Bacterial pan-genomics

Transcription

1 Bacterial pan-genomics Dave Ussery 4 th annual workshop on Comparative Microbial Genomics and Taxonomy Petropolois, Brazil Lecture #3 Wednesday, 5 August, 2009

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3 What we DO know What we DON'T know Outline What can we do about it? New genes New gene families Core genome Pan genome - E. coli K-12 1 : Ecoli_K12DH10B 2 : Ecoli_K12MG : Ecoli_K12W : Ecoli_B 5 : Ecoli_B03 6 : Ecoli_B171 7 : Ecoli_B7A 8 : Ecoli_F11 9 : Ecoli_H : Ecoli_HS 11 : Ecoli_101!1 12 : Ecoli_ : Ecoli_ : Ecoli_ : Ecoli_ : Ecoli_CFT : Ecoli_E : Ecoli_E22 19 : Ecoli_E : Ecoli_E24377A 21 : Ecoli_ED1a 22 : Ecoli_IAI1 23 : Ecoli_IAI39 24 : Ecoli_LANL_ECA 25 : Ecoli_LANL_ECF 26 : Ecoli_O103Oslo 27 : Ecoli_O157_EC : Ecoli_O157_EC : Ecoli_O157_EC : Ecoli_O157_EC : Ecoli_O157_EC : Ecoli_O157_EC : Ecoli_O157_EC : Ecoli_O157_EC : Ecoli_O157_EC : Ecoli_O157_EC : Ecoli_O157_EC : Ecoli_O157_EC : Ecoli_O157_EDL : Ecoli_O157_Sakai 41 : Ecoli_ : Ecoli_RS : Ecoli_S88 44 : Ecoli_SE11 45 : Ecoli_SMS35 46 : Ecoli_UMN : Ecoli_UTI89 48 : Ecoli_VR50 49 : Ecoli_APEC01 50 : Sflex_ : Sflex_2a : Sflex_ : Sboyd_Sb : Sdyse_Sd : Ssone_Ss : Ealbe_TW : Eferg_35469T

4 Genome analysis of multiple pathogenic isolates of Streptococcus agalactiae: Implications for the microbial pan-genome Hervé Tettelin a,b, Vega Masignani b,c, Michael J. Cieslewicz b,d,e, Claudio Donati c, Duccio Medini c, Naomi L. Ward a,f, Samuel V. Angiuoli a, Jonathan Crabtree a, Amanda L. Jones g, A. Scott Durkin a, Robert T. DeBoy a, Tanja M. Davidsen a, Marirosa Mora c, Maria Scarselli c, Immaculada Margarit y Ros c, Jeremy D. Peterson a, Christopher R. Hauser a, Jaideep P. Sundaram a, William C. Nelson a, Ramana Madupu a, Lauren M. Brinkac a, Robert J. Dodson a, Mary J. Rosovitz a, Steven A. Sullivan a, Sean C. Daugherty a, Daniel H. Haft a, Jeremy Selengut a, Michelle L. Gwinn a, Liwei Zhou a, Nikhat Zafar a, Hoda Khouri a, Diana Radune a, George Dimitrov a, Kisha Watkins a, Kevin J. B. O Connor h, Shannon Smith i, Teresa R. Utterback i, Owen White a, Craig E. Rubens g, Guido Grandi c, Lawrence C. Madoff e,j, Dennis L. Kasper e,j, John L. Telford c, Michael R. Wessels d,e, Rino Rappuoli c,k,l, and Claire M. Fraser a,b,k,m a Institute for Genomic Research, 9712 Medical Center Drive, Rockville, MD 20850; c Chiron Vaccines, Via Fiorentina 1, Siena, Italy; d Division of Infectious Diseases, Children s Hospital, 300 Longwood Avenue, Boston, MA 02115; e Harvard Medical School, Boston, MA 02115; f Center of Marine Biotechnology, University of Maryland Biotechnology Institute, 701 East Pratt Street, Baltimore, MD 21202; g Children s Hospital and Regional Medical Center, 307 Westlake Avenue N, Seattle, WA 98109; h The Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218; i J. Craig Venter Institute, 5 Research Place, Rockville, MD 20850; j Channing Laboratory, Brigham and Women s Hospital, 181 Longwood Avenue, Boston, MA 02115; and m George Washington University Medical Center, 2300 Eye Street NW, Washington, DC t t Contributed by Rino Rappuoli, August 5, 2005 Fig. 2. GBS core genome. The number of shared genes is plotted as a function of the number n of strains sequentially added (see Materials and Methods). For each n, circles are the 8! [(n 1)! (8 n)!] values obtained for Proc Natl Acad Sci USA, 102: (2005). Fig. 3. GBS pan-genome. The number of specific genes is plotted as a function of the number n of strains sequentially added (see Materials and Methods). For each n, circles are the 8! [(n 1)! (8 n)!] values obtained for G p t w i t c c t i g t ( F ( 7 s t s s

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6 Table 12.1 The number of sequenced genomes and ongoing projects of various bacterial genera and the current numbers of available multiple genomes per species. The top-scoring genera only are listed Sequencing projects Number of species Number of Number of finished With projects finished With projects Genus projects in progress genomes in progress Streptococcus Clostridium Burkholderia Bacillus Salmonella Escherichia Vibrio Mycobacterium Listeria Yersinia Mycoplasma Shewanella Pseudomonas Borrelia Haemophilus Staphylococcus Campylobacter Synechococcus Francisella Lactobacillus Rickettsia As of 5 August, 2009 D.W. Ussery et al., Computing for Comparative Microbial Genomics, Computational Biology 8, DOI / _14, Springer-Verlag London Limited 2009

7 # genomes sequenced Streptococcus Burkholderia Bacillus Clostridium Vibrio Mycobacterium Salmonella Listeria Escherichia Mycoplasma Shewanella Pseudomonas Yersinia Haemophilus Staphylococcus Campylobacter Synechococcus Francisella Lactobacillus Rickettsia Projects 104 ongoing

8 News and Views Nature 412, (9 August 2001) doi: / Genome sequencing: The ABC of symbiosis J. Allan Downie and J. Peter W. Young It is a truth universally acknowledged, that there are only two kinds of bacteria. One is Escherichia coli and the other is not. Anything that E. coli does is a universal truth about bacteria; anything it does not do must be a specialization.

9 E. coli CFT073 Escherichia coli, strain K-12, isolate W3110 4,641,433 bp 4390 genes E. coli O157 E. coli E2348 5,528,445 bp 5,231,428 bp 4,227,846 bp

10 A_Crenarchaeota (n=12) A_Euryarchaeota (n=26) A_Nanoarchaeota (n=1) B_Acidobacteria (n=2) B_Actinobacteria (n=39) B_Aquificae (n=1) B_BacteroidetesChlorobi (n=12) B_Chlamydiae (n=11) B_Chloroflexi (n=2) B_Cyanobacteria (n=23) B_DeinococcusThermus (n=4) B_Firmicutes (n=106) B_Fusobacteria (n=1) B_Planctomycetes (n=1) B_Proteobacteria_Alpha (n=56) B_Proteobacteria_Beta (n=43) B_Proteobacteria_Delta (n=14) B_Proteobacteria_Epsilon (n=11) B_Proteobacteria_Gamma (n=115) B_Spirochaetes (n=9) B_Thermotogae (n=1) Size distribution of Prokaryotic genomes (n=490) E. E. coli coli Genome Size (Mbases)

11 Core genes E. coli core genes in 32 genomes ~1560 core genes counts n genomes Genome Biology, 2007, 8:R267doi: /gb r267

12 Specific genes E. coli pan-genome based on 32 genomes 11,862 gene families ~79 new genes/genome (cp. 441 new genes/genome, predicted previously) counts n genomes Genome Biology, 2007, 8:R267doi: /gb r267

13 9,797 unique to E. coli and Shigella 9,797 core families E. coli and Shigella strain-specific families 2,041 Total of 11,838 E. coli and Shigella gene families (32 genome sequences) 2,041 E. coli and Shigella core families

14 # Gene Families 4,000 3,000 2,000 1,000 0 How large is the E. coli pan-genome? Can we estimate this? Distribution of 11,838 E. coli and Shigella gene families 3798 unique genes 2041 genes found in all 32 genomes # Homologs per Family

15 IS2 transposase orfb Size of gene families in 53 Ecoli + Shigella genomes From: carsten@cbs.dtu.dk Subject: A question for the Ecoli expert Date: 5 August :59:56 GMT-03:00 To: dave@cbs.dtu.dk Hi Dave, Was looking at the Ecoli pangenome. There is a transposase which seems ridiculously ubiquitous. In 52 Ecoli/Shigella genomes I find individual instances of this bugger with the coregenome script. I've been into the genbank annotations and at first glance this thing appears genuine. Is this described somewhere? I've found it listed as "IS2 transposase orfb", but it may go under other names as well. -- /Carsten

16 IS2 transposase orfb Size of gene families in 53 Ecoli + Shigella genomes Size of gene families in 45 Ecoli genomes largest gene families

17 1 E. coli K-12 MG E. coli K-12 W E. coli K-12 DH10B E. coli CFT #genes # new genes # new gene families pan- core K-12 MG1655 K-12 W3110 K-12 DH10B CFT073

18 number of gene families K12 New genes New gene families Core genome Pan genome new E. coli genomes 1 : E. coli K12MG16 2 : E. coli O157RIM 3 : E. coli O157EDL 4 : S. flexneri 2a 30 5 : E. coli CFT073 6 : S. flexneri 2a 24 7 : S. sonnei Ss046 8 : S. boydii Sb227 9 : S. dysenteriae S 10 : E. coli W : E. coli UTI89 12 : E. coli : S. flexneri : E. coli APEC O 15 : E. coli : E. coli : E. coli B : E. coli B7A 19 : E. coli E22 20 : E. coli E : E. coli F11 22 : E. coli HS 23 : E. coli B 24 : E. coli E : S. boydii BS51 26 : E. coli : E. coli B03 28 : E. coli E : E. coli H : E. coli RS218

19 K-12 New genes New gene families Core genome Pan genome O157 Shigella E. fergusonii E. albertii 1 : Ecoli_K12DH10B 2 : Ecoli_K12MG : Ecoli_K12W : Ecoli_B 5 : Ecoli_B03 6 : Ecoli_B171 7 : Ecoli_B7A 8 : Ecoli_F11 9 : Ecoli_H : Ecoli_HS 11 : Ecoli_101!1 12 : Ecoli_ : Ecoli_ : Ecoli_ : Ecoli_ : Ecoli_CFT : Ecoli_E : Ecoli_E22 19 : Ecoli_E : Ecoli_E24377A 21 : Ecoli_ED1a 22 : Ecoli_IAI1 23 : Ecoli_IAI39 24 : Ecoli_LANL_ECA 25 : Ecoli_LANL_ECF 26 : Ecoli_O103Oslo 27 : Ecoli_O157_EC : Ecoli_O157_EC : Ecoli_O157_EC : Ecoli_O157_EC : Ecoli_O157_EC : Ecoli_O157_EC : Ecoli_O157_EC : Ecoli_O157_EC : Ecoli_O157_EC : Ecoli_O157_EC : Ecoli_O157_EC : Ecoli_O157_EC : Ecoli_O157_EDL : Ecoli_O157_Sakai 41 : Ecoli_ : Ecoli_RS : Ecoli_S88 44 : Ecoli_SE11 45 : Ecoli_SMS35 46 : Ecoli_UMN : Ecoli_UTI89 48 : Ecoli_VR50 49 : Ecoli_APEC01 50 : Sflex_ : Sflex_2a : Sflex_ : Sboyd_Sb : Sdyse_Sd : Ssone_Ss : Ealbe_TW : Eferg_35469T Plot made for E. coli alliance (by DU) on 19 January, 2009

20 Microbial comparative pan-genomics using binomial mixture models Lars Snipen 1, Trygve Almøy 1 and David W. Ussery 2 1 Biostatistics, Department of Chemistry, Biotechnology and Food Sciences, Norwegian University of Life Sciences, Ås, Norway 2 Centre for Biological Sequence Analysis, Technical University of Denmark, Lyngby, Denmark Lars Snipen - lars.snipen@umb.no; Trygve Almøy - trygve.almoy@umb.no; David W. Ussery - dave@cbs.dtu.dk; Corresponding author Abstract BMC Genomics 2009, 10: in the press [August 2009] Background: The size of the core- and pan-genome of bacterial species is a topic of increasing interest due to the growing number of sequenced prokaryote genomes, many from the same species. Attempts to estimate these quantities have been made, using regression methods or mixture models. We extend the latter approach by using statistical ideas developed for capture-recapture problems in ecology and epidemiology. Results: We estimate core- and pan-genome sizes for 16 different bacterial species. The results reveal a complex dependency structure for most species, manifested as heterogeneous detection probabilities. Estimated pangenome sizes range from small (around 2600 gene families) in Buchnera aphidicola to large (around gene families) in Escherichia coli. Results for Echerichia coli show that as more data become available, a larger diversity is estimated, indicating an extensive pool of rarely occurring genes in the population. Conclusions: Analyzing pan-genomics data with binomial mixture models is a way to handle dependencies between genomes, which we find is always present. A bottleneck in the estimation procedure is the annotation of rarely occurring genes.

21 Table 1: Number of genomes refer to completed genomes at NCBI [8] at the end of January Number of components is the optimal choice of mixture components. The black bars indicate pan-genome coverage, i.e. the current sample pan-genome size as a fraction of the estimated pan-genome size. Species Genomes Components Coverage Campylobacter jejuni 5 3 Coxiella burnetii 5 3 Acinetobacter baumannii 6 4 Buchnera aphidicola 6 3 Helicobacter pylori 6 3 Rhodopseudomonas palustris 6 3 Streptococcus pneumoniae 6 3 Yersinia pestis 7 4 Francisella tularensis 7 4 Bacillus cereus 8 4 Clostridium botulinum 8 3 Prochlorococcus marinus 12 4 Streptococcus pyogenes 13 5 Salmonella enterica 14 5 Staphylococcus aureus 14 6 Escherichia coli 22 6 BMC Genomics 2009, 10: in the press [August 2009]

22 Escherichia coli Bacillus cereus Rhodopseudomonas palustris Salmonella enterica Clostridium botulinum Prochlorococcus marinus Acinetobacter baumannii Yersinia pestis Staphylococcus aureus Streptococcus pyogenes Streptococcus pneumoniae Coxiella burnetii Campylobacter jejuni Helicobacter pylori Francisella tularensis Buchnera aphidicola c c c c c c c c c c c c c c c c + predict core observed core obs. genome observed pan c Chao lower pan X predict pan - 90% naive bootstrap Figure Number of gene families BMC Genomics 2009, 10: in the press [August 2009]

23 Number of gene families Number of gene families Francisella tularensis θ g = Number of genomes ( ) G K π k ρ g k g (1 ρ k) G g, g =0,..., G (1) k=1 where is the mixing proportion and is the de- Escherichia coli Number of genomes BMC Genomics 2009, 10: in the press [August 2009]

24 Probability Probability Number of genomes Number of genomes Probability Probability Number of genomes Number of genomes Figure 5: An illustration of a three component binomial mixture model when G = 10. The upper left panel shows the binomial PDF (red) for the detection probability ρ 1 =1.0, i.e. the core genes who are always detected in any genome. In the upper right panel a second component has a binomial PDF (green) where ρ 2 =0.9, i.e. these genes are often detected. In the lower left panel a third component (blue) has ρ 3 =0.05 and genes of this class are rarely observed. The lower right panel shows the combination with mixing proportions π 1 =0.2, π 2 =0.1 and π 3 =0.7. BMC Genomics 2009, 10: in the press [August 2009]

25 Francisella tularensis Coxiella burnetii Yersinia pestis Streptococcus pneumoniae Helicobacter pylori Clostridium botulinum Rhodopseudomonas palustris Streptococcus pyogenes Prochlorococcus marinus Staphylococcus aureus Campylobacter jejuni Acinetobacter baumannii Buchnera aphidicola Salmonella enterica Bacillus cereus Escherichia coli Relative contribution Figure 3 BMC Genomics 2009, 10: in the press [August 2009]

26 Number of gene families Number of genomes sampled BMC Genomics 2009, 10: in the press [August 2009]

27 Fraction of proteins in set E. coli str. K-12 substr. MG1655 All proteins (4131) Unique proteins (2614) Not matching SWISS-PROT (16) Matching SWISS-PROT (2592) E. coli singletons (6195) Length of protein (aa)

28 gene family tree for 25 E. coli genomes Manhattan Distance (# gene families)

29 The Burkholderia Pan- and Core Genome 223 x Salmonella E. coli / Shigella Yersinia x x x Pseudomonas x Vibrio Pan-genome Core genome Novel genes Novel gene families 5 5 Fig The pan-genome and core genome for five different Proteobacterial genera. The Salmonella graph represents one species (Salmonella enterica), whereas the E. coli/shigella figure contains both E. coli and four different Shigella species. The other graphs represent multiple species per genus. All graphs are drawn on the same scale D.W. Ussery et al., Computing for Comparative Microbial Genomics, Computational Biology 8, DOI / _14, Springer-Verlag London Limited 2009

30 New genes New gene families Core genome Pan genome Streptococcus [26 genomes] 1=Spyogenes_MGAS315 2=Spyogenes_MGAS2096 3=Spyogenes_MGAS =Spyogenes_MGAS =Spyogenes_MGAS =Spyogenes_MGAS6180 7=Spyogenes_MGAS5005 8=Spyogenes_MGAS9429 9=Spyogenes_MGAS =Spyogenes_M1_GAS 11=Spyogenes_SSI-1 12=Spyogenes_Manfredo 13=Spneumoniae_R6 14=Spneumoniae_TIGR4 15=Spneumoniae_D39 16=Sthermophilus_CNRZ =Sthermophilus_LMD-9 18=Sthermophilus_LMG =Ssuis_98HAH33 20=Ssuis_05ZYH33 21=Sagalactiae_NEM316 22=Sagalactiae_A909 23=Sagalactiae_2603V_R 24=Ssanguinis_SK36 25=Smutans_UA159 26=Sgordonii_Challis_CH

31 Number of genes and gene families Pan-genome Core genome Novel genes Burkholderia [56 genomes] Novel gene families B. cepacia complex Pseudomallei group B. cenocepacia B. ambifaria B. dolosa B. vietnamiensis B. mulltivorans B. ubonensis B. lata B. pseudomallei B. mallei Genomes (n=56) B. oklahomensis B. thailandensis B. graminis B. phytofirmans B. phymatum Burkholderia H160 B. xenovorans

32 New genes New gene families Core genome Pan genome Bacillus [24 genomes] 1=Banthracis_A1055 2=Banthracis_Ames0581 3=Banthracis_Ames 4=Banthracis_Australia94 5=Banthracis_CNEVA =Banthracis_Kruger_B 7=Banthracis_Sterne 8=Banthracis_USA6153 9=Banthracis_Vollum 10=Bcereus_AH187 11=Bcereus_AH820 12=Bcereus_ATCC10987_Main 13=Bcereus_ATCC14579_Main 14=Bcereus_E33L_Main 15=Bcereus_G =Bcereus_NVH =Bsubtilis_168 18=Bthuringiensis_ =Bthuringiensis_AlHakam 20=Bamyloliquefaciens_FZB42 21=Bclausii_KSM-K16 22=Bhalodurans_C =Blicheniformis_ATCC =Bpumilus_SAFR

33 *!*** "*** #*** $*** %*** &*** +,-./,0,1 +,-./,0, ,1 789,./,084, " # $ % & ' ( )!*!!!"!#!$!%!&!'!(!) "*

34 New genes New gene families Core genome Pan genome Clostridium [15 genomes] 1=Cperfringens_13 2=Cperfringens_ATCC =Cperfringens_SM101 4=Cbotulinum_A_ATCC_ =Cbotulinum_F_Langeland 6=Cdifficile_630 7=Ctetani_E88 8=Cacetobutylicum_ATCC824 9=Cbeijerincki_NCIMB_ =Ccellulolyticum_H10 11=Cnovyi_NT 12=Cphytofermentans_ISDg 13=Csp_OhILAs 14=Cthermocellum_ATCC =Ckluyveri_DSM

35 e+02 Newgenes Coregenome Pangenome Vibrio [28 genomes] V.cholerae V.parahaemolyticus V.vulnificus P. profundum V.profundum Others

36 New genes New gene families Core genome Pan genome Mycobacterium [18 genomes] 1=Mtuberculosis_CDC1551 2=Mtuberculosis_C 3=Mtuberculosis_F11 4=Mtuberculosis_H37Ra 5=Mtuberculosis_H37Rv 6=Mtuberculosis_Haarlem 7=Msp_JLS 8=Msp_KMS 9=Msp_MCS 10=Mavium_104 11=Mavium_K-10 12=Mbovis_AF2122_97 13=Mbovis_BCG 14=Mgilvum_PYR-GCK 15=Mleprae_TN 16=Msmegmatis_MC2_155 17=Mulcerans_Agy99 18=Mvanbaalenii_PYR

37 Genus length %AT core pan #genomes Streptococcus ~2 Mbp 62% , [63] Burkholderia 5-10 Mbp 31% , [55] Bacillus 4-5 Mbp 63% , [48] Clostridium 2-5 Mbp 72% , [43] Vibrio 4-6 Mbp 53% , [35] Mycobacterium 3-5 Mbp 43% , [30] Salmonella ~5 Mbp 48% , [30] Yersinia ~5 Mbp 52% , [23]

38 Unknown 5.3% Unk. conserved 5.3% Partial Info. 4.0% Phage/IS in common pseudo in both leader pep. cell process lipo RNA enzymes pc carrierpsstruct mem pf pm factor pr regul pe pt trans

39 Of Proteins, Genomes, and Proteomes Code COGs Domains Description Information storage and processing J A K L B , ,271 10, Translation, ribosomal structure and biogenesis RNA processing and modification Transcription Replication, recombination and repair Chromatin structure and dynamics Cellular processes and signaling D Y V T M N Z W U O C G E F H I P Q R S ,678 2,380 7,683 7,853 2, ,743 6, ,816 14,939 3,922 6,582 5,201 9,232 4,055 22,721 13,883 Cell cycle control, cell division, chromosome partitioning Nuclear structure Defense mechanisms Signal transduction mechanisms Cell wall/membrane/envelope biogenesis Cell motility Cytoskeleton Extra-cellular structures Intracellular trafficking, secretion, vesicular transport Post-translational modification, protein turnover, chaperones Metabolism Energy production and conversion Carbohydrate transport and metabolism Amino acid transport and metabolism Nucleotide transport and metabolism Coenzyme transport and metabolism Lipid transport and metabolism Inorganic ion transport and metabolism Secondary metabolites biosynth., transport and catabolism Poorly characterized General function prediction only Function unknown Fig Cluster of Orthologous Genes codes for functionally related gene categories. The table was reproduced from the NCBI website as it appeared in March 2008 (Obtained from

40 E. coli K-12 genome unknown not in COGs Metabolism Information Cell proc. Translation RNA processing and modification Transcription Replication, recombination and repair Cell cycle control, mitosis and mei Defense mechanisms Signal transduction mechanisms Cell wall/membrane biogenesis Cell motility Pili and flagella Intracellular trafficking and secret Posttranslational modification Energy production and conversion Carbohydrate transport and metabolism Amino acid transport and metabolism Nucleotide transport and metabolism Coenzyme transport and metabolism Lipid transport and metabolism Inorganic ion transport and metabolism Secondary metab biosynt, transp catab General function prediction only Function unknown not in COGs

41 E. coli core E. coli pan not in COGs Information Information unknown Cell proc. not in COGS Cell proc. Metabolism unknown unknown Metabolism Translation RNA processing and modification Transcription Replication, recombination and repair Cell cycle control, mitosis and mei Defense mechanisms Signal transduction mechanisms Cell wall/membrane biogenesis Cell motility Pili and flagella Intracellular trafficking and secret Posttranslational modification Energy production and conversion Carbohydrate transport and metabolism Amino acid transport and metabolism Nucleotide transport and metabolism Coenzyme transport and metabolism Lipid transport and metabolism Inorganic ion transport and metabolism Secondary metab biosynt, transp catab General function prediction only

42 E. coli K-12 genome Unknown Information Cellular processes Metabolism E. coli core E. coli pan

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