Stepping stones in DNA sequencing

In recent years there have been tremendous advances in our ability to rapidly and cost-effectively sequence DNA. This has revolutionized the fields of genetics and biology, leading to a deeper understanding of the molecular events in life processes. The rapid technological advances have enormously expanded sequencing opportunities and applications, but also imposed strains and challenges on steps prior to sequencing and in the downstream process of handling and analysis of these massive amounts of sequence data. Traditionally, sequencing has been limited to small DNA fragments of approximately one thousand bases (derived from the organism's genome) due to issues in maintaining a high sequence quality and accuracy for longer read lengths. Although many technological breakthroughs have been made, currently the commercially available massively parallel sequencing methods have not been able to resolve this issue. However, recent announcements in nanopore sequencing hold the promise of removing this read-length limitation, enabling sequencing of larger intact DNA fragments. The ability to sequence longer intact DNA with high accuracy is a major stepping stone towards greatly simplifying the downstream analysis and increasing the power of sequencing compared to today. This review covers some of the technical advances in sequencing that have opened up new frontiers in genomics.     

Commercial high-throughput sequencing and its applications in DNA analysis

DNA sequences can be used for the analysis of genetic variation and gene function. The high-throughput sequencing techniques that have been developed over the past three years can read as many as one billion bases per run, and are far less expensive than the traditional Sanger sequencing method. Therefore, the high-throughput sequencing has been applied extensively to genomic analyses, such as screening for mutations, construction of genomic methylation maps, and the study of DNA-protein interactions. Although they have only been available for a short period, high-throughput sequencing techniques are profoundly affecting many of the life sciences, and are opening out new potential avenues of research. With the highly-developed commercial high-throughput sequencing platforms, each laboratory has the opportunity to explore this research field. Therefore, in this paper, we have focused on commercially-popular high-throughput sequencing techniques and the ways in which they have been applied over the past three years.     

高通量测序技术结合正向遗传学手段在基因定位研究中的应用

传统的利用正向遗传学方法的基因定位一般是通过构建遗传连锁图谱进行的,该过程步骤繁琐、耗时耗力,很多情形下定位精确度低、区间大。随着高通量测序技术的快速发展以及测序成本的不断降低,多种简单快捷的利用测序手段定位基因的方法被开发出来,包括对突变体基因组直接测序定位、突变体材料构建混池测序定位和遗传分离群体测序构建图谱定位等,还可以对转录组和部分基因组进行测序定位。这些方法可以在核苷酸水平鉴定突变位点,并已推广到复杂的遗传背景中。近期报道的一些测序定位甚至是在不依赖于参考基因组序列、遗传杂交和连锁信息的情况下完成的,这使得很多非模式物种也能开展正向遗传学研究。本文就这些新技术及其在基因定位中的应用进行了综述。     

A simplified biochemistry for DNA sequencing

A simplified method of DNA sequencing by dideoxy chain termination is developed that approaches a single-step protocol. Utilizing the sequencing advantages contributed by a thermophilic polymerase and a guanine analog, stable sequencing reaction concentrates have been obtained that readily perform the entire sequencing reaction simply by adding prepared DNA to each of the four reaction concentrates required by this method. The mechanics and dynamics of these reactions have been investigated and the capacity of these reactions to withstand normal user variation is demonstrated. This study focuses on one form of this simplified method embodied in the FASTaq DNA sequencing kit.     

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.     

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.     

纳米孔测序技术在环境微生物研究中的应用

高通量测序技术是研究环境微生物的有效手段,而以纳米孔测序为代表的第三代测序技术以其测序读长长、测序速度快、测序数据实时监控、仪器方便携带、无GC偏好性、无需经过PCR扩增等显著优势有力推动了环境微生物研究的发展.本文对纳米孔测序技术的技术原理和特点进行了简要概述,重点介绍了纳米孔测序技术在环境微生物扩增子测序、宏基因组...     

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.     

Nanopore sequencing technology: research trends and applications

Nanopore sequencing is one of the most promising technologies being developed as a cheap and fast alternative to the conventional Sanger sequencing method. Protein or synthetic nanopores have been used to detect DNA or RNA molecules. Although none of the technologies to date has shown single-base resolution for de novo DNA sequencing, there have been several reports of alpha-hemolysin protein nanopores being used for basic DNA analyses, and various synthetic nanopores have been fabricated. This review will examine current nanopore sequencing technologies, including recent developments of new applications.     

Recent patents of nanopore DNA sequencing technology: progress and challenges

DNA sequencing techniques witnessed fast development in the last decades, primarily driven by the Human Genome Project. Among the proposed new techniques, Nanopore was considered as a suitable candidate for the single DNA sequencing with ultrahigh speed and very low cost. Several fabrication and modification techniques have been developed to produce robust and well-defined nanopore devices. Many efforts have also been done to apply nanopore to analyze the properties of DNA molecules. By comparing with traditional sequencing techniques, nanopore has demonstrated its distinctive superiorities in main practical issues, such as sample preparation, sequencing speed, cost-effective and read-length. Although challenges still remain, recent researches in improving the capabilities of nanopore have shed a light to achieve its ultimate goal: Sequence individual DNA strand at single nucleotide level. This patent review briefly highlights recent developments and technological achievements for DNA analysis and sequencing at single molecule level, focusing on nanopore based methods.     

基因测序技术的发展以及对个体化用药水平提高的影响

自基因测序技术发明之时起,就已开始运用在生命科学的研究中,对揭示生命本质的研究起到了关键作用。基因测序技术的运用推动了生命科学的发展,并由此引申了更多的科学问题;人们对未知领域的渴求又推动了基因测序技术的进步,发展出更高速、更低价的新技术。随着测序技术的逐步应用,临床个体化用药的水平有了极大的提高。基因测序技术目前已经成功应用于遗传基因多态性标志物的筛选中,使基因导向的合理用药成为可能;还成功应用于疾病组织突变位点标志物的筛查中,使肿瘤靶向用药成为可能;在病原体耐药基因突变检测中的应用,使基于细菌或病毒耐药突变的个体化用药成为可能。随着测序技术向更高通量、更高精度、更低成本的方向发展,基于基因检测的个体化健康时代将会到来。     

新一代测序技术的研究进展

大规模DNA测序技术是揭秘人类和其它生物遗传密码的重要技术,在分子生物学和基础医学领域有广泛应用。第二代测序技术的出现使DNA测序的通量大幅提高,测序的成本大幅下降,原来只有在大型测序中心才能完成的测序任务现在已经可以在更多的实验室展开。但是,早期的第二代测序技术仍然存在诸如文库构建过程复杂、测序成本依然较高等缺点。为了克服上述缺点,近三年发展了几种新的第二代和第三代测序技术,这些技术不仅继承了早期第二代测序技术通量高的优点,而且在文库构建等方面取得了重要突破,进一步简化了测序操作,降低了测序成本,缩短了测序时间。本文就几种最新的大规模测序技术的原理、特点与发展趋势进行简要介绍。     

ConDeTri--a content dependent read trimmer for Illumina data

During the last few years, DNA and RNA sequencing have started to play an increasingly important role in biological and medical applications, especially due to the greater amount of sequencing data yielded from the new sequencing machines and the enormous decrease in sequencing costs. Particularly, Illumina/Solexa sequencing has had an increasing impact on gathering data from model and non-model organisms. However, accurate and easy to use tools for quality filtering have not yet been established. We present ConDeTri, a method for content dependent read trimming for next generation sequencing data using quality scores of each individual base. The main focus of the method is to remove sequencing errors from reads so that sequencing reads can be standardized. Another aspect of the method is to incorporate read trimming in next-generation sequencing data processing and analysis pipelines. It can process single-end and paired-end sequence data of arbitrary length and it is independent from sequencing coverage and user interaction. ConDeTri is able to trim and remove reads with low quality scores to save computational time and memory usage during de novo assemblies. Low coverage or large genome sequencing projects will especially gain from trimming reads. The method can easily be incorporated into preprocessing and analysis pipelines for Illumina data. AVAILABILITY AND IMPLEMENTATION: Freely available on the web at http://code.google.com/p/condetri.     

High throughput direct end sequencing of BAC clones

Libraries constructed in bacterial artificial chromosome (BAC) vectors have become the choice for clone sets in high throughput genomic sequencing projects primarily because of their high stability. BAC libraries have been proposed as a source for minimally over-lapping clones for sequencing large genomic regions, and the use of BAC end sequences (i.e. sequences adjoining the insert sites) has been proposed as a primary means for selecting minimally overlapping clones for sequencing large genomic regions. For this strategy to be effective, high throughput methods for BAC end sequencing of all the clones in deep coverage BAC libraries needed to be developed. Here we describe a low cost, efficient, 96 well procedure for BAC end sequencing. These methods allow us to generate BAC end sequences from human and Arabidoposis libraries with an average read length of >450 bases and with a single pass sequencing average accuracy of >98%. Application of BAC end sequences in genomic sequen-cing is discussed.     

Enhancing PCR amplification and sequencing using DNA-binding proteins

The polymerase chain reaction (PCR) is a powerful core molecular biology technique, which when coupled to chain termination sequencing allows gene and DNA sequence information to be derived rapidly. A number of modifications to the basic PCR format have been developed in an attempt to increase amplification efficiency and the specificity of the reaction. We have applied the use of DNA-binding protein, gene 32 protein from bacteriophage T4 (T4gp32) to increase amplification efficiency with a number of diverse templates. In addition, we have found that using single-stranded DNA-binding protein (SSB) or recA protein in DNA sequencing reactions dramatically increases the resolution of sequencing runs. The use of DNA-binding proteins in amplification and sequencing may prove to be generally applicable in improving the yield and quality of a number of templates from various sources.     

基于第二代测序技术的基因资源挖掘

转录组研究一直是生命科学研究的一个重要方向,在第二代测序技术问世以前,已经产生了一些行之有效的转录组研究方法,但这些方法存在一定的局限性。第二代测序技术的出现不仅使转录组研究很快进入了高速发展期,同时也为遗传资源的挖掘提供了一套全新的技术平台。本文简要介绍了第二代测序技术的化学原理和特性,重点阐述了利用第二代测序技术进行转录组测序,从而在此基础上挖掘遗传资源的研究。     

第三代测序技术的研究进展与临床应用

Sanger测序法,又称第一代测序技术,作为测序金标准推动了人类基因组“工作框架图”的绘制,但通量低、成本高的缺点限制了其进一步大规模应用。第二代测序技术,又称下一代测序技术,因其通量高、成本低等优点使得基因测序在基础研究与临床诊疗中得到广泛应用,但短读长一直是其不可回避的技术短板。第三代测序技术的出现,因其具有长读长优势,为基因序列上复杂重复区域解析与高质量基因组组装提供了新的技术手段。近年来,第三代测序技术进一步发展与完善,同时在肿瘤、免疫、生殖等相关领域逐步体现出临床应用价值。本文将综述第三代测序技术的研究进展与临床应用。     

Genomic sequencing of single microbial cells from environmental samples

Recently developed techniques allow genomic DNA sequencing from single microbial cells [Lasken RS: Single-cell genomic sequencing using multiple displacement amplification. Curr Opin Microbiol 2007, 10:510-516]. Here, we focus on research strategies for putting these methods into practice in the laboratory setting. An immediate consequence of single-cell sequencing is that it provides an alternative to culturing organisms as a prerequisite for genomic sequencing. The microgram amounts of DNA required as template are amplified from a single bacterium by a method called multiple displacement amplification (MDA) avoiding the need to grow cells. The ability to sequence DNA from individual cells will likely have an immense impact on microbiology considering the vast numbers of novel organisms, which have been inaccessible unless culture-independent methods could be used. However, special approaches have been necessary to work with amplified DNA. MDA may not recover the entire genome from the single copy present in most bacteria. Also, some sequence rearrangements can occur during the DNA amplification reaction. Over the past two years many research groups have begun to use MDA, and some practical approaches to single-cell sequencing have been developed. We review the consensus that is emerging on optimum methods, reliability of amplified template, and the proper interpretation of 'composite' genomes which result from the necessity of combining data from several single-cell MDA reactions in order to complete the assembly. Preferred laboratory methods are considered on the basis of experience at several large sequencing centers where >70% of genomes are now often recovered from single cells. Methods are reviewed for preparation of bacterial fractions from environmental samples, single-cell isolation, DNA amplification by MDA, and DNA sequencing.