genetic research, enabling discoveries of important biological or medical benefit. Sequencing projects have traditionally used long (400800 base pair) reads, but the existence of reference sequences for the human and many other genomes makes it possible to develop new, fast approaches to re-sequencing, whereby shorter reads are compared to a reference to identify intraspecies genetic variation. Here we report an approach that generates several billion bases of accurate nucleotide sequence per experiment at low cost. Single molecules of DNA are attached to a flat surface, amplified in situ and used as templates for synthetic sequencing with fluorescent reversible terminator deoxyribonucleotides. Images of the surface are analysed to generate high-quality sequence.We demonstrate application of this approach to human genome sequencing on flow-sorted X chromosomes and then scale the approach to determine the genome sequence of a male Yoruba from Ibadan, Nigeria. We build an accurate consensus sequence from.303 average depth of paired 35-base reads. We characterize four million single-nucleotide polymorphisms and four hundred thousand structural variants, many of which were previously unknown. Our approach is effective for accurate, rapid and economical whole-genome re-sequencing and many other biomedical applications.
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paper_title: Whole-genome re-sequencing

paper_content:

DNA sequencing can be used to gain important information on genes, genetic variation and gene function for biological and medical studies. The growing collection of publicly available reference genome sequences will underpin a new era of whole genome re-sequencing, but sequencing costs need to fall and throughput needs to rise by several orders of magnitude. Novel technologies are being developed to meet this need by generating massive amounts of sequence that can be aligned to the reference sequence. The challenge is to maintain the high standards of accuracy and completeness that are hallmarks of the previous genome projects. One or more new sequencing technologies are expected to become the mainstay of future research, and to make DNA sequencing centre stage as a routine tool in genetic research in the coming years.
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paper_title: Single-molecule PCR using water-in-oil emulsion.

paper_content:

Polymerase chain reaction (PCR) using a single molecule of DNA is very useful for analysis, detection and cloning of the desired DNA fragment. We developed a simple PCR method utilizing a water-in-oil (W/O) emulsion that included numerous droplets of reaction mixture in bulk oil phase. These droplets, which were stable even at high temperatures, functioned as micro-reactors. This allows the effective concentration of template DNA to be increased, even for low concentrations of template DNA. The present method consists of a two-step thermal cycle. The first step was carried out using the W/O emulsion. During this step, the template DNA was amplified in the limited volume of the droplets in the W/O emulsion. The W/O emulsion was broken and the second PCR step was carried out. This method can be easily applied to amplify a single DNA molecule.
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paper_title: An integrated semiconductor device enabling non-optical genome sequencing

paper_content:

The seminal importance of DNA sequencing to the life sciences, biotechnology and medicine has driven the search for more scalable and lower-cost solutions. Here we describe a DNA sequencing technology in which scalable, low-cost semiconductor manufacturing techniques are used to make an integrated circuit able to directly perform non-optical DNA sequencing of genomes. Sequence data are obtained by directly sensing the ions produced by template-directed DNA polymerase synthesis using all-natural nucleotides on this massively parallel semiconductor-sensing device or ion chip. The ion chip contains ion-sensitive, field-effect transistor-based sensors in perfect register with 1.2 million wells, which provide confinement and allow parallel, simultaneous detection of independent sequencing reactions. Use of the most widely used technology for constructing integrated circuits, the complementary metal-oxide semiconductor (CMOS) process, allows for low-cost, large-scale production and scaling of the device to higher densities and larger array sizes. We show the performance of the system by sequencing three bacterial genomes, its robustness and scalability by producing ion chips with up to 10 times as many sensors and sequencing a human genome.
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paper_title: Next generation sequencing technology: Advances and applications

paper_content:

Impressive progress has been made in the field of Next Generation Sequencing (NGS). Through advancements in the fields of molecular biology and technical engineering, parallelization of the sequencing reaction has profoundly increased the total number of produced sequence reads per run. Current sequencing platforms allow for a previously unprecedented view into complex mixtures of RNA and DNA samples. NGS is currently evolving into a molecular microscope finding its way into virtually every fields of biomedical research. In this chapter we review the technical background of the different commercially available NGS platforms with respect to template generation and the sequencing reaction and take a small step towards what the upcoming NGS technologies will bring. We close with an overview of different implementations of NGS into biomedical research. This article is part of a Special Issue entitled: From Genome to Function.
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paper_title: From Sensors to Systems: CMOS-Integrated Electrochemical Biosensors

paper_content:

Novel electronic detection techniques are being increasingly sought as components of highly scalable technologies for high-throughput biosensing applications. Among the techniques being considered, electrochemical detection offers an attractive alternative. Advancement in nanoscale electrochemistry makes this an opportune moment to consider the prospects of its integration with CMOS processes. This paper focuses on the new properties and challenges that emerge from the downscaling of electrode dimensions, focusing, in particular, on redox-cycling-based approaches to nanoscale electrochemical devices. We explore the possibilities of interfacing arrays of such devices with CMOS process technology to create highly parallelized integrated platforms. We cite selective examples to provide a qualitative overview of the general design constraints that attend any system-level integration process. We also discuss several challenges that limit the scalability of such platforms and that need to be overcome to create reliable and robust CMOS-integrated electrochemical biosensing platforms.
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paper_title: Progress in ion torrent semiconductor chip based sequencing.

paper_content:

In order for next-generation sequencing to become widely used as a diagnostic in the healthcare industry, sequencing instrumentation will need to be mass produced with a high degree of quality and economy. One way to achieve this is to recast DNA sequencing in a format that fully leverages the manufacturing base created for computer chips, complementary metal-oxide semiconductor chip fabrication, which is the current pinnacle of large scale, high quality, low-cost manufacturing of high technology. To achieve this, ideally the entire sensory apparatus of the sequencer would be embodied in a standard semiconductor chip, manufactured in the same fab facilities used for logic and memory chips. Recently, such a sequencing chip, and the associated sequencing platform, has been developed and commercialized by Ion Torrent, a division of Life Technologies, Inc. Here we provide an overview of this semiconductor chip based sequencing technology, and summarize the progress made since its commercial introduction. We described in detail the progress in chip scaling, sequencing throughput, read length, and accuracy. We also summarize the enhancements in the associated platform, including sample preparation, data processing, and engagement of the broader development community through open source and crowdsourcing initiatives.
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paper_title: Piet Bergveld - 40 years of ISFET technology: from neuronal sensing to DNA sequencing

paper_content:

This article presents a personal account of the life and scientifi c journey of Professor Piet Bergveld, the inventor and founding father of the Ion-Sensitive Field Effect Transistor (ISFET). The interview gives a unique overview of how ISFET technology has evolved over the years, and the challenges faced during the development from its initial use in neuronal sensing to the technology we see today, which has huge potential in the current era of genetic technology.
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paper_title: A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers

paper_content:

BackgroundNext generation sequencing (NGS) technology has revolutionized genomic and genetic research. The pace of change in this area is rapid with three major new sequencing platforms having been released in 2011: Ion Torrent’s PGM, Pacific Biosciences’ RS and the Illumina MiSeq. Here we compare the results obtained with those platforms to the performance of the Illumina HiSeq, the current market leader. In order to compare these platforms, and get sufficient coverage depth to allow meaningful analysis, we have sequenced a set of 4 microbial genomes with mean GC content ranging from 19.3 to 67.7%. Together, these represent a comprehensive range of genome content. Here we report our analysis of that sequence data in terms of coverage distribution, bias, GC distribution, variant detection and accuracy.ResultsSequence generated by Ion Torrent, MiSeq and Pacific Biosciences technologies displays near perfect coverage behaviour on GC-rich, neutral and moderately AT-rich genomes, but a profound bias was observed upon sequencing the extremely AT-rich genome of Plasmodium falciparum on the PGM, resulting in no coverage for approximately 30% of the genome. We analysed the ability to call variants from each platform and found that we could call slightly more variants from Ion Torrent data compared to MiSeq data, but at the expense of a higher false positive rate. Variant calling from Pacific Biosciences data was possible but higher coverage depth was required. Context specific errors were observed in both PGM and MiSeq data, but not in that from the Pacific Biosciences platform.ConclusionsAll three fast turnaround sequencers evaluated here were able to generate usable sequence. However there are key differences between the quality of that data and the applications it will support.
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paper_title: Real-time DNA sequencing from single polymerase molecules.

paper_content:

We present single-molecule, real-time sequencing data obtained from a DNA polymerase performing uninterrupted template-directed synthesis using four distinguishable fluorescently labeled deoxyribonucleoside triphosphates (dNTPs). We detected the temporal order of their enzymatic incorporation into a growing DNA strand with zero-mode waveguide nanostructure arrays, which provide optical observation volume confinement and enable parallel, simultaneous detection of thousands of single-molecule sequencing reactions. Conjugation of fluorophores to the terminal phosphate moiety of the dNTPs allows continuous observation of DNA synthesis over thousands of bases without steric hindrance. The data report directly on polymerase dynamics, revealing distinct polymerization states and pause sites corresponding to DNA secondary structure. Sequence data were aligned with the known reference sequence to assay biophysical parameters of polymerization for each template position. Consensus sequences were generated from the single-molecule reads at 15-fold coverage, showing a median accuracy of 99.3%, with no systematic error beyond fluorophore-dependent error rates.
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paper_title: Zero-Mode Waveguides for Single-Molecule Analysis at High Concentrations

paper_content:

Optical approaches for observing the dynamics of single molecules have required pico- to nanomolar concentrations of fluorophore in order to isolate individual molecules. However, many biologically relevant processes occur at micromolar ligand concentrations, necessitating a reduction in the conventional observation volume by three orders of magnitude. We show that arrays of zero-mode waveguides consisting of subwavelength holes in a metal film provide a simple and highly parallel means for studying single-molecule dynamics at micromolar concentrations with microsecond temporal resolution. We present observations of DNA polymerase activity as an example of the effectiveness of zero-mode waveguides for performing single-molecule experiments at high concentrations.
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paper_title: Parallel confocal detection of single molecules in real time

paper_content:

The confocal detection principle is extended to a highly parallel optical system that continuously analyzes thousands of concurrent sample locations. This is achieved through the use of a holographic laser illumination multiplexer combined with a confocal pinhole array before a prism dispersive element used to provide spectroscopic information from each confocal volume. The system is demonstrated to detect and identify single fluorescent molecules from each of several thousand independent confocal volumes in real time.
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paper_title: Single-molecule PCR using water-in-oil emulsion.

paper_content:

Polymerase chain reaction (PCR) using a single molecule of DNA is very useful for analysis, detection and cloning of the desired DNA fragment. We developed a simple PCR method utilizing a water-in-oil (W/O) emulsion that included numerous droplets of reaction mixture in bulk oil phase. These droplets, which were stable even at high temperatures, functioned as micro-reactors. This allows the effective concentration of template DNA to be increased, even for low concentrations of template DNA. The present method consists of a two-step thermal cycle. The first step was carried out using the W/O emulsion. During this step, the template DNA was amplified in the limited volume of the droplets in the W/O emulsion. The W/O emulsion was broken and the second PCR step was carried out. This method can be easily applied to amplify a single DNA molecule.
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paper_title: Third-generation sequencing and the future of genomics

paper_content:

Third-generation long-range DNA sequencing and mapping technologies are creating a renaissance in high-quality genome sequencing. Unlike second-generation sequencing, which produces short reads a few hundred base-pairs long, third-generation single-molecule technologies generate over 10,000 bp reads or map over 100,000 bp molecules. We analyze how increased read lengths can be used to address longstanding problems in de novo genome assembly, structural variation analysis and haplotype phasing.
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paper_title: Direct sequencing of small genomes on the Pacific Biosciences RS without library preparation

paper_content:

We have developed a sequencing method on the Pacific Biosciences RS sequencer (the PacBio) for small DNA molecules that avoids the need for a standard library preparation. To date this approach has been applied toward sequencing single-stranded and double-stranded viral genomes, bacterial plasmids, plasmid vector models for DNA-modification analysis, and linear DNA fragments covering an entire bacterial genome. Using direct sequencing it is possible to generate sequence data from as little as 1 ng of DNA, offering a significant advantage over current protocols which typically require 400-500 ng of sheared DNA for the library preparation.
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paper_title: Ion-Channel Genosensor for the Detection of Specific DNA Sequences Derived from Plum Pox Virus in Plant Extracts

paper_content:

A DNA biosensor for detection of specific oligonucleotides sequences of Plum Pox Virus (PPV) in plant extracts and buffer is proposed. The working principles of a genosensor are based on the ion-channel mechanism. The NH2-ssDNA probe was deposited onto a glassy carbon electrode surface to form an amide bond between the carboxyl group of oxidized electrode surface and amino group from ssDNA probe. The analytical signals generated as a result of hybridization were registered in Osteryoung square wave voltammetry in the presence of [Fe(CN)6]3−/4− as a redox marker. The 22-mer and 42-mer complementary ssDNA sequences derived from PPV and DNA samples from plants infected with PPV were used as targets. Similar detection limits of 2.4 pM (31.0 pg/mL) and 2.3 pM (29.5 pg/mL) in the concentration range 1–8 pM were observed in the presence of the 22-mer ssDNA and 42-mer complementary ssDNA sequences of PPV, respectively. The genosensor was capable of discriminating between samples consisting of extracts from healthy plants and leaf extracts from infected plants in the concentration range 10–50 pg/mL. The detection limit was 12.8 pg/mL. The genosensor displayed good selectivity and sensitivity. The 20-mer partially complementary DNA sequences with four complementary bases and DNA samples from healthy plants used as negative controls generated low signal.
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paper_title: The evolution of nanopore sequencing

paper_content:

The “$1,000 Genome” project has been drawing increasing attention since its launch a decade ago. Nanopore sequencing, the third-generation, is believed to be one of the most promising sequencing technologies to reach four gold standards set for the “$1,000 Genome” while the second-generation sequencing technologies are bringing about a revolution in life sciences, particularly in genome sequencing-based personalized medicine. Both of protein and solid-state nanopores have been extensively investigated for a series of issues, from detection of ionic current blockage to field-effect-transistor (FET) sensors. A newly released protein nanopore sequencer has shown encouraging potential that nanopore sequencing will ultimately fulfill the gold standards. In this review, we address advances, challenges, and possible solutions of nanopore sequencing according to these standards.
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paper_title: Three decades of nanopore sequencing

paper_content:

A long-held goal in sequencing has been to use a voltage-biased nanoscale pore in a membrane to measure the passage of a linear, single-stranded (ss) DNA or RNA molecule through that pore. With the development of enzyme-based methods that ratchet polynucleotides through the nanopore, nucleobase-by-nucleobase, measurements of changes in the current through the pore can now be decoded into a DNA sequence using an algorithm. In this Historical Perspective, we describe the key steps in nanopore strand-sequencing, from its earliest conceptualization more than 25 years ago to its recent commercialization and application.
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paper_title: Advances in nanopore sequencing technology.

paper_content:

Much tremendous break through have been obtained in recent years for nanopore sequencing to achieve the goal of $1000 genome. As a method of single molecule sequencing, nanopore sequencing can discriminate the individual molecules of the target DNA strand rapidly due to the current blockages by translocating the nucleotides through a nano-scale pore. Both the protein-pores and solid-state nanopore channels which called single nanopore sequencing have been studied widely for the application of nanopore sequencing technology. This review will give a detail representation to protein nanopore and solid-state nanopore sequencing. For protein nanopore sequencing technology, we will introduce different nanopore types, device assembly and some challenges still exist at present. We will focus on more research fields for solid-state nanopore sequencing in terms of materials, device assembly, fabricated methods, translocation process and some specific challenges. The review also covers some of the technical advances in the union nanopore sequencing, which include nanopore sequencing combine with exonuclease, hybridization, synthesis and design polymer.
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paper_title: Recent advances in nanopore sequencing

paper_content:

The prospect of nanopores as a next-generation sequencing (NGS) platform has been a topic of growing interest and considerable government-sponsored research for more than a decade. Oxford Nanopore Technologies recently announced the first commercial nanopore sequencing devices, to be made available by the end of 2012, while other companies (Life, Roche, IBM) are also pursuing nanopore sequencing approaches. In this paper, the state of the art in nanopore sequencing is reviewed, focusing on the most recent contributions that have or promise to have NGS commercial potential. We consider also the scalability of the circuitry to support multichannel arrays of nanopores in future sequencing devices, which is critical to commercial viability.
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paper_title: A first look at the Oxford Nanopore MinION sequencer

paper_content:

Oxford Nanopore's third-generation single-molecule sequencing platform promises to decrease costs for reagents and instrumentation. After a 2-year hiatus following the initial announcement, the first devices have been released as part of an early access program. We explore the performance of this platform by resequencing the lambda phage genome, and amplicons from a snake venom gland transcriptome. Although the handheld MinION sequencer can generate more than 150 megabases of raw data in one run, at most a quarter of the resulting reads map to the reference, with less than average 10% identity. Much of the sequence consists of insertion/deletion errors, or is seemingly without similarity to the template. Using the lambda phage data as an example, although the reads are long, averaging 5 kb, at best 890 ± 1932 bases per mapped read could be matched to the reference without soft clipping. In the course of a 36 h run on the MinION, it was possible to resequence the 48 kb lambda phage reference at 16× coverage. Currently, substantially larger projects would not be feasible using the MinION. Without increases in accuracy, which would be required for applications such as genome scaffolding and phasing, the current utility of the MinION appears limited. Library preparation requires access to a molecular laboratory, and is of similar complexity and cost to that of other next-generation sequencing platforms. The MinION is an exciting step in a new direction for single-molecule sequencing, though it will require dramatic decreases in error rates before it lives up to its promise.
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paper_title: Continuous base identification for single-molecule nanopore DNA sequencing.

paper_content:

A single-molecule method for sequencing DNA that does not require fluorescent labelling could reduce costs and increase sequencing speeds. An exonuclease enzyme might be used to cleave individual nucleotide molecules from the DNA, and when coupled to an appropriate detection system, these nucleotides could be identified in the correct order. Here, we show that a protein nanopore with a covalently attached adapter molecule can continuously identify unlabelled nucleoside 5'-monophosphate molecules with accuracies averaging 99.8%. Methylated cytosine can also be distinguished from the four standard DNA bases: guanine, adenine, thymine and cytosine. The operating conditions are compatible with the exonuclease, and the kinetic data show that the nucleotides have a high probability of translocation through the nanopore and, therefore, of not being registered twice. This highly accurate tool is suitable for integration into a system for sequencing nucleic acids and for analysing epigenetic modifications.
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paper_title: The potential and challenges of nanopore sequencing

paper_content:

A nanopore-based device provides single-molecule detection and analytical capabilities that are achieved by electrophoretically driving molecules in solution through a nano-scale pore. The nanopore provides a highly confined space within which single nucleic acid polymers can be analyzed at high throughput by one of a variety of means, and the perfect processivity that can be enforced in a narrow pore ensures that the native order of the nucleobases in a polynucleotide is reflected in the sequence of signals that is detected. Kilobase length polymers (single-stranded genomic DNA or RNA) or small molecules (e.g., nucleosides) can be identified and characterized without amplification or labeling, a unique analytical capability that makes inexpensive, rapid DNA sequencing a possibility. Further research and development to overcome current challenges to nanopore identification of each successive nucleotide in a DNA strand offers the prospect of 'third generation' instruments that will sequence a diploid mammalian genome for ∼$1,000 in ∼24 h.
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paper_title: Electrochemical Sensors Based on Molecularly Imprinted Polymers

paper_content:

Over the past two decades, molecularly imprinted polymers (MIPs) have attracted broad interest from scientists engaged in sensor development. This attention can be explained by the serious potentia ...
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paper_title: Electrochemical DNA sensors

paper_content:

Electrochemistry-based sensors offer sensitivity, selectivity and low cost for the detection of selected DNA sequences or mutated genes associated with human disease. DNA-based electrochemical sensors exploit a range of different chemistries, but all take advantage of nanoscale interactions between the target in solution, the recognition layer and a solid electrode surface. Numerous approaches to electrochemical detection have been developed, including direct electrochemistry of DNA, electrochemistry at polymer-modified electrodes, electrochemistry of DNA-specific redox reporters, electrochemical amplifications with nanoparticles, and electrochemical devices based on DNA-mediated charge transport chemistry.
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paper_title: Graphene Based Electrochemical Sensors and Biosensors: A Review

paper_content:

Graphene, emerging as a true 2-dimensional material, has received increasing attention due to its unique physicochemical properties (high surface area, excellent conductivity, high mechanical strength, and ease of functionalization and mass production). This article selectively reviews recent advances in graphene-based electrochemical sensors and biosensors. In particular, graphene for direct electrochemistry of enzyme, its electrocatalytic activity toward small biomolecules (hydrogen peroxide, NADH, dopamine, etc.), and graphenebased enzyme biosensors have been summarized in more detail; Graphene-based DNA sensing and environmental analysis have been discussed. Future perspectives in this rapidly developing field are also discussed.
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paper_title: Aptamer-Based Analysis: A Promising Alternative for Food Safety Control

paper_content:

Ensuring food safety is nowadays a top priority of authorities and professional players in the food supply chain. One of the key challenges to determine the safety of food and guarantee a high level of consumer protection is the availability of fast, sensitive and reliable analytical methods to identify specific hazards associated to food before they become a health problem. The limitations of existing methods have encouraged the development of new technologies, among them biosensors. Success in biosensor design depends largely on the development of novel receptors with enhanced affinity to the target, while being stable and economical. Aptamers fulfill these characteristics, and thus have surfaced as promising alternatives to natural receptors. This Review describes analytical strategies developed so far using aptamers for the control of pathogens, allergens, adulterants, toxins and other forbidden contaminants to ensure food safety. The main progresses to date are presented, highlighting potential prospects for the future.
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Title: DNA Sequencing Sensors: An Overview

Section 1: Introduction
Description 1: Provide an introduction to the history and significance of DNA sequencing, starting from the discovery of DNA as the genetic material, to the development and evolution of sequencing technologies. 

Section 2: Short-Read Sequencing
Description 2: Describe the methodologies and technologies used in short-read sequencing platforms, including key examples like the Roche 454, AB SOLiD, Illumina, and Ion Torrent platforms. Highlight the principles and detection methods used.

Section 3: Single-Molecule Real-Time Long Read Sequencing
Description 3: Discuss the advancements in third-generation sequencing technologies, focusing on single-molecule real-time sequencing platforms like Pacific Biosciences and Oxford Nanopore. Explain how these platforms differ from short-read sequencing in their approach and technology.

Section 4: 454 Roche Platform
Description 4: Provide a detailed description of the Roche 454 platform, explaining its pyrosequencing methodology, detection system, and unique features.

Section 5: AB SOLiD Platform
Description 5: Explain the sequencing by oligonucleotide ligation and detection (SOLiD) platform, focusing on its fluorescence detection methodology and how it differentiates from other sequencing techniques.

Section 6: Illumina Platform
Description 6: Describe the Illumina platform's use of fluorescence-labeled nucleotide analogs and bridge PCR for DNA sequencing, along with its detection technologies.

Section 7: Ion Torrent Platform
Description 7: Outline the technology behind the Ion Torrent platform, emphasizing its semiconductor technology and non-optical sensors for sequencing.

Section 8: Pacific Bioscience Platform
Description 8: Discuss the single-molecule real-time sequencing (SMRT) technology utilized by Pacific Biosciences, explaining the role of nanosensors and zero-mode waveguides (ZMW).

Section 9: Oxford Nanopore Platform
Description 9: Detail the nanopore-based DNA sequencing technology of the Oxford Nanopore platform, describing how it detects DNA sequence through changes in voltage as molecules pass through pores.

Section 10: Conclusions
Description 10: Summarize the evolution and importance of DNA sequencing sensors, highlighting the ongoing advancements in the field and the future potential of fourth-generation sequencing technologies.

Section 11: Conflicts of Interest
Description 11: Mention any potential conflicts of interest disclosed by the authors.