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.     

Comparison of sequence reads obtained from three next-generation sequencing platforms

Next-generation sequencing technologies enable the rapid cost-effective production of sequence data. To evaluate the performance of these sequencing technologies, investigation of the quality of sequence reads obtained from these methods is important. In this study, we analyzed the quality of sequence reads and SNP detection performance using three commercially available next-generation sequencers, i.e., Roche Genome Sequencer FLX System (FLX), Illumina Genome Analyzer (GA), and Applied Biosystems SOLiD system (SOLiD). A common genomic DNA sample obtained from Escherichia coli strain DH1 was applied to these sequencers. The obtained sequence reads were aligned to the complete genome sequence of E. coli DH1, to evaluate the accuracy and sequence bias of these sequence methods. We found that the fraction of "junk" data, which could not be aligned to the reference genome, was largest in the data set of SOLiD, in which about half of reads could not be aligned. Among data sets after alignment to the reference, sequence accuracy was poorest in GA data sets, suggesting relatively low fidelity of the elongation reaction in the GA method. Furthermore, by aligning the sequence reads to the E. coli strain W3110, we screened sequence differences between two E. coli strains using data sets of three different next-generation platforms. The results revealed that the detected sequence differences were similar among these three methods, while the sequence coverage required for the detection was significantly small in the FLX data set. These results provided valuable information on the quality of short sequence reads and the performance of SNP detection in three next-generation sequencing platforms.     

高通量测序技术在细菌耐药中的应用

细菌耐药已成为威胁全球人类公共健康的重要因素之一,快速、准确明确细菌耐药的特性、机制及传播特征对疾病治疗及控制耐药菌的传播具有重要意义。高通量测序技术可以同时平行检测多个基因序列的状态,已广泛应用于细菌耐药检测。目前高通量测序技术在细菌耐药领域的应用主要有:全基因组测序技术、目标区域测序技术和宏基因组测序技术。所采用的测序平台主要为Illumina、Ion Torrent、BGI等二代测序和Pacific Biosciences、Oxford Nonopore 等三代测序平台。通过细菌耐药基因预测细菌耐药表型的准确性在很大程度上依赖于成熟的专业耐药基因数据库,各种通用型、特异型及隐马尔可夫模型耐药基因数据库的建立和完善,为高通量测序技术在细菌耐药领域的应用提供了坚实的基础。本文简要介绍了高通量测序技术、数据分析方法及相应测序平台在细菌耐药领域中的应用进展,并同时介绍了细菌耐药数据库的现状。     

高通量测序技术在农作物全基因组序列测定中的应用概览

近几年飞速发展的高通量测序技术(next generation sequencing,NGS)在生命科学研究的各个领域充分展现了其低成本、高通量和应用面广等优势。在现代农业生物技术领域,利用高通量测序技术,科学家们不仅能更经济而高效对农作物、模式植物或不同栽培品种进行深入的全基因组测序、重测序,也可以对成百上千的栽培品种进行高效而准确的遗传差异分析、分子标记分析、连锁图谱分析、表观遗传学分析、转录组分析,进而改进农作物的育种技术,加快新品种的育种研究。其中,获得农作物的全基因组序列是其他研究和分析的基础。本文通过介绍近年来发表的一些利用高通量测序技术进行的农作物全基因组测定和组装的工作,展示高通量测序技术在现代农业生物技术领域的广泛前景以及其建立起来的研究基础。     

The impact of next-generation sequencing on genomics

This article reviews basic concepts,general applications,and the potential impact of next-generation sequencing(NGS)technologies on genomics,with particular reference to currently available and possible future platforms and bioinformatics.NGS technologies have demonstrated the capacity to sequence DNA at unprecedented speed,thereby enabling previously unimaginable scientific achievements and novel biological applications.But,the massive data produced by NGS also presents a significant challenge for data storage,analyses,and management solutions.Advanced bioinformatic tools are essential for the successful application of NGS technology.As evidenced throughout this review,NGS technologies will have a striking impact on genomic research and the entire biological field.With its ability to tackle the unsolved challenges unconquered by previous genomic technologies,NGS is likely to unravel the complexity of the human genome in terms of genetic variations,some of which may be confined to susceptible loci for some common human conditions.The impact of NGS technologies on genomics will be far reaching and likely change the field for years to come.     

基于测序技术的畜禽基因组学研究进展

人类通过数千年的驯化和近代以来有计划性的育种,形成了当今多样化的畜禽品种,从而提供丰富的动物源性蛋白满足人类需求。在过去的100年里,数量遗传学应用于动物育种领域引发了畜禽育种技术的革命,但畜禽机体遗传发育体系相当复杂,一些性状仍然难以通过基于系谱的育种值进行高效选育,遗传潜能尚未充分发掘。人类基因组计划带来的理念和技术极大促进了畜禽基因组学的发展,使得人们可以从全基因组水平精准定位功能变异,挖掘功能元件的生物学意义,为畜禽分子设计育种提供重要的理论基础。本文对近10年来猪(Sus scrofa)、牛(Bos taurus)、牦牛(Bos grunniens)、山羊(Capra hircus)、绵羊(Ovis aries)、鸡(Gallus gallus)、鸭(Anas platyrhynchos)和鹅(Anser cygnoides)等主要畜禽的基因组学研究进展进行综述,分别从参考基因组构建和群体基因组学分析两个方面进行论述,并对畜禽基因组未来的研究工作进行了展望。     

合成基因组学：设计与合成的艺术

随着基因组相关技术(测序、编辑、合成等)和知识(功能基因组学)的日益成熟,合成基因组学在本世纪迎得了发展的契机。病毒、原核生物的全基因组相继被化学合成并支持生命的存活,第1个真核生物合成基因组计划已经完成过半,人类基因组编写计划提上日程。在基因组合成的实践过程中,研究者们不断探索对基因组进行重编和设计所应遵循的规则,提高从头合成、组装和替换基因组的技术手段。合成基因组在工业、环境、健康和基础研究领域有着广阔的应用前景,同时也带来了相应的伦理问题。结合在Sc2.0计划中的基因组合成研究和近期合成基因组学所取得的重大进展,本文综述了基因组设计和合成相关的科学、技术和伦理内容,并探讨了未来发展所面对的挑战。作为合成生物学最重要的领域之一,合成基因组学方兴未艾。     

单细胞基因组学分析的技术前沿

基因组学已经深刻地改变了生命科学的诸多领域的面貌。目前它的主要内容是新的全基因组碱基序列的测定和在全基因组范围内鉴定那些在不同水平上影响生命活动的基因群的功能和相互作用。为达此要求,近年出现的第二代测序(深度测序)技术和基因芯片技术发挥了关键作用,但是两者都需要足够的高质量的核酸样品。所以,在只有或只能用单细胞或极少量细胞的情况下,如果没有特殊手段,上述分析往往不能常规、方便地进行。文章以DNA扩增为主线,综合阐述了目前在单细胞(特别是微生物)全基因组测序和大基因组的靶向重测序,以及对单细胞或微量细胞进行的基于深度测序或芯片杂交的功能基因组分析,如转录组、ChIP和DNA的CpG甲基化分析等的最新策略和技术,评价了单细胞基因组测序和功能基因组学各技术的特点并对发展前景进行了展望。     

人类基因组结构变异检测方法

本研究介绍了基因组结构变异检测的生物信息学基本方法和前沿技术。对基于第二代测序技术的四种检测方法(读对方法,读深方法,分裂片段方法和序列拼接方法)的原理和特点进行了详细解读,分析了第二代测序技术应用在检测结构变异上的特点与发展趋势。最后介绍了三代测序、Linked-reads和光学物理图谱等新技术在基因组结构变异检测中的应用,论述了融合新技术的结构变异检测方法的特点与优势。     

Translational genomics for plant breeding with the genome sequence explosion

The use of next‐generation sequencers and advanced genotyping technologies has propelled the field of plant genomics in model crops and plants and enhanced the discovery of hidden bridges between genotypes and phenotypes. The newly generated reference sequences of unstudied minor plants can be annotated by the knowledge of model plants via translational genomics approaches. Here, we reviewed the strategies of translational genomics and suggested perspectives on the current databases of genomic resources and the database structures of translated information on the new genome. As a draft picture of phenotypic annotation, translational genomics on newly sequenced plants will provide valuable assistance for breeders and researchers who are interested in genetic studies.     

高通量测序技术及其应用

高通量测序技术是DNA测序发展历程的一个里程碑,它为现代生命科学研究提供了前所未有的机遇。详细介绍了以454、Solexa和SOLiD为代表的第二代高通量测序技术,以HeliScope TIRM和Pacific Biosciences SMRT为代表的单分子测序技术,以及最近Life Science公司推出的Ion Personal Genome Machine (PGM)测序技术等高通量测序技术的最新进展。在此基础上,阐述了高通量测序技术在基因组测序、转录组测序、基因表达调控、转录因子结合位点的检测以及甲基化等研究领域的应用。最后,讨论了高通量测序技术在成本和后续数据分析等方面存在的问题及其未来的发展前景。     

实现“终极版”核苷酸测序仪的技术要素

20世纪70年代发明的第一代DNA测序技术,尽管测序通量有限,却成功地保证了“人类基因组计划”的实施;世纪之交出现的下一代(第二代) DNA测序技术经历了通量飞跃,为各种精准医学项目的实施提供了保障;目前的第三代技术,尽管通量居二代之后,但读长和单分子测序优势也让其有立足之本;第四代测序技术的基本标志是不经过cDNA (以RNA为模版合成的互补DNA),无PCR扩增,而直接测定单分子RNA序列,以及确定单分子RNA上的修饰核苷酸位点。从技术的角度看,第三、四代技术有一定技术要素共享(比如在单分子水平测定DNA序列),但是就测序对象而言,第四代应该属于“终极版”核苷酸测序仪：可以从单细胞出发,既能测定DNA序列,也可以测定RNA序列,也可以直接确定修饰核苷酸位点。因此,要实现这个“终极版”核苷酸测序仪,就要调动相关核心技术要素,而这些要素毫无疑问地会涉及物理、化学、工程学、生物学、半导体科学、计算机科学等领域的前沿技术,包括纳米科学、单分子光学、单分子拉曼光谱、单分子核磁共振、单分子酶学、人工智能等所谓的“硬科技”。其功能是从单细胞的裂解开始,经微纳结构实现组分分流后,直接导入RNA序列测定单元,定量分析细胞RNA分子的种类、数量、序列和修饰核苷酸位点的存在频率。本文系统介绍了第四代测序仪的可能技术要素,以及应用需求和新研究范式。     

Direct Chloroplast Sequencing: Comparison of Sequencing Platforms and Analysis Tools for Whole Chloroplast Barcoding

Direct sequencing of total plant DNA using next generation sequencing technologies generates a whole chloroplast genome sequence that has the potential to provide a barcode for use in plant and food identification. Advances in DNA sequencing platforms may make this an attractive approach for routine plant identification. The HiSeq (Illumina) and Ion Torrent (Life Technology) sequencing platforms were used to sequence total DNA from rice to identify polymorphisms in the whole chloroplast genome sequence of a wild rice plant relative to cultivated rice (cv. Nipponbare). Consensus chloroplast sequences were produced by mapping sequence reads to the reference rice chloroplast genome or by de novo assembly and mapping of the resulting contigs to the reference sequence. A total of 122 polymorphisms (SNPs and indels) between the wild and cultivated rice chloroplasts were predicted by these different sequencing and analysis methods. Of these, a total of 102 polymorphisms including 90 SNPs were predicted by both platforms. Indels were more variable with different sequencing methods, with almost all discrepancies found in homopolymers. The Ion Torrent platform gave no apparent false SNP but was less reliable for indels. The methods should be suitable for routine barcoding using appropriate combinations of sequencing platform and data analysis.     

Application of next-generation sequencing for the identification of herbal products

Conventional Sanger Sequencing for authentication of herbal products is difficult since they are mixture of herbs with fragmented DNA. Next-generation sequencing (NGS) techniques give massive parallelization of sequencing reaction to generate multiple reads with various read length, thus different components in herbal products with fragmented DNA can be identified. NGS is especially suitable for animal derived products with the lack of effective markers for chemical analysis. Currently, second generation sequencing such as Illumina Sequencing and Ion Torrent Sequencing, and third generation sequencing such as PacBio Sequencing and Nanopore Sequencing are representative NGS platforms. The constructed library is first sequenced to obtain a pool of genomic data, followed by bioinformatics analysis and comparison with DNA database. NGS also facilitates the determination of contaminant which is essential for quality control regulation in Good Manufacturing Practice (GMP) factory. In this article, we provide an overview on NGS, summarize the cases on the use of NGS to identify herbal products, discuss the key technological challenges and provide perspectives on future directions for authentication and quality control of herbal products.