Illumina二代测序在微生物领域的应用

Illumina二代测序在微生物领域的应用
以Illumina技术为特色的微生物学研究论文回顾
目录
目录 (2)
简介 (2)
嵌合现象 (3)
适应性 (4)
核心基因组 (5)
实验室中的进化 (6)
抗生素耐药性和毒力 (6)
流行病与传播 (8)
微生物鉴定........................................................................................................................... ........................................
(10)
定向进化和生物工程 10生物燃料和生物修复 (11)
病毒 (12)
病毒检测与鉴定 (12)
疫苗生产 (13)
酵母 (14)
深入研究的微生物 (15)
大肠杆菌 (15)
金黄色葡萄球菌 (16)
链球菌 (17)
分枝杆菌 (17)
沙门氏菌 (18)
参考文献 (19)
简介
新一代测序特别适合微生物实验室，因其基因组小，且数据分析相对简单。

测序与其他实验室方法相比极具吸引力的差异在于结果可直接关联到基因组位点，以及生物学影响的可能解释。

这代表我们向实验结果的解释和生物系统的了解又迈进了一大步。

第二个优势在于它能够测定基因组中的单碱基变化，而无需任何背景知识。

单碱基分辨率让我们能够追踪微生物在短期内对环境的适应性，包括实验室和外部环境。

这些优势如此突出，让我们很难想象在可预见的未来的生物学实验室会没有一台测序仪。

近期的研究表明，微生物的基因组在适应实验室和临床的环境上异常活跃。

从历史上看，全球流行病的蔓延往往需要几年的时间来跟踪。

有了单碱基分辨率的细菌基因组新一代测序1，有望在几周内快速追溯局部地区人群、医院甚至家庭中的流行病原因。

本回顾重点介绍了近期利用Illumina测序技术来快速追踪天然的、
实验室的和临床中的遗传适应的案例。

1新一代测序（NGS）和大规模并行测序（MPS）常常交替使用，均指代高通量测序技术。

边合成边测序（SBS）特指Illumina的测序技术。

嵌合现象
水平基因转移是遗传物质在细菌之间的移动，而非通过传代。

2 这可通过几种机制发生，但最终结果是嵌合现
象，其中微生物的大部分是由遗传自其祖先的序列组成，而一些DNA片段则来自环境中的其他生物。

由于这种遗传物质的变化不是通过传代，故无法预测和定位。

这让微生物基因组的分析更具挑战性。

测序是准确定位嵌合基因组的唯一工具。

功能基因的水平转移，甚至显著的基因组重排，都可能无法由仅代表基因组一小部分的
标记方法所发现。

综述
Joseph, S. J. and Read T. D. (2012) Genome-wide recombin ati on in Chlamydia tracho mati s. Nat Genet 44: 364-366 Rajendhran, J. and Gunasekaran P. (2011) Microbial phylogeny and diversity: small subunit ribosomal RNA sequence analysis and beyond. Microbiol Res 166: 99-110
参考文献
2 Maiden, M. C. (1998) Horizontal gene ti c exchange, evolu ti on, and spread of an ti bio ti c resistance in bacteria. Clin Infect Dis 27 Suppl 1: S12-20
Gonzalez-Escalona, N., Strain E. A., De Jesus A. J., Jones J. L. and Depaola A. (2011) Genome sequence of the clinical O4:K12 serotype Vibrio parahaemol yti cus strain 10329. J Bacteriol 193: 3405-3406
Izumiya, H., Sekizuka T., Nakaya H., Taguchi M., Oguchi A., et al. (2011) Whole-genome analysis of Salmonella enterica serovar Typhimurium T000240 reveals the acquisi ti on of a genomic island involved in mul ti drug resistance via IS1 deri vativ es on the chromosome. An ti microb Agents Chemother 55: 623-630 适应性
3 Gerstein, A. C. and O tt o, S. P. (2011) Cryp ti c Fitness Advantage: Diploids Invade Haploid Popula ti ons Despite Lacking Any Apparent Advantage as Measured by Standard Fitness Assays. PLoS ONE 6:
4 Bachmann, H., Starrenburg, M. J., Molenaar, D., Kleerebezem, M. and van Hylckama Vlieg, J. E. (2012) Microbial domes ti ca ti on signatures of Lactococcus lac ti s can be
reproduced by experimental evolu ti on. Genome Res 22: 115-124
5 Comas, I., Borrell, S., Roetzer, A., Rose, G., Malla, B., et al. (2011) Whole-genome sequencing of rifampicin-resistant Mycobacterium tuberculosis strains iden ti?es compensatory muta ti ons in RNA polymerase genes. Nat Genet 44: 106-110
6 Casali, N., Nikolayevskyy, V., Balabanova, Y., Ignatyeva, O., Kontsevaya, I., et al. (2012) Microevolu ti on of extensively drug-resistant tuberculosis in Russia. Genome Res 22: 735-745
7 Kvitek, D. J. and Sherlock, G. (2011) Reciprocal sign epistasis between frequently experimentally evolved adap ti ve muta ti ons causes a rugged ?tness landscape. PLoS Genet 7: e1002056
8 Moses, A. M. and Davidson, A. R. (2011) In vitro evolu ti on goes deep. Proc Natl Acad Sci U S A 108: 8071-8072
9 Han, T. X., Xu, X. Y., Zhang, M. J., Peng, X. and Du, L. L. (2010) Global ?tness pro ?ling of ?ssion yeast dele ti
on strains by barcode sequencing. Genome Biol 11: R60 10 Eckert, S. E., Dziva, F., Chaudhuri, R. R., Langridge, G. C., Turner, D. J., et al. (2011) Retrospec ti ve applica ti on of transposon-directed inser ti on site sequencing to a library of signature-tagged mini-Tn5Km2 mutants of Escherichia coli O157:H7 screened in ca tt le. J Bacteriol 193: 1771-1776
微生物群体在单一群体的最佳适应性与非最佳变异的维持上有着权衡，当条件变化时，非最佳变异将存活。

3,4 例如抗生素压力下的微生物群体，对抗生素有耐药性的微生物将存活，即使它们长得更慢，且表现出较低的适应性。

5,6 适应性图谱描述了可能的突变轨迹，其中谱系以一种循序渐进的方式从低适应性区域的基因型向更高适应性的区域进化。

7
一种更为系统的方法利用定点突变在培养或通过动物传播之前向一群微生物中引入突变，接着开展高通量测序，以检测幸存者中的突
变。

这种方法利用了高通量测序的优势，更加高效。

8,9
利用定点突变在培养或通过动物传播之前向一群微生物中引入突变，接着开展高通量测
序，以检测幸存者中的突变。

10
Eckert, S. E., Dziva F., Chaudhuri R. R., Langridge G. C., Turner D. J., et al. (2011) Retrospec ti ve applic ati on of transposon-directed inser ti on site sequencing to a library of signature-tagged mini-Tn5Km2 mutants of Escherichia coli O157:H7 screened in c att le. J Bacteriol 193: 1771-1776
Lee, C. Y., Kam Y. W., Fric J., Malleret B., Koh E. G. L., et al. (2011) Chikungunya Virus Neutraliz ati on An ti gens and Direct Cell-to-Cell Transmission Are Revealed by Human An ti body-Escape Mutants. PLoS Pathog 7:
Hietpas, R. T., Jensen J. D. and Bolon D. N. (2011) From the Cover: Experimental illumin ati on of a ?tness landscape. Proceedings of the N ati onal Academy of Sciences of the United States of America 108: 7896-7901
Nair, D., Memmi G., Hernandez D., Bard J., Beaume M., et al. (2011) Whole-Genome Sequencing of Staphylococcus aureus Strain RN4220, a Key Laboratory Strain Used in Virulence Research, Iden ti?es Muta ti ons That A ?ect Not Only Virulence Factors but Also the Fitness of the Strain. J Bacteriol 193: 2332-2335
在一个分类群中，有一套进化上保守的核心基因，为群中的所有
成员所共享。

这个核心基因组对生存很关键。

群中的个体菌株还有附属基因，它们促成了微环境特异的表型，如毒力和耐药性。

如果我们要分类或修饰微生物，区分核心和附属基因就很重要。

比较一个分类群的成员，可鉴定出保守基因的核心基因组。

引入随机突变并追踪突变体的存活能力也可从实验上确定核心基因组。

如果关键基因失活，那么微生物将无法在培养条件下存活。

这种方法非常灵活，且信息量很大。

核心基因组综述
Laing, C. R., Zhang Y., Thomas J. E. and Gannon V. P. (2011) Everything at once: comp
arati ve analysis of the genomes of bacterial pathogens. Vet Microbiol 153: 13-26
参考文献
实验室中的进化
11 Conrad, T. M., Joyce, A. R., Applebee, M. K., Barre tt , C. L., Xie, B., et al. (2009) Whole-genome resequencing of Escherichia coli K-12 MG1655 undergoing short-term laboratory evolu ti on
in lactate minimal media reveals ?exible selec ti on of adap ti ve muta ti ons. Genome Biol 10: R118 12 Charusan ti , P., Conrad, T. M., Knight, E. M., Venkataraman, K., Fong, N. L., et al. (2010) Gene ti c basis of growth adapta ti on of Escherichia coli a ft er dele ti on of pgi, a major metabolic gene. PLoS Genet 6: e1001186 13 Barrick, J. E. and Lenski, R. E. (2009) Genome-wide muta ti onal diversity in an evolving popula ti on of Escherichia coli. Cold Spring Harb Symp Quant Biol 74: 119-129 14 Harris, D. R., Pollock, S. V., Wood, E. A., Goi ?on, R. J., Klingele, A. J., et al. (2009) Directed evolu ti on of ionizing radia ti on resistance in Escherichia coli. J Bacteriol 191: 5240-5252
在实验室中，模式生物基因组中的突变随时间积累。

11,12 因此，不同实验室中同一模式生物的基因组也可能相异，实验逐渐变得很难重复。

全基因组测序已成为一种强大的工具，可常规监控基因组，并像其他需要记录和控制的实验变量一样对待它们。

基于中性进化的模型，我们预计细菌在一个稳定的实验室环境中随时间以稳定的速度积累中性突变。

在现实中，适应性和中性进化之间的相互作用要复杂得多。

一项涉及40,000多代大肠杆菌的长期研究表明，几乎所有的突变似乎都是有益的，在表面上稳定的实验室群体中，中性突变随时间发生急剧变化。

13 40,000多代后积累的突变数量似乎也没有饱和，这是值得注意的，也有些令人不安。

14
抗生素耐药性和毒力
抗生素耐药性的发生可视为定向进化的一个特殊例子。

因抗生素耐药菌株的日益流行，它已成为深入研究的主题。

多个耐药菌的培养物。

此病原体经过下列抗生素的检测：VA：万古霉素，RD：利福平，CIP：环丙沙星，STX：磺胺甲基异恶唑，MY：克林霉素，E：红霉素-
15 Chua, K. Y. L., Seemann, T., Harrison, P. F., Monagle, S., Korman, T. M., et al. (2011) The Dominant Australian Community-Acquired Methicillin-Resistant Staphylococcus aureus Clone ST93-IV [2B] Is Highly Virulent and Gene ti cally Dis ti nct. PLoS ONE 6: 16 Howden, B. P., McEvoy, C. R. E., Allen, D. L., Chua, K., Gao, W., et al. (2011) Evolu ti on of Mul ti drug Resistance during Staphylococcus aureus Infec ti on Involves Muta ti on of the Essen ti al Two Component Regulator WalKR. PLoS Pathog 7: e1002359 17 Toprak, E., Veres, A., Michel, J. B., Chait, R., Hartl, D. L., et al. (2011) Evolu ti onary paths to an ti bio ti c resistance under dynamically sustained drug selec ti on. Nat Genet 44: 101-105 由于细菌基因组的嵌合特性，它可从环境中快速获得抗生素抗性基因或毒力相关基因。

这些基因的获得常常与血清型或基因标记无关。

研究表明，全基因组新一代测序是一种理想的工具，可全面且明确地追踪抗生素抗性和毒力相关基因。

研究抗生素耐药性和毒力变化的传统方法是将各次爆发和流行期间所获得的分离株进行比较。

15,16 新方法追踪微生物对实验室中持续抗生素压力的适应。

它利用新一代测序技术来快速且经济高效地检测单核苷酸变化。

这种方法的优势在于实验室环境内的所有变量都能小心控制，且结果与生物学功能直接相关。

17
Howden, B. P., McEvoy C. R. E., Allen D. L., Chua K., Gao W., et al. (2011) Evolu ti on of Mul ti drug Resistance during
Staphylococcus aureus In fecti on Involves Mu tati on of the Essen ti al Two Component Regulator WalKR. PLoS Pathog 7: e1002359 Peleg, A. Y., Miyakis S., Ward D. V., Earl A. M., Rubio A., et al. (2012) Whole genome characteriz ati on of the mechanisms of daptomycin resistance in clinical and laboratory derived isolates of Staphylococcus aureus. PLoS ONE 7: e28316
Lieberman, T. D., Michel J. B., Aingaran M., Po tt er-Bynoe G., Roux D., et al. (2011) Parallel bacterial evolu ti on within mul ti ple p ati ents iden ti?es candidate pathogenicity genes. Nat Genet 43: 1275-1280
Thomas, J. C., Figueira M., Fennie K. P., Laufer A. S., Kong Y., et al. (2011) Streptococcus pneumoniae clonal complex 199: gene ti c diversity and ti ssue-speci ?c virulence. PLoS ONE 6: e18649
Harvey, R. M., Stroeher U. H., Ogunniyi A. D., Smith-Vaughan H. C., Leach A. J., et al. (2011) A variable region within the genome of Streptococcus pneumoniae contributes to strain-strain vari ati on in virulence. PLoS ONE 6: e19650
流行病与传播
综述
Lenski, R. E. (2011) Chance and necessity in the evolu ti on of a bacterial pathogen. Nat Genet 43: 1174-1176
Editorial, N. B. (2011) Outbreak genomics. Nat Biotechnol 29: 769
参考文献
18
Croucher, N. J. (2009) From small reads do mighty genomes grow. Nat Rev Microbiol 7: 621 19 Reeves, P. R., Liu, B., Zhou, Z., Li, D., Guo, D., et al. (2011) Rates of muta ti on and host transmission for an Escherichia coli clone over 3 years. PLoS ONE 6: e26907 20 Harris, S. R., Feil, E. J., Holden, M. T., Quail, M. A., Nickerson, E. K., et al. (2010) Evolu ti on of MRSA during hospital transmission and intercon ti nental spread. Science 327: 469-474 21 Hendriksen, R. S., Price, L. B., Schupp, J. M., Gillece, J. D., Kaas, R. S., et al. (2011) Popula ti on Gene ti cs of Vibrio cholerae from Nepal in 2010: Evidence on the Origin of the Hai ti an Outbreak. MBio 2: 22 Mutreja, A., Kim, D. W., Thomson, N. R., Connor, T. R., Lee, J. H., et al. (2011) Evidence for several waves of global transmission in the seventh cholera pandemic. Nature 477: 462-465 23 Grad, Y. H., Lipsitch, M., Feldgarden, M., Arachchi, H. M., Cerqueira, G. C., et al. (2012) Genomic epidemiology of the Escherichia coli O104:H4 outbreaks in Europe, 2011. Proc Natl Acad Sci U S A 109: 3065-3070 24 Howden, B. P., McEvoy, C. R. E., Allen, D. L., Chua, K., Gao, W., et al. (2011) Evolu ti on of Mul ti drug Resistance during Staphylococcus aureus Infec ti on Involves Muta ti on of the Essen ti al Two Component Regulator WalKR. PLoS Pathog 7: e1002359
流行病与微生物传播在传统上都是通过血清学或其他标记来追踪的，它们只监控微生物基因组中任意的一小部分。

由于微生物基因组的嵌合性质，只监控一部分基因组的方法会相对不可靠、不灵敏。

相比之下，新一代测序可追踪基因组中的每个碱基，彻底改变我们对这些过程的了解。

18 如今，即使是家庭19或医院20内的相对短期的传播也能追踪，爆发源也能确定。

21,22,23 这允许我们更快速更有针对性地应对爆发。

另一个好处是，爆发过程中积累的突变能够提供有关抗生素耐药性的潜在发展和毒力变化的信息。

24
Reeves et al.26 report the transmission of a a uropathogenic E. coli clone that persisted for 3 years within a household, including a dog. The data imply at least 6 host transfer events.
Fi tti paldi, N., Beres S. B., Olsen R. J., Kapur V., Shea P. R., et al. (2012) Full-Genome Dissec ti on of an Epidemic of Severe Invasive Disease Caused by a Hypervirulent, Recently Emerged Clone of Group A Streptococcus. Am J Pathol Grad, Y. H., Lipsitch M., Feldgarden M., Arachchi H. M., Cerqueira G. C., et al. (2012) Genomic epidemiology of the Escherichia coli O104:H4 outbreaks in Europe, 2011. Proc Natl Acad Sci U S A 109: 3065-3070 Mutreja, A., Kim D. W., Thomson N. R.,
Connor T. R., Lee J. H., et al. (2011) Evidence for several waves of global transmission in the seventh cholera pandemic. Nature 477: 462-465
Gardy, J. L., Johnston J. C., Ho Sui S. J., Cook V. J., Shah L., et al. (2011) Whole-genome sequencing and social-network analysis of a tuberculosis outbreak. N Engl J Med 364: 730-739
25 Johnson, J. R., Clabots, C. and Kuskowski, M. A. (2008) Mul ti ple-host sharing, long-term persistence, and virulence of Escherichia coli clones from human and animal household members. J Clin Microbiol 46: 4078-4082 26 Reeves, P. R., Liu, B., Zhou, Z., Li, D., Guo, D., et al. (2011) Rates of mutation and host transmission for an Escherichia coli clone over 3 years. PLoS ONE 6: e26907
微生物鉴定
传统的微生物鉴定依靠临床症状和一些先验知识来确定微生物种类。

一些病例不太典型，就难以鉴定，微生物检
测的不可知性让新一代测序成为研究这些病例的有用工具。

测序技术不需要先验知识来鉴定微生物。

Avasthi, T. S., Devi S. H., Taylor T. D., Kumar N., Baddam R., et al. (2011) Genomes of two chronological isolates (Helicobacter pylori 2017 and 2018) of the West African Helicobacter pylori strain 908 obtained from a single p ati ent.
J Bacteriol 193: 3385-3386
定向进化和生物工程
定向进化作为传统生物工程技术的一个很有希望的补充正在兴起。

微生物极其快速地适应环境中的变化。

通过系统改变环境，研究人员可追踪基因表达的变化以及生物体为实现理想特性而掺入的突变，例如在半乳糖上生长的适应性进化。

在重复实验中，生物体可能通过改变不同的通路而实现相同的理想特性。

这提供了一系列可能的生物工程方案。

Gibbons, H. S., Broomall S. M., McNew L. A., Daligault H., Chapman C., et al. (2011) Genomic signatures of strain selec ti on and enhancement in Bacillus atrophaeus var. globigii, a historical biowarfare simulant. PLoS ONE 6: e17836 Kvitek, D. J. and Sherlock G. (2011) Reciprocal sign epistasis between frequently experimentally evolved adap ti ve mu tati ons causes a rugged ?t ness landscape. PLoS Genet 7: e1002056
生物燃料和生物修复
寻找新的微生物以进行生物燃料生产和环境的生物修复，通常是利用宏基因组方法开展的。

然而，进化压力和实验室中的操作可能在改善新发现候选微生物上非常有效。

一个污水处理厂
Radakovits, R., Jinkerson R. E., Fuerstenberg S. I., Tae H., Se tt lage R. E., et al. (2012) Dr aft genome sequence and gene ti c transfo rmati on of the oleaginous alga Nannochloropis gaditana. Nat Commun 3: 686
Tremblay, P. L., Summers Z. M., Glaven R. H., Nevin K. P., Zengler K., et al. (2011) A c-type cytochrome and a transcrip ti onal regulator responsible for enhanced extracellular electron transfer in Geobacter sulfurreducens revealed by adap ti ve evolu ti on. Environ Microbiol 13: 13-23
27 Eckerle, L. D., Becker, M. M., Halpin, R. A., Li, K., Venter, E., et al. (2010) In ?delity of SARS-CoV Nsp14-exonuclease mutant virus replica ti on is revealed by complete genome sequencing. PLoS Pathog 6: e1000896 28 Murray, C. L., Oh, T. S. and Rice, C. M. (2011) Keeping Track of Viruses. Microbial Forensics (Second Edi ti on) 137-153 29 Yozwiak, N. L., Skewes-Cox, P., Stenglein, M.
D., Balmaseda, A., Harris,
E., et al. (2012) Virus iden ti?ca ti on in unknown tropical febrile illness cases using deep sequencing. PLoS Negl Trop Dis 6: e1485 30 Jiang, Z., Jhunjhunwala, S., Liu, J., Haverty, P. M., Kennemer, M. I., et al. (2012) The e ?ects of hepa titi s B virus integra ti on into the genomes of hepatocellular carcinoma pa ti ents. Genome Res 22: 593-601 31 Kriesel, J. D., Hobbs, M. R., Jones, B. B., Milash, B., Nagra, R. M., et al. (2012) Deep sequencing for the detec ti on of virus-like sequences in the brains of pa ti ents with mul ti ple sclerosis: detec ti on of
GBV-C in human brain. PLoS ONE 7: e31886 病毒
病毒检测与鉴定
病毒独特的复制策略让它成为一个特殊的挑战，难以在实验室中追踪其进化。

在大部分活细胞中，错误掺入的发生率为每10亿个碱基中一个，而在某些病毒中，错误发生率常常为每一千个碱基中一个。

这是因为使用了无校正活性（RdRp或RT）或修复活性有限的酶。

27 不仅突变更频繁地发生，许多拷贝也快速地产生。

在单个生命周期内，病毒可能复制数百次，甚至数千次，而细胞只传了一代产生两个子代细胞。

28
病毒感染的不明确症状让它们成为诊断挑战。

此外，它们的遗传灵活性又让它们难以鉴定。

近期Yozwiak等开展的研究证明了新一代测序在检测和鉴定病毒上的能力，这些病毒逃脱了传统技术的检测。

29 这种检测能力引发了在肿瘤30和慢性病31中寻找病毒感染的兴趣。

高灵敏度以及不需要先验知识，让新一代测序成为病毒检测与鉴定的主要工具。

Blasdell, K. R., Voysey R., Bulach D., Joubert D. A., Tesh R. B., et al. (2012) Kotonkan and Obodhiang viruses: African
ephemeroviruses with large and complex genomes. Virology 425: 143-153
Dunowska, M., Biggs P. J., Zheng T. and Perro tt M. R. (2012) Iden ti?c a ti on of a novel nidovirus associated with a neurological disease of the Australian brushtail possum (Trichosurus vulpecula). Vet Microbiol 156: 418-424
Flaherty, P., Natsoulis G., Muralidharan O., Winters M., Buenrostro J., et al. (2012) Ultrasensi ti ve detec ti on of rare mu tati ons using next-genera ti on targeted resequencing. Nucleic Acids Res 40: e2
Jiang, Z., Jhunjhunwala S., Liu J., Haverty P. M., Kennemer M.
I., et al. (2012) The e?ects of hep atiti s B virus integ rati on into the genomes of hepatocellular carcinoma pa ti ents. Genome Res 22: 593-601
Kriesel, J. D., Hobbs M. R., Jones B. B., Milash B., Nagra R. M., et al. (2012) Deep sequencing for the detec ti on of virus like sequences in the brains of p ati ents with mul ti ple sclerosis: detec ti on of GBV-C in human brain. PLoS ONE 7: e31886 Tapparel, C., Cordey S., Junier T., Farinelli L., Van Belle S., et al. (2011) Rhinovirus genome varia ti on during chronic upper and lower respiratory tract in fecti ons. PLoS ONE 6: e21163 疫苗生产
活病毒疫苗的生产需要严格的质量控制，以确保疫苗的安全性。

主要的风险之一是RNA病毒的内在遗传不稳定性，它可能导致生产过程中有毒回复突变的积累。

研究人员在实验室中面临着这样的挑战，人巨细胞病毒就是一个很好的例子。

常用的人巨细胞病毒（HCMV）变种T owne和AD169已广泛分布，并作为候选疫苗开发。

多年来，其详细的历史已变得模糊，而人们逐渐清楚这些菌株的生物学性质并不保守。

这些遗传差异可能影响实验研究的解释，也无疑会影响它们在疫苗开发中的应用。

32
32 Bradley, A. J., Lurain, N. S., Ghazal, P., Trivedi, U., Cunningham, C., et al. (2009) High-throughput sequence analysis of variants of human cytomegalovirus strains Towne and AD169. J Gen Virol 90: 2375-2380
Checkley, A. M., Wyllie D. H., Scriba T. J., Golubchik T., Hill A. V., et al. (2011) Iden ti?c a ti on of an ti gens speci ?c to non-tuberculous mycobacteria: the Mce family of proteins as a target of T cell immune responses. PLoS ONE 6: e26434
33 Araya, C. L., Payen, C., Dunham, M. J. and Fields, S. (2010) Whole-genome sequencing of a laboratory-evolved yeast strain. BMC Genomics 11: 88 34 Qi, J., Wijeratne, A. J., Tomsho, L. P., Hu, Y., Schuster, S. C., et al. (2009) Characteriza ti on of meio ti c crossovers and gene conversion by whole-
genome sequencing in Saccharomyces cerevisiae. BMC Genomics 10: 475
酵母
在面对环境压力时，酵母基因组能够发生异常复杂的遗传变化。

酿酒酵母（S. cerevisiae）菌株DBY10147经过约188代硫酸盐有限的连续培养后，分离出一个菌株，开展深度测序。

作者发现了之前芯片研究未发现的单核苷酸多态性和拷贝数扩增。

33
新一代测序技术不依赖参考基因组，这在参考基因组不可用或不正确的许多情况下是个优势。

例如，作者对酵母S288C（12倍覆盖）和RM11（15倍覆盖）的两个菌株进行测序，发现公开序列有803和1104个错误。

34 参考基因组中的任何错误和遗漏都将设计成基于这些基因组的芯片或tiling芯片。

Berry, D. B., Guan Q., Hose J., Haroon S., Gebbia M., et al. (2011) Mul ti ple means to the same end: the gene ti c basis of acquired stress resistance in yeast. PLoS Genet 7: e1002353 Libkind, D., Hi tti nger C. T., Valerio E., Goncalves C., Dover J., et al. (2011) Microbe domes ti c ati on and the iden ti?c a ti on of the wild gene ti c stock of lager-brewing yeast. Proc Natl Acad Sci U S A 108: 14539-14544
Parts, L., Cubillos F. A., Warringer J., Jain K., Salinas F., et al. (2011) Revealing the gene ti c structure of a trait by sequencing a popul ati on under selec ti on. Genome Res 21: 1131-1138
Warringer, J., Zorgo E., Cubillos F. A., Zia A., Gjuvsland A., et al. (2011) Trait vari ati on in yeast is de?ned by popul ati on history. PLoS Genet 7: e1002111
深入研究的微生物
大肠杆菌
据观察，一些大肠杆菌感染的分离株表现出物种内的多样性。

一些证据表明，这种微异质性是来自感染过程的多样化，而非多个分离株的感染。

观察到的多样性类似于实验性进化研究所获得的结果。

无
论是何种机制导致多样性，结果强调了在决定抗生素疗法前开展更广泛分离株检测的必要性。

Grad, Y. H., Lipsitch M., Feldgarden M., Arachchi H. M., Cerqueira G. C., et al. (2012) Genomic epidemiology of the Escherichia coli O104:H4 outbreaks in Europe, 2011. Proc Natl Acad Sci U S A 109: 3065-3070
Loman, N. J., Misra R. V., Dallman T. J., Constan ti nidou C., Gharbia S. E., et al. (2012) Performance comparison of benchtop high-throughput sequencing pl atf orms. Nat Biotechnol Benson, R. W., Norton M. D., Lin I., Du Comb W. S. and Godoy V. G. (2011) An ac ti ve site aro mati c triad in Escherichia coli DNA Pol IV coordinates cell survival and mutagenesis in di?erent DNA damaging agents. PLoS ONE 6: e19944
Sahl, J. W., Steinsland H., Redman J. C., Angiuoli S. V., Nataro J. P., et al. (2011) A comp arati ve genomic analysis of diverse clonal types of enterotoxigenic Escherichia coli reveals pathovar-speci?c conserva ti on. Infect Immun 79: 950-960
金黄色葡萄球菌
35
Nubel, U., Dordel, J., Kurt, K., Strommenger, B., Westh, H., et al. (2010) A ti mescale for evolu ti on, popula ti on expansion, and spa ti al spread of an emerging clone of methicillin-resistant Staphylococcus aureus. PLoS Pathog 6: e1000855 36 Corvaglia, A. R., Francois, P., Hernandez, D., Perron, K., Linder, P., et al. (2010) A type III-like restric ti on endonuclease func ti ons as a major barrier to horizontal gene transfer in clinical Staphylococcus aureus strains. Proc Natl Acad Sci U S A 107: 11954-11958 抗生素耐药性的蔓延通常需要长期追踪。

例如，长期连续采样的耐甲氧西林金黄色葡萄球菌（MRSA）（ST225）的D NA序列让研究人员能够估计，ST225在1990年左右开始分离（1987至1994年），而欧洲分
支在1995年开始扩散（1991至1999年），几年后才确认新克隆。

35
抗生素耐药性的蔓延也因水平基因转移的可能性而复杂化。

一些临床MRSA菌株缺乏类似III型的限制性内切酶系统，因此对其他物种（如大肠杆菌）DNA的水平转移超敏。

例如，敏感的金黄色葡萄球菌菌株可能很容易从肠球菌中获得耐受万古霉素的基因。

36 宿主跳跃是遗传适应的最明显例子之一。

大部分来自肉用仔鸡的金黄色葡萄球菌分离株是一次人向家禽宿主跳跃的后代，那次跳跃大约发生在38年前（范围，30至63年前），是波兰独有的人ST5克隆谱系的一个亚型。

这代表了一种重要的新动物病原体的进化史，它经历了快速的家禽宿主适应和洲际传播，是研究人类活动对动物病原体的出现和蔓延的影响的一个新范例。

37
37 Lowder, B. V., Guinane, C. M., Ben Zakour, N. L., Weinert, L.
A., Conway-Morris, A., et al. (2009) Recent human-to-poultry host jump, adapta ti on, and pandemic spread of Staphylococcus aureus. Proc Natl Acad Sci U S A 106: 19545-19550 Lama, A., Pane-Farre J., Chon T., Wiersma A. M., Sit C. S., et al. (2012) Response of Methicillin-Resistant Staphylococcus aureus to Amicoumacin A. PLoS ONE 7: e34037
Peleg, A. Y., Miyakis S., Ward D. V., Earl A. M., Rubio A., et al. (2012) Whole genome characteriz ati on of the mechanisms of daptomycin resistance in clinical and laboratory derived isolates of Staphylococcus aureus. PLoS ONE 7: e28316
Pozzi, C., Waters E. M., Rudkin J. K., Schae ?er C. R., Lohan A. J., et al. (2012) Methicillin Resistance Alters the Bio ?lm Phenotype and A tt enuates Virulence in Staphylococcus aureus Device-Associated In fecti ons. PLoS Pathog 8:
Price, L. B., Stegger M., Hasman H., Aziz M., Larsen J., et al. (2012) Staphylococcus aureus CC398: host adap tati on and emergence of methicillin resistance in livestock. MBio 3:
Young, B. C., Golubchik T., Ba tt y E. M., Fung R., Larner-Svensson H., et al. (2012) Evolu ti onary dynamics of Staphylococcus aureus during progression from carriage to disease. Proc Natl Acad Sci U S A 109: 4550-4555
McAdam, P. R., Holmes A., Templeton K. E. and Fitzgerald J. R. (2011) Adap ti ve evolu ti on of Staphylococcus aureus during chronic endobronchial infec ti on of a cys ti c ?brosis p ati ent. PLoS ONE 6: e24301
Harris, S. R., Feil E. J., Holden M. T., Quail M. A., Nickerson E. K., et al. (2010) Evolu ti on of MRSA during hospital transmission and intercon ti nental spread. Science 327: 469-474
链球菌
链球菌是一种革兰氏阳性菌，属于厚壁菌门，其基因组大小约为200万个碱基。

38
Thomas, J. C., Figueira M., Fennie K. P., Laufer A. S., Kong Y., et al. (2011) Streptococcus pneumoniae clonal complex 199: gene ti c diversity and ti ssue-speci?c virulence. PLoS ONE 6: e18649
Harvey, R. M., Stroeher U. H., Ogunniyi A. D., Smith-Vaughan H. C., Leach A. J., et al. (2011) A variable region within the genome of Streptococcus pneumoniae contributes to strain-strain vari ati on in virulence. PLoS ONE 6: e19650
Shea, P. R., Beres S. B., Flores A. R., Ewbank A. L., Gonzalez-Lugo J. H., et al. (2011) Dis ti nct signatures of diversifying selec ti on revealed by genome analysis of respiratory tract and invasive bacterial popula ti ons. Proc Natl Acad Sci U S A 108: 5039-5044
分枝杆菌
类似真菌的分枝杆菌引发了各种疾病，从结核病到麻风病。

它的基因组大小约为400万个碱基对，包含3959个基因，很容易通过新
一代测序技术来测序。

Ghosh, P., Hsu C., Alyamani E. J., Shehata M. M., Al-Dubaib M.
A., et al. (2012) Genome-wide analysis of the emerging in fecti on with Mycobacterium avium subspecies paratuberculosis in the Arabian camels (Camelus dromedarius). PLoS ONE 7: e31947 Koser, C. U., Niemann S., Summers D. K. and Archer J. A. (2012) Overview of errors in the reference sequence and anno tati on of Mycobacterium tuberculosis H37Rv, and vari ati on amongst its isolates. Infect Genet Evol 12: 807-810
Checkley, A. M., Wyllie D. H., Scriba T. J., Golubchik T., Hill A. V., et al. (2011) Iden ti?c a ti on of an ti gens speci?c to non-tuberculous mycobacteria: the Mce family of proteins as a target of T cell immune responses. PLoS ONE 6: e26434
Comas, I., Borrell S., Roetzer A., Rose G., Malla B., et al. (2011) Whole-genome sequencing of rifampicin-resistant Mycobacterium tuberculosis strains iden ti?es compensatory muta ti ons in RNA polymerase genes. Nat Genet 44: 106-110 Ford, C. B., Lin P. L., Chase M. R., Shah R. R., Iartchouk O., et al. (2011) Use of whole genome sequencing to es ti mate the muta ti on rate of Mycobacterium tuberculosis during latent infec ti on. Nat Genet 43: 482-486
38 Te tt elin, H., Masignani, V., Cieslewicz, M. J., Dona ti, C., Medini, D., et al. (2005) Genome analysis of mul ti ple pathogenic isolates of Streptococcus agalac ti ae: implica ti ons for the
microbial "pan-genome". Proc Natl Acad Sci U S A 102: 13950-13955
Ford, C. B., Lin P. L., Chase M. R., Shah R. R., Iartchouk O., et al. (2011) Use of whole genome sequencing to es ti mate the muta ti on rate of Mycobacterium tuberculosis during latent infec ti on. Nat Genet 43: 482-486
Gardy, J. L., Johnston J. C., Ho Sui S. J., Cook V. J., Shah L., et al. (2011) Whole-genome sequencing and social-network analysis of a tuberculosis outbreak. N Engl J Med 364: 730-739 Gri ?n, J. E., Gawronski J. D., DeJesus M. A., Ioerger T. R., Akerley B. J., et al. (2011) High-Resolu ti on Phenotypic Pro ?ling De ?nes Genes Essen ti al for Mycobacterial Growth and Cholesterol Catabolism. PLoS Pathog 7:
Saunders, N. J., Trivedi U. H., Thomson M. L., Doig C., Laurenson I. F., et al. (2011) Deep resequencing of serial sputum isolates of Mycobacterium tuberculosis during therapeu ti c failure due to poor compliance reveals stepwise muta ti on of key resistance genes on an otherwise stable gene ti c background. J Infect 62: 212-217
39 Holt, K. E., Parkhill, J., Mazzoni, C. J., Roumagnac, P., Weill,
F. X., et al. (2008) High-throughput sequencing provides insights into genome varia ti on and evolu ti on in Salmonella Typhi. Nat Genet 40: 987-993 40 Holt, K. E., Thomson, N. R., Wain, J., Langridge,
G. C., Hasan, R., et al. (2009) Pseudogene accumula ti on in the evolu ti onary histories of Salmonella enterica serovars Paratyphi A and Typhi. BMC Genomics 10: 36 41 Lan, R., Reeves, P. R. and Octavia, S. (2009) Popula ti on structure, origins and evolu ti on of major Salmonella enterica clones. Infect Genet Evol 9: 996-1005 沙门氏菌
Deng, X., Li Z. and Zhang W. (2012) Transcriptome
sequencing of Salmonella enterica serovar Enteri ti dis under desicc ati on and s tarvati on stress in peanut oil. Food Microbiol 30: 311-315
Okoro, C. K., Kingsley R. A., Quail M. A., Kankw ati ra A. M., Feasey N. A., et al. (2012) High-resolu ti on single nucleo ti de polymorphism analysis dis ti nguishes recrudescence and reinfec ti on in recurrent invasive nontyphoidal salmonella typhimurium disease. Clin Infect Dis 54: 955-963
Izumiya, H., Sekizuka T., Nakaya H., Taguchi M., Oguchi A., et al. (2011) Whole-genome analysis of Salmonella enterica serovar Typhimurium T000240 reveals the acquisi ti on of a genomic island involved in mul ti drug resistance via IS1 deri vativ es on the chromosome. An ti microb Agents Chemother 55: 623-630 Jansen, A. M., Hall L. J., Clare S., Goulding D., Holt K. E., et al. (2011) A Salmonella Typhimurium-Typhi Genomic Chimera: A Model to Study Vi Polysaccharide Capsule Func ti on In Vivo. PLoS Pathog 7:
Leschner, S., Deyneko I. V., Lienenklaus S., Wolf K., Bloecker H., et al. (2011) Iden ti?c a ti on of tumor-speci ?c Salmonella Typhimurium promoters and their regulatory logic. Nucleic Acids Res
肠道沙门氏菌（Salmonella enterica）血清型的测序证明了全基因组测序的许多优势。

39,40,41 它也作为一个反面教材，说明了目前表型鉴定（如血清型和噬菌体型）的欺骗性。

根据突变变化，噬菌体型DT104是异质的，呈现出多个序列类型。

这一观察应该不会让人惊讶。

血清型由基因组的一小部分所编码，它们处于完全不同的选择压力下，决定了耐药性和毒力表型。

利用血清型作为毒力或其他特征的替代标记受到严重限制。

此限制很明显，因为DT104的多个耐药变异是世界上许多地方发生流行病的原因。

测序是直接观察基因组并分子分型的唯一明确方法
参考文献12385939
Araya, C. L., Payen, C., Dunham, M. J. and Fields, S. (2010) Whole-genome sequencing of a laboratory-evolved yeast strain. BMC Genomics 11: 88 Avasthi, T. S., Devi, S. H., Taylor, T. D., Kumar, N., Baddam, R., et al. (2011) Genomes of two chronological isolates (Helicobacter pylori 2017 and 2018) of the West African Helicobacter pylori strain 908 obtained from a single patient. J Bacteriol 193: 3385-3386
Bachmann, H., Starrenburg, M. J., Molenaar, D., Kleerebezem, M. and van Hylckama Vlieg, J. E. (2012) Microbial domestication signatures of Lactococcus lactis can be reproduced by experimental evolution. Genome Res 22: 115-124 Barrick, J. E. and Lenski, R. E. (2009) Genome-wide mutational diversity in an evolving population of Escherichia coli. Cold Spring Harb Symp Quant Biol 74: 119-129
Benson, R. W., Norton, M. D., Lin, I., Du Comb, W. S. and Godoy, V. G. (2011) An active site aromatic triad in Escherichia coli DNA Pol IV coordinates cell survival and mutagenesis in di?erent DNA damaging agents. PLoS ONE 6: e19944 Berry, D. B., Guan, Q., Hose, J., Haroon, S., Gebbia, M., et al. (2011) Multiple means to the same end: the genetic basis of acquired stress resistance in yeast. PLoS Genet 7: e1002353
Blasdell, K. R., Voysey, R., Bulach, D., Joubert, D. A., Tesh, R. B., et al. (2012) Kotonkan and Obodhiang viruses: African ephemeroviruses with large and complex genomes. Virology 425: 143-153
Bradley, A. J., Lurain, N. S., Ghazal, P., Trivedi, U., Cunningham, C., et al. (2009) High-throughput sequence analysis of variants of human cytomegalovirus strains Towne and AD169. J Gen Virol 90: 2375-2380。