肠道微生物与疾病关系的研究新进展- 运用454 测序技术分析

由于传统研究方法成本和速度的限制,远远满足不了对微生物群落大规模的研究,以454测序为代表的新一代高通量测序技术凭借低成本、高通量、流动自动化的优势为研究微生物的多样性和组成提供了新的技术平台。本文就近年来454测序技术在研究人体肠道微生物与疾病关系的应用进行了综述。     

新一代测序技术在临床检测中的应用

新一代测序技术在问世不到十年的时间里,大大推动了基因组学研究的发展。新一代测序技术因其高通量、快速、单碱基成本低等特点,自问世以来便广泛应用于生物学基础研究中。近年来,随着各种新测序平台和技术的成熟以及对基因组知识的积累,新一代测序技术在分子检测、临床诊断等应用研究领域也开始崭露头角。本文简要综述了新一代测序技术平台以及在肿瘤、遗传病、复杂性疾病等检测和诊断中的应用和前景。     

高通量测序技术及其在微生物学研究中的应用

20世纪70年代发明的核酸测序技术为基因组学及其相关学科的发展做出了巨大贡献,本世纪初发展的以Illumina公司的HiSeq 2000,ABI公司的SOLID,和Roche公司的454技术为代表的高通量测序技术又为基因组学的发展注入了新活力.本文在阐述这些技术的基础上,着重讨论了新一代测序技术在微生物领域中的应用.     

454测序技术开发微卫星标记的研究进展

第二代测序技术(454测序为例)已成为测定基因组序列的一种成熟技术.454测序亦可应用于目标DNA区域分析,因此可用作微卫星标记的开发.较传统方法而言,具有便捷、高效等特点.目前,运用454技术进行基因组测序或转录组测序开发微卫星标记,用以研究种群生态学、构建遗传图谱等得到越来越多的重视和应用.综述了454测序技术在开发微卫星标记上的应用,并根据其优缺点对其应用前景进行了展望,旨在为应用454测序开发微卫星提供参考.     

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。     

转录组测序技术在整合网络调控中的应用现状及前景

2005年以来,以454焦磷酸测序技术、Solexa边合成边测序技术和SOLiD连续连接合成测序技术为标志的新一代测序技术相继诞生,使得全转录组商业化测序成为可能.针对转录因子与基因、转录因子与miRNA、以及miRNA与基因所构成的整合调控网络,转录组测序技术以高通量、高分辨率、高精度和价格低廉等优势发挥着巨大的作用.从miRNA与蛋白编码基因等层次分别介绍了转录组测序技术在基础研究、临床研究等领域的应用现状及发展前景.     

454测序技术在微生物生态学研究中的应用

以Sanger法(双脱氧核苷酸末端终止法)为代表的第1代测序技术由于其成本高、速度慢、通量低等不足,满足不了大规模测序的要求.进入21世纪后,以Roche 454为代表的第2代测序技术诞生了,454测序法作为一种高通量的测序方法,近年来已被广泛应用于微生物生态学研究中.介绍了该测序技术的原理和操作步骤,结合本实验室的研...     

落叶松干细胞发育模型研究中酌高通量测序技术

后基因组时代的生命科学研究对每一位林业科学工作者来说都充满了挑战。随着“后基因组时代”的到来,国际学术界已经认识到发展低成本、高效率的测序技术的重要,陛。在这样的背景下,高通量测序技术应运而生,成为DNA测序发展历程的一个新的里程碑,为现代生命科学领域的研究提供了前所未有的机遇。其代表性技术平台有Roche的454N序技术、Illumina的Solexa测序技术、microRNA微阵列芯片技术及降解组高通量测序技术。目前,此系列技术在模式植物中挖掘关键基因的研究已经非常成熟。在林木领域,尤其是在尚无参考基因组和基因组信息庞大的裸子植物中的应用逐渐成为研究热点,并已经取得了很大的进展。     

植物内生固氮菌系统发育进化新进展

在植物内生固氮菌系统发育进化关系研究中,常用的方法有形态学与蛋白质水平法、数值分类和自动化鉴定法、化学分类法、分子遗传学方法等。本文简要介绍了常用方法的关键技术,并归纳了它们的优缺点。生物学的研究进入基因组时代后,随着高通量DNA测序技术在微生物学领域应用的迅速发展,全基因组测序被应用到微生物系统发育进化研究中,然而目前并未发现对已测全基因组序列的植物内生固氮菌进行系统总结。本文在对已测序植物内生固氮菌进行归纳的基础上,又详细研究了基于基因组数据的几种具有代表性的新方法(ANI分析法、最大唯一匹配指数法、核心基因组分析、组分矢量法、基因流动性分析),并结合目前系统发育进化研究常用方法,对植物内生固氮菌系统发育进化研究趋势进行总结和展望,旨在使植物内生固氮菌的系统发育进化关系研究在精确度、可靠性等方面有所突破。     

使用边扩增边连接技术以两步PCR反应制备两个癌症候选基因的重测序DNA文库

近年来应大规模基因组学和遗传学研究发展的需要,能够提供“高通量、低成本、低劳动量”测序服务的新一代大规模并行测序技术应运而生.辅助其应用的生物信息分析软件和实验方法也层出不穷.而在关联研究如癌症研究中有广泛应用的基于PCR的基因组学和遗传学研究方法,却因其研究的目标区域较短,而较少地受益于采用“鸟枪法测序”策略的新一代测序技术.尽管已提出诸如“芯片捕获法”、“目标选择法”等技术克服这一难题,但这些方法对仪器和实验室操作的额外要求限制了它们的应用和推广.本实验提出一种能简便克服这一难题的“边扩增边连接方法”（Ligation by Amplification,LBA）：使用一对“公用接头”和两步PCR反应将多个目标区域随机连接起来,其随机连接而成的长链产物可被简易地随机片段化并在新一代测序仪上测序.使用专门设计的、包含公用接头的LBA引物,将人类基因组保守编码序列中两个癌症相关基因：BRCA1和BRCA2的外显子进行扩增,并将所得到的70个扩增产物在一步PCR过程中进行边扩增边连接.所得的长链LBA产物经随机片段化后制备成可被传统Sanger测序法和新一代边合成边测序技术测序的DNA文库,分别在ABI3730xl测序仪和Illumina/Solexa Genome Analyzer测序仪上进行测序.对目标序列的覆盖度、覆盖深度和单核苷酸多态性检测的有效性的生物信息学分析证明：运用本方法可高效地在新一代测序仪上进行基于PCR的重测序和遗传多态性研究,推动各种基于PCR的基因组学和遗传学研究的发展.     

The development and impact of 454 sequencing

The 454 Sequencer has dramatically increased the volume of sequencing conducted by the scientific community and expanded the range of problems that can be addressed by the direct readouts of DNA sequence. Key breakthroughs in the development of the 454 sequencing platform included higher throughput, simplified all in vitro sample preparation and the miniaturization of sequencing chemistries, enabling massively parallel sequencing reactions to be carried out at a scale and cost not previously possible. Together with other recently released next-generation technologies, the 454 platform has started to democratize sequencing, providing individual laboratories with access to capacities that rival those previously found only at a handful of large sequencing centers. Over the past 18 months, 454 sequencing has led to a better understanding of the structure of the human genome, allowed the first non-Sanger sequence of an individual human and opened up new approaches to identify small RNAs. To make next-generation technologies more widely accessible, they must become easier to use and less costly. In the longer term, the principles established by 454 sequencing might reduce cost further, potentially enabling personalized genomics.     

Next-generation phylogeography: a targeted approach for multilocus sequencing of non-model organisms

The field of phylogeography has long since realized the need and utility of incorporating nuclear DNA (nDNA) sequences into analyses. However, the use of nDNA sequence data, at the population level, has been hindered by technical laboratory difficulty, sequencing costs, and problematic analytical methods dealing with genotypic sequence data, especially in non-model organisms. Here, we present a method utilizing the 454 GS-FLX Titanium pyrosequencing platform with the capacity to simultaneously sequence two species of sea star (Meridiastra calcar and Parvulastra exigua) at five different nDNA loci across 16 different populations of 20 individuals each per species. We compare results from 3 populations with traditional Sanger sequencing based methods, and demonstrate that this next-generation sequencing platform is more time and cost effective and more sensitive to rare variants than Sanger based sequencing. A crucial advantage is that the high coverage of clonally amplified sequences simplifies haplotype determination, even in highly polymorphic species. This targeted next-generation approach can greatly increase the use of nDNA sequence loci in phylogeographic and population genetic studies by mitigating many of the time, cost, and analytical issues associated with highly polymorphic, diploid sequence markers.     

The Accuracy,Feasibility and Challenges of Sequencing Short Tandem Repeats Using Next-Generation Sequencing Platforms

To date we have little knowledge of how accurate next-generation sequencing (NGS) technologies are in sequencing repetitive sequences beyond known limitations to accurately sequence homopolymers. Only a handful of previous reports have evaluated the potential of NGS for sequencing short tandem repeats (microsatellites) and no empirical study has compared and evaluated the performance of more than one NGS platform with the same dataset. Here we examined yeast microsatellite variants from both long-read (454-sequencing) and short-read (Illumina) NGS platforms and compared these to data derived through Sanger sequencing. In addition, we investigated any locus-specific biases and differences that might have resulted from variability in microsatellite repeat number, repeat motif or type of mutation. Out of 112 insertion/deletion variants identified among 45 microsatellite amplicons in our study, we found 87.5% agreement between the 454-platform and Sanger sequencing in frequency of variant detection after Benjamini-Hochberg correction for multiple tests. For a subset of 21 microsatellite amplicons derived from Illumina sequencing, the results of short-read platform were highly consistent with the other two platforms, with 100% agreement with 454-sequencing and 93.6% agreement with the Sanger method after Benjamini-Hochberg correction. We found that the microsatellite attributes copy number, repeat motif and type of mutation did not have a significant effect on differences seen between the sequencing platforms. We show that both long-read and short-read NGS platforms can be used to sequence short tandem repeats accurately, which makes it feasible to consider the use of these platforms in high-throughput genotyping. It appears the major requirement for achieving both high accuracy and rare variant detection in microsatellite genotyping is sufficient read depth coverage. This might be a challenge because each platform generates a consistent pattern of non-uniform sequence coverage, which, as our study suggests, may affect some types of tandem repeats more than others.     

Identification of Novel Viruses Using VirusHunter -- an Automated Data Analysis Pipeline

Quick and accurate identification of microbial pathogens is essential for both diagnosis and response to emerging infectious diseases. The advent of next-generation sequencing technology offers an unprecedented platform for rapid sequencing-based identification of novel viruses. We have developed a customized bioinformatics data analysis pipeline, VirusHunter, for the analysis of Roche/454 and other long read Next generation sequencing platform data. To illustrate the utility of VirusHunter, we performed Roche/454 GS FLX titanium sequencing on two unclassified virus isolates from the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA). VirusHunter identified sequences derived from a novel bunyavirus and a novel reovirus in the two samples respectively. Further sequence analysis demonstrated that the viruses were novel members of the Phlebovirus and Orbivirus genera. Both Phlebovirus and Orbivirus genera include many economic important viruses or serious human pathogens.     

Perspectives of DNA microarray and next-generation DNA sequencing technologies

DNA microarray and next-generation DNA sequencing technologies are important tools for high-throughput genome research, in revealing both the structural and functional characteristics of genomes. In the past decade the DNA microarray technologies have been widely applied in the studies of functional genomics, systems biology and pharmacogenomics. The next-generation DNA sequencing method was first introduced by the 454 Company in 2003, immediately followed by the establishment of the Solexa and Solid techniques by other biotech companies. Though it has not been long since the first emergence of this technology, with the fast and impressive improvement, the application of this technology has extended to almost all fields of genomics research, as a rival challenging the existing DNA microarray technology. This paper briefly reviews the working principles of these two technologies as well as their application and perspectives in genome research. Supported by the National High-Tech Research Program of China (Grant No.2006AA020704) and Shanghai Science and Technology Commission (Grant No. 05DZ22201)     

Comparison of Normalization Methods for Construction of Large,Multiplex Amplicon Pools for Next-Generation Sequencing

Constructing mixtures of tagged or bar-coded DNAs for sequencing is an important requirement for the efficient use of next-generation sequencers in applications where limited sequence data are required per sample. There are many applications in which next-generation sequencing can be used effectively to sequence large mixed samples; an example is the characterization of microbial communities where ≤1,000 sequences per samples are adequate to address research questions. Thus, it is possible to examine hundreds to thousands of samples per run on massively parallel next-generation sequencers. However, the cost savings for efficient utilization of sequence capacity is realized only if the production and management costs associated with construction of multiplex pools are also scalable. One critical step in multiplex pool construction is the normalization process, whereby equimolar amounts of each amplicon are mixed. Here we compare three approaches (spectroscopy, size-restricted spectroscopy, and quantitative binding) for normalization of large, multiplex amplicon pools for performance and efficiency. We found that the quantitative binding approach was superior and represents an efficient scalable process for construction of very large, multiplex pools with hundreds and perhaps thousands of individual amplicons included. We demonstrate the increased sequence diversity identified with higher throughput. Massively parallel sequencing can dramatically accelerate microbial ecology studies by allowing appropriate replication of sequence acquisition to account for temporal and spatial variations. Further, population studies to examine genetic variation, which require even lower levels of sequencing, should be possible where thousands of individual bar-coded amplicons are examined in parallel.Emergent technologies that generate DNA sequence data are designed primarily to perform resequencing projects at reasonable cost. The result is a substantial decrease in per base costs from traditional methods. However, these next-generation platforms do not readily accommodate projects that require obtaining moderate amounts of sequence from large numbers of samples. These platforms also have per run costs that are significant and generally preclude large numbers of single-sample, nonmultiplexed runs. One example of research that is not readily supported is rRNA-directed metagenomics study of some human clinical samples or environmental rRNA analysis of samples from communities with low community diversity that require only thousands of sequences. Thus, strategies to utilize next-generation DNA sequencers efficiently for applications that require lower throughput are critical to capitalize on the efficiency and cost benefits of next-generation sequencing platforms.Directed metagenomics based on amplification of rRNA genes is an important tool to characterize microbial communities in various environmental and clinical settings. In diverse environmental samples, large numbers of sequences are required to fully characterize the microbial communities (15). However, a lower number of sequences is generally adequate to answer specific research questions. In addition, the levels of diversity in human clinical samples are usually lower than what is observed in environmental samples (for example, see reference 7).The Roche 454 genome sequencer system FLX pyrosequencer (which we will refer to as 454 FLX hereafter) is the most useful platform for rRNA-directed metagenomics because it currently provides the longest read lengths of any next-generation sequencing platform (1, 14). Computational analysis has shown that the 250-nucleotide read length (available from the 454 FLX-LR chemistry) is adequate for identification of bacteria if the amplified region is properly positioned within variable regions of the small-subunit rRNA (SSU-rRNA) gene (9, 10).In this study, we used the 454 FLX-LR genome sequencing platform and chemistry, which provides >400,000 sequences of ∼250 bp per run. After we conducted this study, a new reagent set (454 FLX-XLR titanium chemistry) was released, which further increases reads to >1,000,000 and read lengths to >400 bp (Roche). The 454 FLX platform dramatically reduces per base costs of obtaining sequence, and physical separation into between 2 and 16 lanes is available; this physical separation on the plate reduces sequencing output overall, up to 40% comparing 2 lanes versus 16 lanes. For applications where modest sequencing depth (∼1,000 sequences per sample) is adequate to address research questions, physical separation does not allow adequate sample multiplexing because even a 1/16 454 FLX-LR plate run is expected to produce ∼15,000 reads. Further, the utility of the platform as a screening tool at 16-plex is limited by cost per run.A solution to make next-generation sequencing economical for projects such as rRNA-directed metagenomics is to use bar-coded primers to multiplex amplicon pools so they can be sequenced together and computationally separated afterward (6). To successfully accomplish this strategy, precise normalization of the DNA concentrations of the individual amplicons in the multiplex pools is essential for effective multiplex sequencing when large numbers of pooled samples are sequenced in parallel. There are several potential methods available for normalizing concentrations of amplicons included in multiplex pools, but the relative and absolute performance of each approach has not been compared.In this study, we present a direct quantitative comparison of three available methods for amplicon pool normalization for downstream next-generation sequencing. The central goal of the study was to identify the most effective method for normalizing multiplex pools containing >100 individual amplicons. We evaluated each pooling approach by 454 sequencing and compared the observed frequencies of sequences from different pooled bar-coded amplicons. From these data, we determined the efficacy of each method based on the following factors: (i) how well normalized the sequences within the pool were, (ii) the proportion of samples failing to meet a minimum threshold of sequences per sample, and (iii) the overall efficiency (speed and labor required) of the process to multiplex samples.     

Next-Generation Sequencing in the Mycology Lab

New state-of-the-art techniques in sequencing offer valuable tools in both detection of mycobiota and in understanding of the molecular mechanisms of resistance against antifungal compounds and virulence. Introduction of new sequencing platform with enhanced capacity and a reduction in costs for sequence analysis provides a potential powerful tool in mycological diagnosis and research. In this review, we summarize the applications of next-generation sequencing techniques in mycology.     

Rapid identification of thousands of copperhead snake (Agkistrodon contortrix) microsatellite loci from modest amounts of 454 shotgun genome sequence

Optimal integration of next-generation sequencing into mainstream research requires re-evaluation of how problems can be reasonably overcome and what questions can be asked. One potential application is the rapid acquisition of genomic information to identify microsatellite loci for evolutionary, population genetic and chromosome linkage mapping research on non-model and not previously sequenced organisms. Here, we report on results using high-throughput sequencing to obtain a large number of microsatellite loci from the venomous snake Agkistrodon contortrix, the copperhead. We used the 454 Genome Sequencer FLX next-generation sequencing platform to sample randomly ∼27 Mbp (128 773 reads) of the copperhead genome, thus sampling about 2% of the genome of this species. We identified microsatellite loci in 11.3% of all reads obtained, with 14 612 microsatellite loci identified in total, 4564 of which had flanking sequences suitable for polymerase chain reaction primer design. The random sequencing-based approach to identify microsatellites was rapid, cost-effective and identified thousands of useful microsatellite loci in a previously unstudied species.