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Phylogenomics and the diversity and the diversification of microbes

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Talk by Jonathan Eisen at the California Academy of Sciences Dec 16, 2010

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Phylogenomics and the diversity and the diversification of microbes

  1. 1. Phylogenomics and the Diversity and Diversification of Microbes Jonathan A. Eisen UC Davis Calacademy Talk December 16, 2010 1Monday, November 26, 12
  2. 2. Phylogenomics of Novelty Origin of New Genome Functions and Dynamics Processes •Evolvability •New genes •Repair and recombination •Changes in old genes processes •Changes in pathways •Intragenomic variation Species Evolution •Phylogenetic history •Vertical vs. horizontal descent •Needed to track gain/loss of processes, infer convergenceMonday, November 26, 12
  3. 3. Phylogenomic Analysis • Evolutionary reconstructions greatly improve genome analyses • Genome analysis greatly improves evolutionary reconstructions • There is a feedback loop such that these should be integrated 3Monday, November 26, 12
  4. 4. Outline • Introduction • Phylogenomic Tales – Selecting genomes for sequencing – Species evolution – Predicting functions of genes – Uncultured microbes 4Monday, November 26, 12
  5. 5. Outline • Introductino • Phylogenomic Tales – Selecting genomes for sequencing – Species evolution – Predicting functions of genes – Uncultured microbes • All of these going to be told in context of a recent project “A Genomic Encyclopedia of Bacteria and Archaea” (aka GEBA) 5Monday, November 26, 12
  6. 6. Fleischmann et al. 1995 6Monday, November 26, 12
  7. 7. Whole Genome Shotgun Sequencing 7Monday, November 26, 12
  8. 8. Whole Genome Shotgun Sequencing 7Monday, November 26, 12
  9. 9. Whole Genome Shotgun SequencingWarner Brothers, Inc. 7Monday, November 26, 12
  10. 10. Whole Genome Shotgun Sequencing shotgunWarner Brothers, Inc. 7Monday, November 26, 12
  11. 11. Whole Genome Shotgun Sequencing shotgunWarner Brothers, Inc. 7Monday, November 26, 12
  12. 12. Whole Genome Shotgun Sequencing shotgunWarner Brothers, Inc. sequence 7Monday, November 26, 12
  13. 13. Whole Genome Shotgun Sequencing shotgunWarner Brothers, Inc. sequence 7Monday, November 26, 12
  14. 14. Assemble Fragments 8Monday, November 26, 12
  15. 15. Assemble Fragments sequencer output 8Monday, November 26, 12
  16. 16. Assemble Fragments sequencer output 8Monday, November 26, 12
  17. 17. Assemble Fragments sequencer output assemble fragments 8Monday, November 26, 12
  18. 18. Assemble Fragments sequencer output assemble fragments Closure & Annotation 8Monday, November 26, 12
  19. 19. From http://genomesonline.org 9Monday, November 26, 12
  20. 20. 10Monday, November 26, 12
  21. 21. 11Monday, November 26, 12
  22. 22. 12Monday, November 26, 12
  23. 23. Human commensals 13Monday, November 26, 12
  24. 24. Genome Sequences Have Revolutionized Microbiology • Predictions of metabolic processes • Better vaccine and drug design • New insights into mechanisms of evolution • Genomes serve as template for functional studies • New enzymes and materials for engineering and synthetic biology 14Monday, November 26, 12
  25. 25. From http://genomesonline.org 15Monday, November 26, 12
  26. 26. rRNA Tree of Life Bacteria Archaea Eukaryotes FIgure from Barton, Eisen et al. “Evolution”, CSHL Press. Based on tree from Pace NR, 2003.Monday, November 26, 12
  27. 27. As of 2002 Proteobacteria TM6 OS-K • At least 40 Acidobacteria Termite Group OP8 phyla of Nitrospira Bacteroides bacteria Chlorobi Fibrobacteres Marine GroupA WS3 Gemmimonas Firmicutes Fusobacteria Actinobacteria OP9 Cyanobacteria Synergistes Deferribacteres Chrysiogenetes NKB19 Verrucomicrobia Chlamydia OP3 Planctomycetes Spriochaetes Coprothmermobacter OP10 Thermomicrobia Chloroflexi TM7 Deinococcus-Thermus Dictyoglomus Aquificae Thermudesulfobacteria Thermotogae OP1 Based on OP11 Hugenholtz, 2002 17Monday, November 26, 12
  28. 28. As of 2002 Proteobacteria TM6 OS-K • At least 40 Acidobacteria Termite Group OP8 phyla of Nitrospira Bacteroides bacteria Chlorobi Fibrobacteres Marine GroupA • Genome WS3 Gemmimonas Firmicutes sequences are Fusobacteria Actinobacteria mostly from OP9 Cyanobacteria Synergistes three phyla Deferribacteres Chrysiogenetes NKB19 Verrucomicrobia Chlamydia OP3 Planctomycetes Spriochaetes Coprothmermobacter OP10 Thermomicrobia Chloroflexi TM7 Deinococcus-Thermus Dictyoglomus Aquificae Thermudesulfobacteria Thermotogae OP1 Based on OP11 Hugenholtz, 2002 18Monday, November 26, 12
  29. 29. As of 2002 Proteobacteria TM6 OS-K • At least 40 Acidobacteria Termite Group OP8 phyla of Nitrospira Bacteroides bacteria Chlorobi Fibrobacteres Marine GroupA • Genome WS3 Gemmimonas Firmicutes sequences are Fusobacteria Actinobacteria mostly from OP9 Cyanobacteria Synergistes three phyla Deferribacteres Chrysiogenetes NKB19 • Some other Verrucomicrobia Chlamydia OP3 phyla are Planctomycetes Spriochaetes only sparsely Coprothmermobacter OP10 Thermomicrobia sampled Chloroflexi TM7 Deinococcus-Thermus Dictyoglomus Aquificae Thermudesulfobacteria Thermotogae OP1 Based on OP11 Hugenholtz, 2002 19Monday, November 26, 12
  30. 30. Proteobacteria2002 TM6 OS-K Acidobacteria • At least 40 Termite Group phyla of OP8 Nitrospira Bacteroides bacteria Chlorobi Fibrobacteres Marine GroupA • Genome WS3 Gemmimonas sequences are Firmicutes Fusobacteria mostly from Actinobacteria OP9 three phyla Cyanobacteria Synergistes Deferribacteres Chrysiogenetes • Some other NKB19 Verrucomicrobia phyla are only Chlamydia OP3 Planctomycetes sparsely Spriochaetes Coprothmermobacter sampled OP10 Thermomicrobia Chloroflexi TM7 Deinococcus-Thermus Dictyoglomus Aquificae Thermudesulfobacteria Thermotogae OP1 Based on Hugenholtz, OP11 2002Monday, November 26, 12
  31. 31. Need for Tree Guidance Well Established • Common approach within some eukaryotic groups • Many small projects funded to fill in some bacterial or archaeal gaps • Phylogenetic gaps in bacterial and archaeal projects commonly lamented in literature 21Monday, November 26, 12
  32. 32. Proteobacteria• NSF-funded TM6 OS-K • At least 40 Tree of Life Acidobacteria Termite Group phyla of OP8 Project Nitrospira Bacteroides bacteria Chlorobi• A genome Fibrobacteres Marine GroupA • Genome WS3 from each of Gemmimonas sequences are Firmicutes eight phyla Fusobacteria mostly from Actinobacteria OP9 Cyanobacteria Synergistes three phyla Deferribacteres Chrysiogenetes NKB19 • Some other Verrucomicrobia Chlamydia OP3 phyla are only Planctomycetes Spriochaetes sparsely Coprothmermobacter OP10 Thermomicrobia sampled Chloroflexi TM7 Deinococcus-Thermus • Solution I: Dictyoglomus Aquificae sequence moreEisen & Ward, PIs Thermudesulfobacteria Thermotogae OP1 phyla OP11 22Monday, November 26, 12
  33. 33. Organisms Selected Phylum Species selected Chrysiogenes Chrysiogenes arsenatis (GCA) Coprothermobacter Coprothermobacter proteolyticus (GCBP) Dictyoglomi Dictyoglomus thermophilum (GD T ) Thermodesulfobacteria Thermodesulfobacterium commune (GTC) Nitrospirae Thermodesulfovibrio yellowstonii (GTY) Thermomicrobia Thermomicrobium roseum (GTR ) Deferribacteres Geovibrio thiophilus (GGT) Synergistes Synergistes jonesii (GSJ) 23Monday, November 26, 12
  34. 34. Bacterial aTOL Project AIMS • Improve resolution of deep branches in the bacterial tree • Launch biological studies of these phyla • Leverage data for interpreting environmental surveys 24Monday, November 26, 12
  35. 35. Monday, November 26, 12
  36. 36. Proteobacteria• NSF-funded TM6 OS-K • At least 40 Tree of Life Acidobacteria Termite Group phyla of bacteria OP8 Project Nitrospira • Genome Bacteroides• A genome Chlorobi Fibrobacteres sequences are Marine GroupA from each of WS3 Gemmimonas mostly from eight phyla Firmicutes Fusobacteria three phyla Actinobacteria OP9 Cyanobacteria • Some other Synergistes Deferribacteres Chrysiogenetes phyla are only NKB19 Verrucomicrobia sparsely Chlamydia OP3 Planctomycetes sampled Spriochaetes Coprothmermobacter • Still highly OP10 Thermomicrobia Chloroflexi biased in terms TM7 Deinococcus-Thermus Dictyoglomus of the tree AquificaeEisen & Ward, PIs Thermudesulfobacteria Thermotogae OP1 OP11Monday, November 26, 12
  37. 37. Major Lineages of Actinobacteria 2.5 Actinobacteria 2.5.1 Acidimicrobidae 2.5.1 Acidimicrobidae 2.5.1.1 Unclassified 2.5.1.2 "Microthrixineae 2.5.1.1 Unclassified 2.5.1.3 Acidimicrobineae 2.5.1.3.1 Unclassified 2.5.1.2 "Microthrixineae 2.5.1.3.2 Acidimicrobiaceae 2.5.1.4 BD2-10 2.5.1.3 Acidimicrobineae 2.5.1.5 EB1017 2.5.2 Actinobacteridae 2.5.1.4 BD2-10 2.5.2.1 Unclassified 2.5.2.10 Ellin306/WR160 2.5.1.5 EB1017 2.5.2.11 Ellin5012 2.5.2.12 Ellin5034 2.5.2 Actinobacteridae 2.5.2.13 Frankineae 2.5.2.13.1 Unclassified 2.5.2.1 Unclassified 2.5.2.13.2 Acidothermaceae 2.5.2.10 Ellin306/WR160 2.5.2.13.3 2.5.2.13.4 Ellin6090 Frankiaceae 2.5.2.11 Ellin5012 2.5.2.13.5 2.5.2.13.6 Geodermatophilaceae Microsphaeraceae 2.5.2.12 Ellin5034 2.5.2.13.7 2.5.2.14 Sporichthyaceae Glycomyces 2.5.2.13 Frankineae 2.5.2.15 2.5.2.15.1 Intrasporangiaceae Unclassified 2.5.2.14 Glycomyces 2.5.2.15.2 2.5.2.15.3 Dermacoccus Intrasporangiaceae 2.5.2.15 Intrasporangiaceae 2.5.2.16 2.5.2.17 Kineosporiaceae Microbacteriaceae 2.5.2.16 Kineosporiaceae 2.5.2.17.1 2.5.2.17.2 Unclassified Agrococcus 2.5.2.17 Microbacteriaceae 2.5.2.17.3 2.5.2.18 Agromyces Micrococcaceae 2.5.2.18 Micrococcaceae 2.5.2.19 2.5.2.2 Micromonosporaceae Actinomyces 2.5.2.19 Micromonosporaceae 2.5.2.20 2.5.2.20.1 Propionibacterineae Unclassified 2.5.2.2 Actinomyces 2.5.2.20.2 2.5.2.20.3 Kribbella Nocardioidaceae 2.5.2.20 Propionibacterineae 2.5.2.20.4 2.5.2.21 Propionibacteriaceae Pseudonocardiaceae 2.5.2.21 Pseudonocardiaceae 2.5.2.22 2.5.2.22.1 Streptomycineae Unclassified 2.5.2.22 Streptomycineae 2.5.2.22.2 2.5.2.22.3 Kitasatospora Streptacidiphilus 2.5.2.23 Streptosporangineae 2.5.2.23 2.5.2.23.1 Streptosporangineae Unclassified 2.5.2.3 Actinomycineae 2.5.2.23.2 2.5.2.23.3 Ellin5129 Nocardiopsaceae 2.5.2.4 Actinosynnemataceae 2.5.2.23.4 2.5.2.23.5 Streptosporangiaceae Thermomonosporaceae 2.5.2.5 Bifidobacteriaceae 2.5.2.3 Actinomycineae 2.5.2.4 Actinosynnemataceae 2.5.2.6 Brevibacteriaceae 2.5.2.5 Bifidobacteriaceae 2.5.2.6 Brevibacteriaceae 2.5.2.7 Cellulomonadaceae 2.5.2.7 Cellulomonadaceae 2.5.2.8 Corynebacterineae 2.5.2.8 Corynebacterineae 2.5.2.8.1 Unclassified 2.5.2.8.2 Corynebacteriaceae 2.5.2.9 Dermabacteraceae 2.5.2.8.3 Dietziaceae 2.5.2.8.4 Gordoniaceae 2.5.3 Coriobacteridae 2.5.2.8.5 Mycobacteriaceae 2.5.2.8.6 Rhodococcus 2.5.3.1 Unclassified 2.5.2.8.7 Rhodococcus 2.5.2.8.8 Rhodococcus 2.5.3.2 Atopobiales 2.5.2.9 Dermabacteraceae 2.5.2.9.1 Unclassified 2.5.3.3 Coriobacteriales 2.5.2.9.2 Brachybacterium 2.5.2.9.3 Dermabacter 2.5.3.4 Eggerthellales 2.5.3 Coriobacteridae 2.5.3.1 Unclassified 2.5.4 OPB41 2.5.3.2 Atopobiales 2.5.3.3 Coriobacteriales 2.5.5 PK1 2.5.3.4 Eggerthellales 2.5.4 OPB41 2.5.6 Rubrobacteridae 2.5.5 PK1 2.5.6 Rubrobacteridae 2.5.6.1 Unclassified 2.5.6.1 Unclassified 2.5.6.2 "Thermoleiphilaceae 2.5.6.2 "Thermoleiphilaceae 2.5.6.2.1 Unclassified 27 2.5.6.2.2 Conexibacter 2.5.6.3 MC47 2.5.6.2.3 XGE514 2.5.6.3 MC47 2.5.6.4 Rubrobacteraceae 2.5.6.4 RubrobacteraceaeMonday, November 26, 12
  38. 38. Proteobacteria• NSF-funded TM6 OS-K • At least 40 Tree of Life Acidobacteria Termite Group phyla of bacteria OP8 Project Nitrospira • Genome Bacteroides• A genome Chlorobi Fibrobacteres sequences are Marine GroupA from each of WS3 Gemmimonas mostly from eight phyla Firmicutes Fusobacteria three phyla Actinobacteria OP9 Cyanobacteria • Some other Synergistes Deferribacteres Chrysiogenetes phyla are only NKB19 Verrucomicrobia sparsely Chlamydia OP3 Planctomycetes sampled Spriochaetes Coprothmermobacter • Same trend in OP10 Thermomicrobia Chloroflexi Archaea TM7 Deinococcus-Thermus Dictyoglomus AquificaeEisen & Ward, PIs Thermudesulfobacteria Thermotogae OP1 OP11Monday, November 26, 12
  39. 39. Proteobacteria• NSF-funded TM6 OS-K • At least 40 Tree of Life Acidobacteria Termite Group phyla of bacteria OP8 Project Nitrospira • Genome Bacteroides• A genome Chlorobi Fibrobacteres sequences are Marine GroupA from each of WS3 Gemmimonas mostly from eight phyla Firmicutes Fusobacteria three phyla Actinobacteria OP9 Cyanobacteria • Some other Synergistes Deferribacteres Chrysiogenetes phyla are only NKB19 Verrucomicrobia sparsely Chlamydia OP3 Planctomycetes sampled Spriochaetes Coprothmermobacter • Same trend in OP10 Thermomicrobia Chloroflexi Eukaryotes TM7 Deinococcus-Thermus Dictyoglomus AquificaeEisen & Ward, PIs Thermudesulfobacteria Thermotogae OP1 OP11Monday, November 26, 12
  40. 40. Proteobacteria• NSF-funded TM6 OS-K • At least 40 Tree of Life Acidobacteria Termite Group phyla of bacteria OP8 Project Nitrospira • Genome Bacteroides• A genome Chlorobi Fibrobacteres sequences are Marine GroupA from each of WS3 Gemmimonas mostly from eight phyla Firmicutes Fusobacteria three phyla Actinobacteria OP9 Cyanobacteria • Some other Synergistes Deferribacteres Chrysiogenetes phyla are only NKB19 Verrucomicrobia sparsely Chlamydia OP3 Planctomycetes sampled Spriochaetes Coprothmermobacter • Same trend in OP10 Thermomicrobia Chloroflexi Viruses TM7 Deinococcus-Thermus Dictyoglomus AquificaeEisen & Ward, PIs Thermudesulfobacteria Thermotogae OP1 OP11Monday, November 26, 12
  41. 41. Proteobacteria• GEBA TM6 OS-K • At least 40 Acidobacteria• A genomic Termite Group OP8 phyla of bacteria encyclopedia Nitrospira Bacteroides • Genome Chlorobi of bacteria Fibrobacteres Marine GroupA sequences are and archaea WS3 Gemmimonas mostly from Firmicutes Fusobacteria three phyla Actinobacteria OP9 Cyanobacteria • Some other Synergistes Deferribacteres Chrysiogenetes phyla are only NKB19 Verrucomicrobia sparsely Chlamydia OP3 Planctomycetes sampled Spriochaetes Coprothmermobacter OP10 • Solution: Really Thermomicrobia Chloroflexi Fill in the Tree TM7 Deinococcus-Thermus Dictyoglomus Aquificae ThermudesulfobacteriaEisen & Ward, PIs Thermotogae OP1 OP11Monday, November 26, 12
  42. 42. http://www.jgi.doe.gov/programs/GEBA/pilot.html 32Monday, November 26, 12
  43. 43. GEBA Pilot Project Overview • Identify major branches in rRNA tree for which no genomes are available • Identify those with a cultured representative in DSMZ • DSMZ grew > 200 of these and prepped DNA • Sequence and finish 100+ (covering breadth of bacterial/archaea diversity) • Annotate, analyze, release data • Assess benefits of tree guided sequencing • 1st paper Wu et al in Nature Dec 2009Monday, November 26, 12
  44. 44. GEBA Pilot Project: Components • Project overview (Phil Hugenholtz, Nikos Kyrpides, Jonathan Eisen, Eddy Rubin, Jim Bristow) • Project management (David Bruce, Eileen Dalin, Lynne Goodwin) • Culture collection and DNA prep (DSMZ, Hans-Peter Klenk) • Sequencing and closure (Eileen Dalin, Susan Lucas, Alla Lapidus, Mat Nolan, Alex Copeland, Cliff Han, Feng Chen, Jan-Fang Cheng) • Annotation and data release (Nikos Kyrpides, Victor Markowitz, et al) • Analysis (Dongying Wu, Kostas Mavrommatis, Martin Wu, Victor Kunin, Neil Rawlings, Ian Paulsen, Patrick Chain, Patrik D’Haeseleer, Sean Hooper, Iain Anderson, Amrita Pati, Natalia N. Ivanova, Athanasios Lykidis, Adam Zemla) • Adopt a microbe education project (Cheryl Kerfeld) • Outreach (David Gilbert) • $$$ (DOE, Eddy Rubin, Jim Bristow) 34Monday, November 26, 12
  45. 45. GEBA Phylogenomic Lesson 1 The rRNA Tree of Life is a Useful Tool for Identifying Phylogenetically Novel Genomes 35Monday, November 26, 12
  46. 46. rRNA Tree of Life Bacteria Archaea Eukaryotes FIgure from Barton, Eisen et al. “Evolution”, CSHL Press. Based on tree from Pace NR, 2003.Monday, November 26, 12
  47. 47. Network of Life Bacteria Archaea Eukaryotes Figure from Barton, Eisen et al. “Evolution”, CSHL Press. Based on tree from Pace NR, 2003.Monday, November 26, 12
  48. 48. “Whole Genome” Concatenation Tree w/ AMPHORA See Wu and Eisen, Genome Biology 2008 9: R151 http://bobcat.genomecenter.ucdavis.edu/AMPHORA/Monday, November 26, 12
  49. 49. Compare PD in TreesMonday, November 26, 12
  50. 50. 40Monday, November 26, 12
  51. 51. PD of rRNA, Genome Trees Similar From Wu et al. 2009 Nature 462, 1056-1060Monday, November 26, 12
  52. 52. GEBA Phylogenomic Lesson 2 rRNA Tree is good but not perfect and better genomic sampling improves phylogenetic inference 42Monday, November 26, 12
  53. 53. 16s Says Hyphomonas is in RhodobacterialesBadger et al.2005 43Monday, November 26, 12
  54. 54. WGT and individual gene trees: Its Related to CaulobacteralesBadger et al.2005 44Monday, November 26, 12
  55. 55. GEBA Phylogenomic Lesson 3 Phylogeny-driven genome selection helps discover new genetic diversityMonday, November 26, 12
  56. 56. Network of Life Bacteria Archaea Eukaryotes FIgure from Barton, Eisen et al. “Evolution”, CSHL Press. Based on tree from Pace NR, 2003.Monday, November 26, 12
  57. 57. Protein Family Rarefaction Curves • Take data set of multiple complete genomes • Identify all protein families using MCL • Plot # of genomes vs. # of protein familiesMonday, November 26, 12
  58. 58. Monday, November 26, 12
  59. 59. Monday, November 26, 12
  60. 60. Monday, November 26, 12
  61. 61. Monday, November 26, 12
  62. 62. Monday, November 26, 12
  63. 63. Synapomorphies existMonday, November 26, 12
  64. 64. Structural Novelty • Of the 17000 protein families in the GEBA56, 1800 are novel in sequence (Wu) • Structural modeling suggests many are structurally novel too (Dhaeseleer) • 372 being crystallized by the PSI (Kerfeld)Monday, November 26, 12
  65. 65. Phylogenetic Distribution Novelty: Bacterial Actin Related Protein C. boidinii  gi57157304 S. cerevisiae  gi14318479 L. starkeyi  gi166080363  S. japonicus  gi213407080 ACTIN A. cliftonii  gi14269497 99 U. pertusa  gi50355609 H. sapiens  gi4501889 M. cerebralis  gi46326807 67 C. cinerea  gi169844021 N. crassa  gi85101929 ARP1 100 I. scapularis  gi215507378  51 100 H. sapiens  gi5031569 65 S. japonicus  gi213404844 100 S. cerevisiae  gi6320175 ARP2 D. melanogaster  gi24642545 100 G. gallus  gi45382569 75 C. neoformans  gi58266690 S. cerevisiae  gi6322525 ARP3 100 D. melanogaster  gi17737543 100 H. sapiens  gi5031573  H. ochraceum  gi227395998 BARP S. cerevisiae  gi1008244  73 P. patens  gi168051992  ARP4 99 A. thaliana  gi18394608  94 S. cerevisiae  gi1301932 100 S. japonicus  gi213408393  ARP5 87 D. discoideum  gi66802418 74 D. melanogaster  gi17737347 97 S. cerevisiae  gi6323114 100 D. hansenii gi21851 1921 ARP6 100 O. sativa  gi182657420  A. thaliana gi1841 1737 ARP7 D. melanogater  gi19920358 100 M. musculus  gi226246593 ARP10 0.5  Haliangium ochraceum DSM 14365 Patrik D’haeseleer, Adam Zemla, Victor Kunin See also Guljamow et al. 2007 Current Biology.Monday, November 26, 12
  66. 66. GEBA Phylogenomic Lesson 4 Phylogeny driven genome selection (and phylogenetics in general) improves genome annotation 52Monday, November 26, 12
  67. 67. Predicting Function • Key step in genome projects • More accurate predictions help guide experimental and computational analyses • Many diverse approaches • All improved both by “phylogenomic” type analyses that integrate evolutionary reconstructions and understanding of how new functions evolve 53Monday, November 26, 12
  68. 68. Most/All Functional Prediction Improves w/ Better Phylogenetic Sampling • Took 56 GEBA genomes and compared results vs. 56 randomly sampled new genomes • Better definition of protein family sequence “patterns” • Greatly improves “comparative” and “evolutionary” based predictions • Conversion of hypothetical into conserved hypotheticals • Linking distantly related members of protein families • Improved non-homology prediction Kostas Natalia Thanos Nikos Iain Mavrommatis Ivanova Lykidis Kyrpides AndersonMonday, November 26, 12
  69. 69. GEBA Phylogenomic Lesson 4 Metadata and individual genome papers important 55Monday, November 26, 12
  70. 70. SIGS http://standardsingenomics.org/ 56Monday, November 26, 12
  71. 71. GEBA Phylogenomic Lesson 5 Improves analysis of genome data from uncultured organisms 57Monday, November 26, 12
  72. 72. Great Plate Count Anomaly Culturing Microscope Count Count 58Monday, November 26, 12
  73. 73. Great Plate Count Anomaly Culturing Microscope Count <<<< Count 59Monday, November 26, 12
  74. 74. Great Plate Count Anomaly DNA Culturing Microscope Count <<<< Count 60Monday, November 26, 12
  75. 75. PCR Saves the Day 61Monday, November 26, 12
  76. 76. rRNA Phylotyping • Collect DNA from environment • PCR amplify rRNA genes using broad (so- called universal) primers • Sequence • Align to others • Infer evolutionary tree • Unknowns “identified” by placement on tree • Some use BLAST, but not as good as phylogeny 62Monday, November 26, 12
  77. 77. Uses of rRNA sequences The Hidden Majority Richness estimates Hugenholtz 2002 Bohannan and Hughes 2003 63Monday, November 26, 12
  78. 78. rRNA: A Phylogenetic Anchor to Determine Who’s Out There Eisen et al. 1992 64Monday, November 26, 12
  79. 79. rRNA: A Phylogenetic Anchor to Determine Who’s Out There Eisen et al. 1992 64Monday, November 26, 12
  80. 80. rRNA: A Phylogenetic Anchor to Determine Who’s Out There Eisen et al. 1992 64Monday, November 26, 12
  81. 81. rRNA: A Phylogenetic Anchor to Determine Who’s Out There Biology not Eisen et similar enough al. 1992 64Monday, November 26, 12
  82. 82. Metagenomics shotgun cloneMonday, November 26, 12
  83. 83. 66Monday, November 26, 12
  84. 84. Example I: Phylotyping with rRNA and other genes 67Monday, November 26, 12
  85. 85. Weighted % of Clones 0 0.1250 0.2500 0.3750 0.5000 Al ph ap ro t eo Be b ac ta pr t er ot e ia G ob am ac m tMonday, November 26, 12 ap er ro ia Ep te si ob lo ac np t er ro ia De t eo lta b ac pr te ot ria eo b C ac ya t er n ob ia ac ter Fi ia rm ic u te Ac s tin ob ac t er C ia hl or ob i C FB Major Phylogenetic Group Sargasso Phylotypes C hl or of le Sp xi iro ch ae te Fu so s De ba in ct er oc ia oc cu s- Eu The ry r ar mu ch s ae C ot re a na rc ha eo ta Shotgun Sequencing Allows Use of Other Markers Venter et al., 2004 68 EFG EFTu rRNA RecA RpoB HSP70
  86. 86. Weighted % of Clones 0 0.1250 0.2500 0.3750 0.5000 Al ph ap ro t eo Be b ac ta pr t er ot e ia G ob am ac m tMonday, November 26, 12 ap er ro ia Ep te si ob lo ac np t er ro ia De t eo lta b ac pr te ot ria eo b C ac ya t er n ob ia ac ter Fi ia rm ic u te Ac s tin ob ac t er C ia hl or ob i without good C FB Major Phylogenetic Group Sargasso Phylotypes C Cannot be done hl or of le Sp xi iro ch ae te Fu so s De ba in ct er oc ia sampling of genomes oc cu s- Eu The ry r ar mu ch s ae C ot re a na rc ha eo ta Shotgun Sequencing Allows Use of Other Markers Venter et al., 2004 69 EFG EFTu rRNA RecA RpoB HSP70
  87. 87. Example II: Binning 70Monday, November 26, 12
  88. 88. Metagenomics ChallengeMonday, November 26, 12
  89. 89. Binning challenge A T B U C V D W E X F Y G Z 72Monday, November 26, 12
  90. 90. Glassy Winged Sharpshooter • Feeds on xylem sap • Vector for Pierce’s Disease • Potential bioterror agent 73Monday, November 26, 12
  91. 91. Xylem and Phloem From Lodish et al. 2000 74Monday, November 26, 12
  92. 92. Wu et al. 2006 PLoS Biology 4: e188. 75Monday, November 26, 12
  93. 93. Sharpshooter Shotgun Sequencing shotgun Collaboration with Nancy Wu et al. 2006 PLoS Biology 4: e188. 76 Moran’s labMonday, November 26, 12
  94. 94. Baumannia is a Vitamin and Cofactor Producing Machine Wu et al. 2006 PLoS Biology 4: e188. 77Monday, November 26, 12
  95. 95. No Amino-Acid Synthesis 78Monday, November 26, 12
  96. 96. 79Monday, November 26, 12
  97. 97. 80Monday, November 26, 12
  98. 98. ??????? 80Monday, November 26, 12
  99. 99. Commonly Used Binning Methods Did not Work Well • Assembly – Only Baumannia generated good contigs • Depth of coverage – Everything else 0-1X coverage • Nucleotide composition – No detectible peaks in any vector we looked at 81Monday, November 26, 12
  100. 100. CFB Phyla 82Monday, November 26, 12
  101. 101. Wu et al. 2006 PLoS Biology 4: e188. 83Monday, November 26, 12
  102. 102. Essential Amino Acid Synthesis Wu et al. 2006 PLoS Biology 4: e188. 84Monday, November 26, 12
  103. 103. Sulcia makes amino acids Baumannia makes vitamins and cofactors Wu et al. 2006 PLoS Biology 4: e188. 85Monday, November 26, 12
  104. 104. 86Monday, November 26, 12
  105. 105. Weighted % of Clones 0 0.1250 0.2500 0.3750 0.5000 Al ph ap ro t eo Be b ac ta pr t er ot e ia G ob am ac m tMonday, November 26, 12 ap er ro ia Ep te si ob lo ac np t er ro ia De t eo lta b ac pr te ot ria eo b C ac ya t er n ob ia ac ter Fi ia rm ic u te Ac s tin ob ac t er C ia hl or ob i without good C FB Major Phylogenetic Group Sargasso Phylotypes C Cannot be done hl or of le Sp xi iro ch ae te Fu so s De ba in ct er oc ia sampling of genomes oc cu s- Eu The ry r ar mu ch s ae C ot re a na rc ha eo ta Shotgun Sequencing Allows Use of Other Markers Venter et al., 2004 87 EFG EFTu rRNA RecA RpoB HSP70

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