DNA sequencing: an overview of solid-state and biological nanopore-based methods

The field of sequencing is a topic of significant interest since its emergence and has become increasingly important over time. Impressive achievements have been obtained in this field, especially in relations to DNA and RNA sequencing. Since the first achievements by Sanger and colleagues in the 1950s, many sequencing techniques have been developed, while others have disappeared. DNA sequencing has undergone three generations of major evolution. Each generation has its own specifications that are mentioned briefly. Among these generations, nanopore sequencing has its own exciting characteristics that have been given more attention here. Among pioneer technologies being used by the third-generation techniques, nanopores, either biological or solid-state, have been experimentally or theoretically extensively studied. All sequencing technologies have their own advantages and disadvantages, so nanopores are not free from this general rule. It is also generally pointed out what research has been done to overcome the obstacles. In this review, biological and solid-state nanopores are elaborated on, and applications of them are also discussed briefly.     

Nanopore sequencing technology: nanopore preparations

For the past decade, nanometer-scale pores have been developed as a powerful technique for sensing biological macromolecules. Various potential applications using these nanopores have been reported at the proof-of-principle stage, with the eventual aim of using them as an alternative to de novo DNA sequencing. Currently, there have been two general approaches to prepare nanopores for nucleic acid analysis: organic nanopores, such as alpha-hemolysin pores, are commonly used for DNA analysis, whereas synthetic solid-state nanopores have also been developed using various conventional and non-conventional fabrication techniques. In particular, synthetic nanopores with pore sizes smaller than the alpha-hemolysin pores have been prepared, primarily by electron-beam-assisted techniques: these are more robust and have better dimensional adjustability. This review will examine current methods of nanopore preparation, ranging from organic pore preparations to recent developments in synthetic nanopore fabrications.     

Next-generation sequencing technologies for environmental DNA research

Since 2005, advances in next-generation sequencing technologies have revolutionized biological science. The analysis of environmental DNA through the use of specific gene markers such as species-specific DNA barcodes has been a key application of next-generation sequencing technologies in ecological and environmental research. Access to parallel, massive amounts of sequencing data, as well as subsequent improvements in read length and throughput of different sequencing platforms, is leading to a better representation of sample diversity at a reasonable cost. New technologies are being developed rapidly and have the potential to dramatically accelerate ecological and environmental research. The fast pace of development and improvements in next-generation sequencing technologies can reflect on broader and more robust applications in environmental DNA research. Here, we review the advantages and limitations of current next-generation sequencing technologies in regard to their application for environmental DNA analysis.     

Single-molecule DNA sequencing technologies for future genomics research

During the current genomics revolution, the genomes of a large number of living organisms have been fully sequenced. However, with the advent of new sequencing technologies, genomics research is now at the threshold of a second revolution. Several second-generation sequencing platforms became available in 2007, but a further revolution in DNA resequencing technologies is being witnessed in 2008, with the launch of the first single-molecule DNA sequencer (Helicos Biosciences), which has already been used to resequence the genome of the M13 virus. This review discusses several single-molecule sequencing technologies that are expected to become available during the next few years and explains how they might impact on genomics research.     

Single polypeptide detection using a translocon EXP2 nanopore

DNA sequencing using nanopores has already been achieved and commercialized; the next step in advancing nanopore technology is towards protein sequencing. Although trials have been reported for discriminating the 20 amino acids using biological nanopores and short peptide carriers, it remains challenging. The size compatibility between nanopores and peptides is one of the issues to be addressed. Therefore, exploring biological nanopores that are suitable for peptide sensing is key in achieving amino acid sequence determination. Here, we focus on EXP2, the transmembrane protein of a translocon from malaria parasites, and describe its pore-forming properties in the lipid bilayer. EXP2 mainly formed a nanopore with a diameter of 2.5 nm assembled from 7 monomers. Using the EXP2 nanopore allowed us to detect poly-L-lysine (PLL) at a single-molecule level. Furthermore, the EXP2 nanopore has sufficient resolution to distinguish the difference in molecular weight between two individual PLL, long PLL (Mw: 30,000–70,000) and short PLL (Mw: 10,000). Our results contribute to the accumulation of information for peptide-detectable nanopores.     

Solid-state nanopores for biosensing with submolecular resolution

Biological cell membranes contain various types of ion channels and transmembrane pores in the 1-100 nm range, which are vital for cellular function. Individual channels can be probed electrically, as demonstrated by Neher and Sakmann in 1976 using the patch-clamp technique [Neher and Sakmann (1976) Nature 260, 799-802]. Since the 1990s, this work has inspired the use of protein or solid-state nanopores as inexpensive and ultrafast sensors for the detection of biomolecules, including DNA, RNA and proteins, but with particular focus on DNA sequencing. Solid-state nanopores in particular have the advantage that the pore size can be tailored to the analyte in question and that they can be modified using semi-conductor processing technology. This establishes solid-state nanopores as a new class of single-molecule biosensor devices, in some cases with submolecular resolution. In the present review, we discuss a few of the most important recent developments in this field and how they might be applied to studying protein-protein and protein-DNA interactions or in the context of ultra-fast DNA sequencing.     

HITAC-seq enables high-throughput cost-effective sequencing of plasmids and DNA fragments with identity

DNA sequencing is vital for many aspects of biological research and diagnostics. Despite the development of second and third generation sequencing technologies, Sanger sequencing has long been the only choice when required to precisely track each sequenced plasmids or DNA fragments. Here, we report a complete set of novel barcoding and assembling system, Highly-parallel Indexed Tagmentation-reads Assembled Consensus sequencing(HITAC-seq), that could massively sequence and track the identities of each individual sequencing sample. With the cost of much less than that of single read of Sanger sequencing,HITAC-seq can generate high-quality contiguous sequences of up to 10 kilobases or longer. The capability of HITAC-seq was confirmed through large-scale sequencing of thousands of plasmid clones and hundreds of amplicon fragments using approximately 100 pg of input DNAs. Due to its long synthetic length, HITACseq was effective in detecting relatively large structural variations, as demonstrated by the identification of a～1.3 kb Copia retrotransposon insertion in the upstream of a likely maize domestication gene. Besides being a practical alternative to traditional Sanger sequencing, HITAC-seq is suitable for many highthroughput sequencing and genotyping applications.     

Automation of Molecular-Based Analyses: A Primer on Massively Parallel Sequencing

Recent advances in genetics have been enabled by new genetic sequencing techniques called massively parallel sequencing (MPS) or next-generation sequencing. Through the ability to sequence in parallel hundreds of thousands to millions of DNA fragments, the cost and time required for sequencing has dramatically decreased. There are a number of different MPS platforms currently available and being used in Australia. Although they differ in the underlying technology involved, their overall processes are very similar: DNA fragmentation, adaptor ligation, immobilisation, amplification, sequencing reaction and data analysis. MPS is being used in research, translational and increasingly now also in clinical settings. Common applications include sequencing of whole genomes, whole exomes or targeted genes for disease-causing gene discovery, genetic diagnosis and targeted cancer therapy. Even though the revolution that is occurring with MPS is exciting due to its increasing use, improving and emerging technologies and new applications, significant challenges still exist. Particularly challenging issues are the bioinformatics required for data analysis, interpretation of results and the ethical dilemma of ‘incidental findings’.     

Distinguishable populations report on the interactions of single DNA molecules with solid-state nanopores

Solid-state nanopores have received increasing interest over recent years because of their potential for genomic screening and sequencing. In particular, small nanopores (2-5 nm in diameter) allow the detection of local structure along biological molecules, such as proteins bound to DNA or possibly the secondary structure of RNA molecules. In a typical experiment, individual molecules are translocated through a single nanopore, thereby causing a small deviation in the ionic conductance. A correct interpretation of these conductance changes is essential for our understanding of the process of translocation, and for further sophistication of this technique. Here, we present translocation measurements of double-stranded DNA through nanopores down to the diameter of the DNA itself (1.8-7 nm at the narrowest constriction). In contrast to previous findings on such small nanopores, we find that single molecules interacting with these pores can cause three distinct levels of conductance blockades. We attribute the smallest conductance blockades to molecules that briefly skim the nanopore entrance without translocating, the intermediate level of conductance blockade to regular head-to-tail translocations, and the largest conductance blockades to obstruction of the nanopore entrance by one or multiple (duplex) DNA strands. Our measurements are an important step toward understanding the conductance blockade of biomolecules in such small nanopores, which will be essential for future applications involving solid-state nanopores.     

A survey of enabling technologies in synthetic biology

Background

Realizing constructive applications of synthetic biology requires continued development of enabling technologies as well as policies and practices to ensure these technologies remain accessible for research. Broadly defined, enabling technologies for synthetic biology include any reagent or method that, alone or in combination with associated technologies, provides the means to generate any new research tool or application. Because applications of synthetic biology likely will embody multiple patented inventions, it will be important to create structures for managing intellectual property rights that best promote continued innovation. Monitoring the enabling technologies of synthetic biology will facilitate the systematic investigation of property rights coupled to these technologies and help shape policies and practices that impact the use, regulation, patenting, and licensing of these technologies.

Results

We conducted a survey among a self-identifying community of practitioners engaged in synthetic biology research to obtain their opinions and experiences with technologies that support the engineering of biological systems. Technologies widely used and considered enabling by survey participants included public and private registries of biological parts, standard methods for physical assembly of DNA constructs, genomic databases, software tools for search, alignment, analysis, and editing of DNA sequences, and commercial services for DNA synthesis and sequencing. Standards and methods supporting measurement, functional composition, and data exchange were less widely used though still considered enabling by a subset of survey participants.

Conclusions

The set of enabling technologies compiled from this survey provide insight into the many and varied technologies that support innovation in synthetic biology. Many of these technologies are widely accessible for use, either by virtue of being in the public domain or through legal tools such as non-exclusive licensing. Access to some patent protected technologies is less clear and use of these technologies may be subject to restrictions imposed by material transfer agreements or other contract terms. We expect the technologies considered enabling for synthetic biology to change as the field advances. By monitoring the enabling technologies of synthetic biology and addressing the policies and practices that impact their development and use, our hope is that the field will be better able to realize its full potential.

     

Single-Stranded DNA within Nanopores: Conformational Dynamics and Implications for Sequencing; a Molecular Dynamics Simulation Study

Engineered protein nanopores, such as those based on α-hemolysin from Staphylococcus aureus have shown great promise as components of next-generation DNA sequencing devices. However, before such protein nanopores can be used to their full potential, the conformational dynamics and translocation pathway of the DNA within them must be characterized at the individual molecule level. Here, we employ atomistic molecular dynamics simulations of single-stranded DNA movement through a model α-hemolysin pore under an applied electric field. The simulations enable characterization of the conformations adopted by single-stranded DNA, and allow exploration of how the conformations may impact on translocation within the wild-type model pore and a number of mutants. Our results show that specific interactions between the protein nanopore and the DNA can have a significant impact on the DNA conformation often leading to localized coiling, which in turn, can alter the order in which the DNA bases exit the nanopore. Thus, our simulations show that strategies to control the conformation of DNA within a protein nanopore would be a distinct advantage for the purposes of DNA sequencing.     

Sequencing enabling design and learning in synthetic biology

The ability to read and quantify nucleic acids such as DNA and RNA using sequencing technologies has revolutionized our understanding of life. With the emergence of synthetic biology, these tools are now being put to work in new ways — enabling de novo biological design. Here, we show how sequencing is supporting the creation of a new wave of biological parts and systems, as well as providing the vast data sets needed for the machine learning of design rules for predictive bioengineering. However, we believe this is only the tip of the iceberg and end by providing an outlook on recent advances that will likely broaden the role of sequencing in synthetic biology and its deployment in real-world environments.     

复杂基因组测序技术研究进展

复杂基因组指的是无法使用常规测序和组装手段直接解析的一类基因组,通常指包含高比例重复序列、高杂合度、极端GC含量、存在难消除异源DNA污染的基因组。为了解决复杂基因组的测序和组装问题,需要分别从基因组测序实验方法、测序技术平台、组装算法与策略3个方面进行深入研究。本文详细介绍了复杂基因组测序组装相关的现有技术与方法,并结合复杂基因组经典实例介绍了复杂基因组测序的技术解决途径和发展历程,可为制订合适的复杂基因组测序策略提供参考。     

Nanopores formed by DNA origami: A review

Nanopores have emerged over the past two decades to become an important technique in single molecule experimental physics and biomolecule sensing. Recently DNA nanotechnology, in particular DNA origami, has been used for the formation of nanopores in insulating materials. DNA origami is a very attractive technique for the formation of nanopores since it enables the construction of 3D shapes with precise control over geometry and surface functionality. DNA origami has been applied to nanopore research by forming hybrid architectures with solid state nanopores and by direct insertion into lipid bilayers. This review discusses recent experimental work in this area and provides an outlook for future avenues and challenges.     

Advanced technologies for genomic analysis in farm animals and its application for QTL mapping

Rapid progress in farm animal breeding has been made in the last few decades. Advanced technologies for genomic analysis in molecular genetics have led to the identification of genes or markers associated with genes that affect economic traits. Molecular markers, large-insert libraries and RH panels have been used to build the genetic linkage maps, physical maps and comparative maps in different farm animals. Moreover, EST sequencing, genome sequencing and SNPs maps are helping us to understand how genomes function in various organisms and further areas will be studied by DNA microarray technologies and proteomics methods. Because most economically important traits in farm animals are controlled by multiple genes and the environment, the main goal of genome research in farm animals is to map and characterize genes determining QTL. There are two main strategies to identify trait loci, candidate gene association tests and genome scan approaches. In recent years, some new concepts, such as RNAi, miRNA and eQTL, have been introduced into farm animal research, especially for QTL mapping and finding QTN. Several genes that influence important traits have already been identified or are close to being identified, and some of them have been applied in farm animal breeding programs by marker-assisted selection.     

Whole-genome re-sequencing

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.     

Use of reverse-phase chromatography in the Maxam-Gilbert method of DNA sequencing A step toward automation

Reverse-phase chromatography has been used for carrying out Maxam-Gilbert sequencing reactions. The DNA loss has been minimized. Time-consuming ethanol precipitation and lyophilization of piperidine have been eliminated. The method has been used successfully in sequencing oligonucleotides as well as large DNA fragments. Based upon these modifications, we have developed a semiautomatic device for carrying out the Maxam-Gilber sequencing reactions. It is also possible to fully automate the Maxam-Gilbert method of DNA sequencing, and we present the diagram of a fully automated set up. The development of such a machine should expedite the sequencing of the large number of genes being cloned.