【专题】细菌基因组学-揭开细菌微观世界

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【进展】新英格兰医学2011-细菌基因组学与感染性疾病

地址：http://www.nejm.org/action/clickThrough?id=2505&url=%2Fdoi%2Ffull%2F10.1056%2FNEJMra1003071%3Fquery%3Dfeatured_infectious-disease&loc=%2Finfectious-disease&pubId=40088338

The pace of technical advancement in microbial genomics has been breathtaking. Since 1995, when the first complete genome sequence of a free-living organism, Haemophilus influenzae, was published,1 1554 complete bacterial genome sequences (the majority of which are from pathogens) and 112 complete archaeal genome sequences have been determined, and more than 4800 and 90, respectively, are in progress.2 A total of 41 complete eukaryotic genome sequences have been determined (19 from fungi), and more than 1100 are in progress. Complete reference genome sequences are available for 2675 viral species, and for some of these species, a large number of strains have been completely sequenced. Nearly 40,000 strains of influenza virus3 and more than 300,000 strains of human immunodeficiency virus (HIV) type 1 have been partially sequenced.4 However, the selection of microbes and viruses for genome sequencing is heavily biased toward the tiny minority that are amenable to cultivation in the laboratory, numerically dominant in particular habitats of interest (e.g., the human body), and associated with disease.

In 2006, investigators reported in-depth metagenomic sequence data from a human mixed microbial community5; in 2007 more than 1000 genes from single cells of cultivation-resistant bacteria were identified.6 Since then, a flood of such data has ensued (Figure 1Figure 1

                               
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Genome Projects and Completed Genomes since 1995.).7-9 Individual investigators can now produce a draft sequence of a bacterial genome containing 4 million base pairs in about a day.10-12 The revolution in DNA-sequencing technology has to a large extent democratized microbial genomics and altered the way infectious diseases are studied.11 However, gene annotation and error correction still take time and effort. Today, the major challenges in microbial genomics are to predict the function of gene products and the behavior of organisms and communities from their sequences and to use genomic data to develop improved tools for managing infectious diseases.

Genomic Diversity

The human body contains remarkable microbial taxonomic richness, with thousands of symbiont species and strains per individual host. Of these, an estimated 90% have not yet been cultivated in the laboratory.13 Differences between closely related strains and species are responsible for virulence, host-species adaptation, and other aspects of lifestyle and account for the individualized nature of the human microbiota. For example, the gene content of pathogenic and nonpathogenic strains of Escherichia coli, as well as different pathogenic types, varies by as much as 36%.14,15 Comparisons of complete genome sequences from multiple strains of the same bacterial species reveal a set of core genes that are common to all strains and a set of dispensable genes that are absent in at least one strain.16 The sum of these genes (i.e., those represented in at least one strain) constitutes the species pangenome.

As compared with the genomes of plants and animals, genomes of microbes are small and usually contain one or two chromosomes, as well as a variable number of plasmids (see Glossary). Yet, approximately 90% of a typical microbial genome encodes proteins or structural RNAs,17 whereas only about 1.1% of the human genome is coding sequence.18 As a result, some complex bacteria have more genes than some simple eukaryotes.

Microbial diversification and adaptation have been accompanied by gene loss and genome reduction, genome rearrangement, horizontal gene transfer, and gene duplication.19,20 The first two of these processes are especially evident in human-specific pathogens, such as Bordetella pertussis (the causative agent of whooping cough),21,22 Tropheryma whipplei (the agent of Whipple's disease),23 and Yersinia pestis (the agent of bubonic plague). A total of 3.7% of Y. pestis genes appear to be inactive, especially those associated with enteropathogenicity.24 The genome of Mycobacterium leprae, the cause of leprosy, provides an even more dramatic example of reductive evolution. Protein-coding genes account for less than half of its genome, whereas inactive and fragmented genes account for most of the remainder.25

Genomic islands are discrete clusters of contiguous genes found in bacterial chromosomes and plasmids, usually between 10,000 and 200,000 base pairs in length with features that suggest a history and origin distinct from other segments of the genome (see Glossary).26,27 Some islands are stably assimilated into the genome; others appear to have been acquired recently and may still be mobile. Genomic islands enhance the fitness of the recipient by providing new, accessory functions, such as pathogenicity, drug resistance, or catabolic functions.

One of the most dramatic examples of short-term genome evolution can be seen in the CRISPR (clustered regularly interspaced short palindromic repeat) loci of bacteria and archaea. CRISPRs serve as a defense against invading phages and plasmids, in a manner akin to adaptive immunity.28 These genomic loci contain segments of phage and plasmid sequences captured from previous encounters. These segments are stored within the CRISPR loci as spacer sequences and are expressed as small RNAs, which then interfere with replication of newly encountered phages and plasmids that bear the same sequences.

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Population Structure, Evolution, and Molecular Epidemiology
Differences in the sequence and structure of genomes from members of a microbial population reflect the composite effects of mutation, recombination, and selection. With the increasing availability of genome sequences, these effects have become better characterized and more effectively exploited so as to understand the history and evolution of microbes and viruses and their sometimes intimate relationships with humans. The resulting insights have practical importance for epidemiologic investigations, forensics, diagnostics, and vaccine development.29

Y. pestis, the cause of the Black Death, arose from a more genetically diverse ancestor that was related to Y. pseudotuberculosis, through genome reduction and gene loss. By analyzing approximately 1200 single-nucleotide polymorphisms (SNPs) and a worldwide collection of strains, the origins of this monomorphic pathogen have been placed between 2600 and 28,000 years ago in China, from which it spread to other areas of the world, giving rise to country-specific lineages.30 All the Y. pestis strains that are found in the United States today are descendants of a single import that probably arrived in San Francisco in 1899. As another example, patterns of early human migration have been traced by comparing genome sequences from contemporary isolates of the chronic gastric pathogen, Helicobacter pylori.31 Transmission of the pathogen is primarily from mother or other household members to baby, and colonization is usually lifelong; thus, pathogen sequences are reasonable markers of host ancestry and host migration. Sequence data for the H. pylori genome indicate the sequential timing and directionality of two distinct waves of human migration into the Pacific region.32 Population mosaicism in H. pylori gene sequences has been used to infer the history of social interactions in human populations.31

The power of full-genome sequencing to discriminate between closely related strains and track real-time evolution of disease-associated clonal isolates offers the possibility of tracing person-to-person transmission and identifying point sources of outbreaks. Using this approach, investigators established a previously unrecognized link among five patients with the same clonal strain of methicillin-resistant Staphylococcus aureus from a hospital in Thailand.33 A study of Vibrio cholerae genome sequences from the October 2010 cholera outbreak in Haiti suggested that the Haitian strains were clonal and more closely related to strains from Bangladesh that were isolated in 2002 and 2008 than to strains isolated in Peru in 1991 and in Mozambique in 2004. The authors concluded that the Haitian outbreak may have originated with the introduction of a V. cholerae strain from South Asia as a result of human activity rather than climatic events or other local environmental factors.12 However, the source of this outbreak has not been fully resolved; genome sequences of environmental strains and additional clinical isolates from Haiti may provide further insight.

A major challenge is the prediction of patterns of evolution and emergence of disease agents. The antigenic evolution of influenza virus is known to follow a punctuated equilibrium model in which periods of relative virus stability around the globe are followed by periods of rapid change, requiring modification of the influenza vaccine. However, it was not clear whether variants arise first in East and Southeast Asia and then seed other geographic regions or whether strains persist locally and evolve simultaneously in a similar fashion. An analysis of the gene encoding hemagglutinin (the major antigenic determinant) from more than 1000 human influenza A (H3N2) isolates that were collected worldwide from 2002 through 2007 produced strong support for external seeding, rather than local persistence, and suggested that the source of seeding is East and Southeast Asia.34 On the basis of whole-genome sequence analysis, the novel 2009 human H1N1 influenza strain was thought to have entered the human population in January of that year after arising from multiple swine virus progenitors that had probably been circulating in swine populations undetected for at least a decade.35 Work of this type will help target efforts regarding influenza virus surveillance more effectively, refine the selection of vaccine strains, and improve predictions of future antigenic characteristics.36 Similar approaches will assist in anticipating the emergence and spread of antibiotic and antiviral resistance.

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Pathogenesis and Symbiosis
Pathogens have received most of the attention in microbial genomics, despite their relative rarity in the microbial world.17,19 As a result, we now have a more complete and deeper understanding of how microbes cause disease and of pathogen emergence, host adaptation, and spread in human populations. The study of microbial genomes reveals four themes with respect to virulence.

First, horizontal gene transfer (see Glossary) has had a major role in the acquisition of genes associated with virulence. Most genes that encode virulence factors are physically segregated in clusters and located within mobile genetic elements. In S. aureus, these genes often occur within phage-related chromosomal islands and encode a variety of superantigens, including the toxin associated with the toxic shock syndrome and staphylococcal enterotoxin B, and encode factors that mediate antibiotic resistance, biofilm induction, and other virulence-associated properties (Figure 2Figure 2
Unexpected Diversity of Virulence Factors for Staphylococcus aureus, as Shown by Comparative Genome Analysis.).37 Genomic islands with similar features occur in other gram-positive bacteria, including streptococcus, enterococcus, and lactococcus species. The emergence of the recent Shiga toxin–producing E. coli clone in Germany was probably the result of horizontal gene transfer, when a toxin-producing phage infected an enteroaggregative E. coli strain.38

Second, symbionts and avirulent relatives of pathogens often contain many of the same virulence-associated genes as do the microbes that typically cause disease.39 The genes that we commonly associate with virulence may have been selected for the advantages they confer in promoting colonization of animal and plant hosts, in avoiding or surviving phagocytosis, and in enhancing competition against symbionts.40-42 For example, the original role for bacterial toxins may have been to protect the bacterium against predation by protozoa and nematodes. The legionella protein IcmT facilitates the escape of the bacterium from human macrophages and also from the far more ancient predator, the free-living amoeba.43 Virulence depends on the choreographed expression of particular combinations of genes at the right place and time in the right host. Commensals and other symbionts also serve as reservoirs of antibiotic resistance genes and genetic diversity.44

A third theme is the surprising diversity of genes associated with mechanisms of virulence. In a study of four closely related fungal species, all of which cause late blight disease but in different host plant species, investigators identified specific regions of the fungal genomes with evidence of accelerated rates of evolution, suggesting that these regions have been under strong positive selective pressure.45 The genes in these regions produce effector molecules (see Glossary) that interact with host plant proteins and elicit host cell death. One of these fungal pathogens, the agent responsible for the 19th-century Irish potato famine, expresses 196 related effectors of unexpected complexity and diversity.46

A fourth theme is genome reduction and pseudogene formation (see Glossary), especially in pathogens with a relatively specialized lifestyle and with restricted numbers and types of habitats, niches, and hosts.20 This is illustrated by an unusual multidrug resistant strain of Salmonella enterica serovar Typhimurium that emerged in sub-Saharan Africa in the early 1990s to become the most common cause of invasive bacterial disease in some regions of that continent. Bacteremia and meningitis are common features of this disease, as they are for typhoid and paratyphoid fevers. The genome sequence of this strain reveals a large number of partially degraded and deleted genes, many of which are also degraded or deleted in the genomes of salmonella serovars Typhi and Paratyphi A.47

The Human Microbiome and Metagenomics
The role of the human indigenous microbiota in human heath and disease has received a great deal of attention in the past 5 years.7,8,13,48 Surveys of bacterial phylogenetic diversity that are based on comparative analyses of ribosomal RNA gene sequences recovered directly from clinical specimens have confirmed habitat- and individual-specific patterns in healthy persons.49,50 Yet, core features of the indigenous microbial communities are conserved in healthy persons.51

A metagenomic analysis (see Glossary) of fecal samples from 124 healthy European subjects identified an average of 536,112 unique genes in each of these samples, 99.1% of which were bacterial and 0.8% of which were archaeal — and a total of 3.3 million unique genes overall, or 150 times the number of genes in the human genome.9 Approximately 38% of an individual's fecal gene pool is shared by at least half of all other individuals. The shared gene products are predicted to mediate degradation of complex sugars, such as pectin and sorbitol, and of glycans harvested from the host diet or intestinal lining, as well as fermentation of mannose, fructose, cellulose, and sucrose (to short-chain fatty acids) and vitamin biosynthesis. These conserved genes constitute an accessory human genome that facilitates dietary energy harvest and nutrition. Alterations in the human microbiome are associated with a number of diseases in which no single organism seems to explain either the presence or the absence of disease. For these diseases (of which Crohn's disease is a leading example), the concept of community as pathogen has been proposed.52 Elucidation of the role played by altered microbial communities in such conditions and the associated mechanisms are likely to emerge from the application of genomic approaches during the next decade.

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Pathogen Discovery and Diagnostics

Genomic approaches have introduced a new era in the discovery and detection of microbial pathogens. The robustness, reliability, and portability of molecular sequence-based data for phylogenetic assessments and for characterization of previously unrecognized pathogens, coupled with technology developments, recommend genomic approaches for both research and routine clinical applications53-63 (Table 1Table 1

                               
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Examples of the Use of Microbial Genomics to Enhance the Management of Infectious Diseases. and Figure 3Figure 3

                               
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Microbial Genomics and Tool Development.; interactive graphic, available with the full text of this article at NEJM.org). Broad-range molecular methods for microbial discovery were introduced two decades ago.54,64 Approaches for targeting differentially abundant or phylogenetically informative molecules have now been joined by less efficient but more powerful methods for broad sequence surveys of clinical and environmental samples with the use of high-density DNA microarrays55,65 and shotgun sequencing56,66 (see Glossary). The advantages of DNA microarrays include the simultaneous detection of diverse sequences with widely varying relative abundance and recovery of captured sequences of interest directly from the microarray. A panviral DNA microarray with oligonucleotides designed from all known viral genera was used to characterize the novel causative agent of the severe acute respiratory syndrome (SARS)55 and has been used to detect viruses in nasopharyngeal aspirates from children with a variety of acute respiratory syndromes.65 The disadvantages of DNA microarrays include their insensitivity to rare microbial sequences in the presence of highly abundant host sequences (i.e., those obtained from host tissues) and their reliance on previous knowledge of microbial sequence diversity for oligonucleotide design.

High-throughput shotgun sequencing offers important new opportunities for the detection and discovery of microbial pathogens. This approach has revealed both previously known viruses (e.g., rotavirus, adenovirus, calicivirus, and astrovirus) and unknown viruses (e.g., novel types of picobirnavirus, enterovirus, TT virus, and norovirus) in fecal samples from children with unexplained acute diarrhea66 and a novel Old World arenavirus that caused fatal disease in three recipients of organs from a single donor.56 Dramatic advances in sequencing technology highlight the need to understand the diversity of microbial sequences in healthy subjects and to develop better methods for distinguishing rare, genuine microbial sequences from sequencing errors.

Sequence-based characterization of pathogens enables the design and development of sensitive and specific diagnostic assays and, in some cases, methods for cultivation of the pathogen. Characterization of the 16S ribosomal RNA gene from the agent of Whipple's disease, T. whipplei, led to a molecular diagnostic assay for this disease agent.67 Subsequent determination of its complete genome sequence23,68 provided additional potential target sequences and the basis for a more sensitive diagnostic test.61 It also provided insight into the metabolic defects of this bacterium, such that cell-free growth medium could be designed to include missing, needed growth factors.69

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Therapeutics and Drug Discovery

Genome sequences provide the blueprint for essential microbial and viral components, the disruption of which can lead to growth inhibition and death. These same sequences can sometimes indicate resistance of the microbe or virus to a particular drug. Although drug susceptibility and resistance are often governed by multiple genetic components, some drug-resistance traits are encoded by single genes and can therefore be easily predicted by detecting or sequencing such genes. Examples include rifampin resistance in M. tuberculosis, methicillin resistance in S. aureus, trimethoprim–sulfamethoxazole resistance in T. whipplei, 70 and resistance to some antiretroviral drugs in HIV. Genome sequences have also provided new targets and leads for the development of new antimicrobials.

The standard of care for the management of HIV infection now includes targeted drug selection with the use of a profile for HIV-drug susceptibility that is derived from the sequence of the infecting HIV species.59 Testing for genotypic resistance is recommended for patients with HIV infection when they enter care and when there is a suboptimal reduction in viral load while they are receiving first- or second-line antiretroviral regimens. Clinically important resistance mutations occur in HIV genes encoding the reverse-transcriptase, protease, envelope, and integrase proteins. Interpretation of these mutant genotypes is facilitated by several databases, including those maintained by the International Antiviral Society–USA71 and a research group at Stanford University.72 Genotypic analysis is cheaper and faster than phenotypic analysis for HIV-drug resistance and is often more sensitive for detecting resistant strains within mixtures of drug-susceptible viruses.73 However, commercial assays of both types do not routinely detect resistant viruses when they are less than 10 to 20% of the overall circulating virus population. With newer sequencing techniques, less abundant strains are easier to detect and characterize. Although the clinical relevance of rare resistant variants is not fully understood, the pretreatment detection of such variants has been shown to have clinical value.58 Traditional phenotypic testing (measuring the ability of the virus to replicate in the presence of the antiviral drug) is still recommended for patients in whom viruses are suspected of having complex drug-resistance mutation patterns.

Schistosomiasis is a chronic and debilitating disease that affects approximately 210 million people in 76 countries around the globe and results in some 280,000 deaths per year in sub-Saharan Africa alone. Praziquantel has been the drug of choice for the treatment of schistosomiasis but is in danger of losing efficacy because of parasite resistance. Schistosoma mansoni is one of three helminths for which there is now a draft genome sequence available to the public.74 Besides enabling the study of gene and protein expression,75 the nuclear genome of S. mansoni and its approximately 11,800 putative genes point to critical compounds and processes on which the worm depends to survive in its host. These compounds and processes reveal potential new drug targets, one of which is a redox enzyme, thioredoxin–glutathione reductase.74 Quantitative high-throughput screening of small-molecule libraries for compounds with activity against the S. mansoni thioredoxin–glutathione reductase has already identified some candidate drugs.62

Microbes produce a wealth of druglike molecules, the vast majority of which remain uncharacterized.76,77 Because many of these molecules are not expressed under typical laboratory conditions, they often escape detection when laboratory culture filtrates are screened for druglike properties. Some of these molecules can now be identified by recognizing the relevant genes in the parent organism's genome with the use of computational tools and detecting the molecules with mass spectroscopy techniques.78,79 Derivative compounds can be designed and tested.

Vaccines

In the same way that genome sequences reveal drug-resistance profiles, vulnerabilities, and synthetic capabilities of microbes and viruses, these sequences also provide clues about antigenic repertoire. This information can be exploited for vaccine design and other immunoprophylactic interventions. Genome-based antigen discovery has also been undertaken for more complex pathogens. One approach, known as reverse vaccinology, involves cloning and expressing all proteins that are predicted (from the organism's complete genome) to be secreted or surface-associated, starting with the complete genome sequence (Figure 3).80 After immunizing mice with each of the proteins, each of the corresponding antiserum samples is tested for its ability to neutralize or kill the original target organism. On the basis of this approach, a small group of proteins from group B meningococcus,81 a pathogen that has so far eluded vaccine development, has shown promise as a candidate multivalent subunit vaccine. A similar approach has been taken with group B streptococcus82 and extraintestinal pathogenic E. coli.83 Protective antigens that are discovered through these sorts of methods may have been previously ignored because they are not immunogenic during natural infections.

Future DirectionsWithout question, the techniques for microbial and viral genome sequencing are becoming increasingly rapid and less expensive. Genome sequencing of a microbe or virus will soon be easier than characterization of its growth-based behavior in the laboratory. In the next 3 to 5 years, direct shotgun sequencing of the DNA and RNA in a clinical sample may become a routine matter. What is less clear is how clinically relevant information will be most effectively extracted from the ensuing massive amounts of data. In the near term, genomic and metagenomic analyses of microbes are most likely to be useful in areas such as the cataloguing and understanding of microbial and viral diversity in the human body, the identification of molecular determinants of virulence and symbiosis, and real-time tracking of particular strains of pathogens. Such analyses will also provide a deeper understanding of how pathogens spread and cause disease and will identify new targets for therapies and antigens for vaccines. Thoughtfully designed clinical and epidemiologic studies will be required to see the full realization of these benefits.

Disclosure forms provided by the author are available with the full text of this article at NEJM.org.

Source InformationFrom the Departments of Medicine and of Microbiology and Immunology, Stanford University, Stanford; and the Veterans Affairs (VA) Palo Alto Health Care System, Palo Alto — both in California.

Address reprint requests to Dr. Relman at VA Palo Alto Health Care System, 154T, Bldg. 101, Rm. B4-185, 3801 Miranda Ave., Palo Alto, CA 94304, or at relman@stanford.edu.

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【谢晓亮】

从单细胞研究到高通量测序

科学网

2011年7月第八期《自然—方法学》刊登了Monya Baker撰写的一篇人物特写，详细介绍了在当期发表的论文 “Fluorogenic DNA sequencing in PDMS microreactors”的主要作者哈佛大学谢晓亮教授的高通量测序技术。全文翻译如下：

在科学界，合情合理的实验也可能会出现令人吃惊的结果。当谈到他的实验室时，谢晓亮把他的主要研究分成三个领域：活体细胞中的动态基因表达研究，单分子酶学和免标记显微成像技术，而现在，又多了一个由于意外而诞生的新领域——高通量测序。

目前常见的测序技术“焦磷酸测序”是通过边合成DNA边测序实现的，当加入新三磷酸核苷酸时，荧光素酶水解三磷酸键所产生的能量会以光的形式发出，然而光信号转瞬即逝，需要检测系统能够灵敏地捕捉到这一瞬间的光信号。 另一种常见的技术是基于荧光的测序，相比之下，它可以产生一个稳定的光信号，但需要很多额外的化学修饰步骤才能产生荧光。在这篇Nature Method的文章中（指Sims, P.A., Greenleaf, W.J., Duan, H. & Xie, X.S.. Nat. Methods 8, 575–580 (2011).），谢晓亮和他的同事们推出了一种新型的测序技术，这种技术兼顾焦磷酸测序的简单流程和荧光检测的稳定信号，这使得高精确度并循环周期短的测序成为可能。

单分子荧光酶学的开端要追溯到十多年前，当时谢晓亮作为美国太平洋西北国家实验室的一位研究员，正在研究表征单个酶分子活性的方法，为此，他和同事曾应用过一个含有可发荧光的吖啶黄素基团的酶。那时，诸如 Helicos和Pacific Bioscience等公司也刚刚宣布了他们的DNA单分子测序计划。谢晓亮对把单分子酶学应用于DNA测序领域很感兴趣，但由于他已经在哈佛就职，这个想法仅仅被搁置于专利层面。“我需要学着做个教授”，谢晓亮说。

谢晓亮偶尔会尝试把基于荧光基团测序的想法推荐给一些研究生或博士后，但是年轻的科学家们通常不大敢尝试这一想法。“提些建议对我来说是很容易的，因为我有很多项目，总有一些会成功的”，谢晓亮解释道，“但是对学生来说这是个很大的赌注，并不是所有人都敢于接受这种挑战。”一位四年级的研究生Peter Sims听说了这个想法，当即接受了这个挑战，尽管当时他完全可以由单分子马达在活细胞的研究来获得学位。 Sims表示这种潜在的高通量测序激发了他的浓厚兴趣，但是对于所需的在核酸上修饰荧光基团的化学工作，他还没有经验。“他当时刚刚涉足于此，才开始学习”，谢晓亮说。谢晓亮和Sims共同商定了一个期限，如果Sims在此之前还没有获得显著的成绩，他就退回到原来的课题上，开始写毕业论文。

捕捉荧光信号就像成功产生荧光一样重要。在博士后William Greenleaf帮助下，他们解决了这个难题。“微反应容器和荧光化学二者的结合，便是这项测序新技术的精髓。”谢晓亮说。Greenleaf设法加工出了这些含有微反应容器的芯片，它是由可以重复密封的聚二甲基硅氧烷（PDMS）聚合物制成。谢晓亮说，没有这种材料，他的实验室的研究人员不可能做出这种尝试。“我想把推广PDMS的功劳归于George Whitesides（George也在哈佛大学工作）”，他说，“基于PDMS我们才能够制作出各式各样的芯片上的实验室，而且他们真的很好用。”

但是研究进展并非一帆风顺。在后来的实验中，含有荧光基团的分子总是会扩散到PDMS 中或是产生一些不可信的伪信号。实验室的另一位成员段海峰加入了他们的小组，负责合成新型的荧光分子。此时，Sims和谢晓亮定下的期限也快到了，但他们仍没有做出很好的结果。

Sims和Greenleaf制定了另外一项计划，但是仅仅是对多拷贝的DNA测序而并非单分子测序。当时谢晓亮正在苏格兰出差，一天深夜他和Sims进行了一次电话长谈，讨论Sims是否应该退回到原来的项目来写毕业论文。谢晓亮回忆道：“那真费了我好大一笔电话费。我说，‘Peter，请你再想想，我们再尽快地尝试一下，如果你真的做到了，学术界将对你的毕业论文产生极大的兴趣。’”几周后，他们果真拿到了数据，并且Sims在他的答辩中成功地阐述了这种测序方法。谢晓亮富有哲理地说：“你开始一直在对着一堵墙作战，后来你稍微改变了方向，这就大不一样了”。Sims也有另外的动机，他曾和谢晓亮开玩笑说，“我做这个只是想毕业。”

虽然这项测序技术本身还是基于DNA扩增的，但谢晓亮希望它能为通用单细胞基因组测序提供一条道路。谢晓亮说：“尽管我们的技术并不是我最初希望的DNA单分子检测，但它依然为单细胞中DNA单分子测序提供了可能。”

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高通量技术为揭开细菌基因组学提供了技术支持。在下面的几个PDF文件中，对高通量测序的几个技术进行了比较和介绍。

toto

这项测序技术本身还是基于DNA扩增的，这些了解一下可以，实在是懒得深入的看，不好意思哈……

蓝鱼o_0

toto 发表于 2011-10-5 00:50

                               
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这项测序技术本身还是基于DNA扩增的，这些了解一下可以，实在是懒得深入的看，不好意思哈……

希望越来越多的人不但关注宏观防控策略，也关注微观领域，在后基因组时代，站在高通量测序的平台上，关注MDRO和healthcare associated virus。

toto

蓝鱼o_0 发表于 2011-10-5 00:57

                               
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希望越来越多的人不但关注宏观防控策略，也关注微观领域，在后基因组时代，站在高通量测序的平台上，关注 ...

这个了解一下就OK，让有条件做分子研究的人去深入研究吧，呵呵！

lzz1806

基因组学的研究是感控学科发展的一个方向，但要使广大院感人知道这一学科的重要性，还有一段很长的路要走……

天天，天蓝

关注微观领域，只能是学习了，接受一些新的知识。

蓝鱼o_0

天天，天蓝 发表于 2011-10-5 09:45

                               
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关注微观领域，只能是学习了，接受一些新的知识。

个人感觉，国内这块研究还是比较薄弱的。
而且分子流行病学研究又是特别耗钱。但是却很重要，就像一个人手掌的小拇指，尽管它的使用频率不是最高。

蓝鱼o_0

lzz1806 发表于 2011-10-5 03:42

                               
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基因组学的研究是感控学科发展的一个方向，但要使广大院感人知道这一学科的重要性，还有一段很长的路要走… ...

您说的一点都没有错。学科的发展应该是多分枝的。
医院感染的分子流行病学研究只是当中的一个小分支。
尽管知道目前开展的不多，论坛关注的也不多，但是作为感染性疾病研究的前沿，还是想给大家展示另外一个侧面。希望越来越多的人参与，研究。

米奇朋克

蓝鱼o_0 发表于 2011-10-5 00:21

                               
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【谢晓亮】

从单细胞研究到高通量测序

微观世界的研究真是很广阔的天地，相信这些新技术、新发现将给宏观的感染预防和控制措施提供强有力的支持！再次感谢蓝鱼老师的介绍。

鬼才

蓝鱼o_0 发表于 2011-10-5 19:57

                               
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您说的一点都没有错。学科的发展应该是多分枝的。
医院感染的分子流行病学研究只是当中的一个小分支。
...

有劳把医院感染分子流行病学的知识多在论坛给予展示，使更多的战友懂得这一学科。

lzz1806

蓝鱼o_0 发表于 2011-10-5 19:57

                               
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您说的一点都没有错。学科的发展应该是多分枝的。
医院感染的分子流行病学研究只是当中的一个小分支。
...

感染控制流行病学前沿领域的知识要加大普及的力度，期待越来越多的有识之士多参与这方面的研讨。

蓝鱼o_0

鬼才 发表于 2011-10-5 21:46

                               
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有劳把医院感染分子流行病学的知识多在论坛给予展示，使更多的战友懂得这一学科。

我会关注这个方面，逐步将一些文献和数据释放，与大家分享。

蓝鱼o_0

为了便于大家阅读，现在将PDF文件传上。值得注意的是，如果大家感兴趣，后面的参考文献是很重要的参照。

lzz1806

已下载了，很好的资料，会认真学习的，谢谢斑斑