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Jonathan Eisen talk at #ievobio 2010
The document discusses the known knowns from the past GEBA project. It notes that as of 2002, genome sequences were mostly from three bacterial phyla, while at least 40 phyla of bacteria were known to exist based on rRNA studies. Some other phyla had only sparse sampling, and the same trend occurred in Archaea and Eukaryotes. More genome sequences were needed from the underrepresented phyla to gain a more comprehensive view of microbial diversity.
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Jonathan Eisen talk at #ievobio 2010
- 1. Phylogenomics of microbes: the dark matter of biology Jonathan A. Eisen UC Davis Talk for iEVOBIO June 29, 2010 Tuesday, June 29, 2010
- 2. Eisen Lab -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 convergence Tuesday, June 29, 2010
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- 4. Social Networking inScience Tuesday, June 29, 2010
- 5. Bacterial evolve Tuesday, June29, 2010
- 6. An homage toDonald Rumsfeld • There are known knowns. These are things we know that we know. • There are known unknowns. That is to say, there are things that we know we don't know. • But there are also unknown unknowns. There are things we don't know we don't know. Tuesday, June 29, 2010
- 7. Outline • Known knowns (background) –rRNA Tree of Life –Genomics –rRNA PCR –Metagenomics • Known unknowns –GEBA project - past –GEBA project - present –GEBA project - future • Unknown unknowns? Tuesday, June 29, 2010
- 8. Known Knowns 1: rRNA Tree of Life Tuesday, June 29, 2010
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- 10. rRNA Tree ofLife Bacteria Archaea Eukaryotes FIgure from Barton, Eisen et al. “Evolution”, CSHL Press. Based on tree from Pace NR, 2003. Tuesday, June 29, 2010
- 11. The Tree ofEukaryotes Life: Three Main Domains The Tree of Life Bacteria Archaea Unrooted Tree of Life from Barton et al. Evolution Tuesday, June 29, 2010
- 12. Known Knowns 2: Genomics and Phylogenomics Tuesday, June 29, 2010
- 13. Fleischmann et al. 1995 Tuesday, June 29, 2010
- 14. Microbial genomes From http://genomesonline.org Tuesday, June 29, 2010
- 15. 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 Tuesday, June 29, 2010
- 16. Microbes Run thePlanet Tuesday, June 29, 2010
- 17. 4. Microbes in the world I: rRNA PCR Perna et al. 2003 Tuesday, June 29, 2010
- 18. Lateral Transfer from Doolittle, 1999 Tuesday, June 29, 2010
- 19. from Lerat etal Tuesday, June 29, 2010
- 20. Known Knowns 3: rRNA PCR Tuesday, June 29, 2010
- 21. Great Plate CountAnomaly Culturing Microscope Count Count Tuesday, June 29, 2010
- 22. Great Plate CountAnomaly Culturing Microscope Count <<<< Count Tuesday, June 29, 2010
- 23. Great Plate CountAnomaly DNA Culturing Microscope Count <<<< Count Tuesday, June 29, 2010
- 24. PCR Revolution Extract DNA PCR w/ Universal rDNA Primers Sequence Align and compare to other rDNAs Phylogenetic OTUs Ecology classification Tuesday, June 29, 2010
- 25. Uses of rDNAPCR Bohannan and Hughes 2003 Hugenholtz 2002 Tuesday, June 29, 2010
- 26. rRNA challenges • Massive amounts of data from next-gen • Need for full automation but –Non overlapping –Alignments not always straightforward –BLAST insufficient –Phylogenetic methods that have been automated still need work • Tree of everything might be useful Tuesday, June 29, 2010
- 27. Known Knowns 4: Metagenomics Tuesday, June 29, 2010
- 28. 4. Microbes in the world I: rRNA PCR Perna et al. 2003 Tuesday, June 29, 2010
- 29. Metagenomics shotgun clone Tuesday, June 29, 2010
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- 33. Metagenomics Challenge Who is out there? What are they doing? Tuesday, June 29, 2010
- 34. rRNA phylotyping frommetagenomics Venter et al., 2004 Tuesday, June 29, 2010
- 35. Shotgun Sequencing AllowsUse of Alternative Anchors (e.g., RecA) Venter et al., 2004 Tuesday, June 29, 2010
- 36. Weighted % ofClones 0 0.1250 0.2500 0.3750 0.5000 Al ph ap ro t eo ba Be ct t er Tuesday, June 29, 2010 ap ia ro t eo G ba ct am er m ia ap ro t eo Ep ba si ct lo er np ia ro t eo De ba lta ct er pr ia ot eo ba ct C er ia ya no ba ct er ia Fi rm ic ut es Ac tin ob ac te ria C hl or ob i Major Phylogenetic Group C FB Sargasso Phylotypes C hl or of le xi Sp iro ch ae te s Fu so ba ct De er in ia oc oc cu s- Th er Eu m ry us ar ch ae ot C a re n ar ch ae ot a Shotgun Sequencing Allows Use of Other Markers Venter et al., 2004 EFG EFTu rRNA RecA RpoB HSP70
- 37. Functional Inference from Metagenomics • Can work well for individual genes • Predicting “community” function is challenging because treating community as a bag of genes does not work well • Better to “compartmentalize” data ... Tuesday, June 29, 2010
- 38. Binning challenge A T B U C V D W E X F Y G Z Tuesday, June 29, 2010
- 39. Binning challenge A T B U C V D W E X F Y G Best binning method: reference genomes Z Tuesday, June 29, 2010
- 40. Reference Genomes Comingfrom Select Environment Tuesday, June 29, 2010
- 41. Binning challenge A T B U C V D W E X F Y G No reference genome? What do you do? Z Tuesday, June 29, 2010
- 42. Binning challenge A T B U C V D W E X F Y G No reference genome? What do you do? Z Assembly? Composition? Get more references? Tuesday, June 29, 2010
- 43. Binning challenge A T B U C V D W E X F Y G No reference genome? What do you do? Z Phylogeny .... Tuesday, June 29, 2010
- 44. Metagenomic challenges • Massive amounts of data from next-gen • Need for full automation but –Data fragmentary –BLAST insufficient –Automation of phylogenetic methods a bit better for protein coding genes b/c alignments better –Reference databases incomplete Tuesday, June 29, 2010
- 45. Known Unknowns 1: GEBA Past Tuesday, June 29, 2010
- 46. Microbial genomes From http://genomesonline.org Tuesday, June 29, 2010
- 47. Proteobacteria 2002 TM6 OS-K Acidobacteria • At least 40 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 Hugenholtz, OP11 2002 Tuesday, June 29, 2010
- 48. 2002 Proteobacteria TM6 OS-K • At least 40 Acidobacteria Termite Group OP8 phyla of Nitrospira Bacteroides bacteria Chlorobi Fibrobacteres Marine GroupA • Genome WS3 Gemmimonas sequences are Firmicutes Fusobacteria mostly from Actinobacteria OP9 Cyanobacteria three phyla Synergistes Deferribacteres Chrysiogenetes NKB19 Verrucomicrobia Chlamydia OP3 Planctomycetes Spriochaetes Coprothmermobacter OP10 Thermomicrobia Chloroflexi TM7 Deinococcus-Thermus Dictyoglomus Aquificae Thermudesulfobacteria Thermotogae OP1 Based on Hugenholtz, OP11 2002 Tuesday, June 29, 2010
- 49. 2002 Proteobacteria TM6 OS-K • At least 40 Acidobacteria Termite Group OP8 phyla of Nitrospira Bacteroides bacteria Chlorobi Fibrobacteres Marine GroupA • Genome WS3 Gemmimonas sequences are Firmicutes Fusobacteria mostly from Actinobacteria OP9 Cyanobacteria three phyla Synergistes Deferribacteres Chrysiogenetes • Some other NKB19 Verrucomicrobia Chlamydia phyla are only OP3 Planctomycetes Spriochaetes sparsely Coprothmermobacter OP10 sampled Thermomicrobia Chloroflexi TM7 Deinococcus-Thermus Dictyoglomus Aquificae Thermudesulfobacteria Thermotogae OP1 Based on Hugenholtz, OP11 2002 Tuesday, June 29, 2010
- 50. 2002 Proteobacteria TM6 OS-K • At least 40 Acidobacteria Termite Group OP8 phyla of Nitrospira Bacteroides bacteria Chlorobi Fibrobacteres Marine GroupA • Genome WS3 Gemmimonas sequences are Firmicutes Fusobacteria mostly from Actinobacteria OP9 Cyanobacteria three phyla Synergistes Deferribacteres Chrysiogenetes • Some other NKB19 Verrucomicrobia Chlamydia phyla are only OP3 Planctomycetes Spriochaetes sparsely Coprothmermobacter OP10 sampled Thermomicrobia Chloroflexi TM7 • Same trend in Deinococcus-Thermus Dictyoglomus Aquificae Archaea Thermudesulfobacteria Thermotogae OP1 Based on Hugenholtz, OP11 2002 Tuesday, June 29, 2010
- 51. 2002 Proteobacteria TM6 OS-K • At least 40 Acidobacteria Termite Group OP8 phyla of Nitrospira Bacteroides bacteria Chlorobi Fibrobacteres Marine GroupA • Genome WS3 Gemmimonas sequences are Firmicutes Fusobacteria mostly from Actinobacteria OP9 Cyanobacteria three phyla Synergistes Deferribacteres Chrysiogenetes • Some other NKB19 Verrucomicrobia Chlamydia phyla are only OP3 Planctomycetes Spriochaetes sparsely Coprothmermobacter OP10 sampled Thermomicrobia Chloroflexi TM7 • Same trend in Deinococcus-Thermus Dictyoglomus Aquificae Eukaryotes Thermudesulfobacteria Thermotogae OP1 Based on Hugenholtz, OP11 2002 Tuesday, June 29, 2010
- 52. The Tree isnot Happy Bacteria Archaea Eukaryotes FIgure from Barton, Eisen et al. “Evolution”, CSHL Press. Based on tree from Pace NR, 2003. Tuesday, June 29, 2010
- 53. Proteobacteria • NSF-funded TM6 • At least 40 phyla OS-K Tree of Life Acidobacteria Termite Group of bacteria OP8 Project Nitrospira • Genome Bacteroides Chlorobi • A genome Fibrobacteres Marine GroupA sequences are from each of WS3 Gemmimonas mostly from eight phyla Firmicutes Fusobacteria three phyla Actinobacteria OP9 Cyanobacteria Synergistes • Some other Deferribacteres Chrysiogenetes phyla are only NKB19 Verrucomicrobia Chlamydia sparsely sampled OP3 Planctomycetes Spriochaetes • Solution I: Coprothmermobacter OP10 sequence more Thermomicrobia Chloroflexi TM7 phyla Deinococcus-Thermus Dictyoglomus Aquificae Eisen & Ward, PIs Thermudesulfobacteria Thermotogae OP1 OP11 Tuesday, June 29, 2010
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- 55. The Tree ofLife is Still Angry Bacteria Archaea Eukaryotes FIgure from Barton, Eisen et al. “Evolution”, CSHL Press. Based on tree from Pace NR, 2003. Tuesday, June 29, 2010
- 56. Proteobacteria TM6 OS-K • At least 100 phyla of bacteria Acidobacteria Termite Group OP8 • Genome sequences are mostly Nitrospira Bacteroides from three phyla Chlorobi Fibrobacteres Marine GroupA • Most phyla with cultured WS3 Gemmimonas Firmicutes species are sparsely sampled Fusobacteria Actinobacteria • Lineages with no cultured OP9 Cyanobacteria Synergistes taxa even more poorly Deferribacteres Chrysiogenetes NKB19 sampled Verrucomicrobia Chlamydia OP3 • Solution - use tree to really Planctomycetes Spriochaetes fill gaps Coprothmermobacter OP10 Thermomicrobia Chloroflexi TM7 Deinococcus-Thermus Dictyoglomus Aquificae Well sampled phyla Thermudesulfobacteria Thermotogae OP1 OP11 Tuesday, June 29, 2010
- 57. A Genomic Encyclopediaof Bacteria and Archaea (GEBA) Tuesday, June 29, 2010
- 58. GEBA Pilot ProjectOverview • Identify major branches in rRNA tree for which no genomes are available • Identify branches with a cultured representative in DSMZ • Grow > 200 of these and prep. DNA • Sequence and finish 100 (covering breadth of bacterial/archaea diversity) • Annotate, analyze, release data • Assess benefits of tree guided sequencing Tuesday, June 29, 2010
- 59. 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) Tuesday, June 29, 2010
- 60. GEBA and Openness • All data released as quickly as possible w/ no restrictions to IMG-GEBA; Genbank, etc • Data also available in Biotorrents (http://biotorrents.net) • Individual genome reports published in OA “Standards in Genome Sciences (SIGS)” • 1st GEBA paper in Nature freely available and published using Creative Commons License Tuesday, June 29, 2010
- 61. Known Unknowns 2: GEBA present Tuesday, June 29, 2010
- 62. GEBA Lesson 1 rRNA Tree is Useful for Identifying Phylogenetically Novel Genomes Tuesday, June 29, 2010
- 63. rRNA Tree ofLife Bacteria Archaea Eukaryotes FIgure from Barton, Eisen et al. “Evolution”, CSHL Press. Based on tree from Pace NR, 2003. Tuesday, June 29, 2010
- 64. Network of Life Bacteria Archaea Eukaryotes Figure from Barton, Eisen et al. “Evolution”, CSHL Press. Based on tree from Pace NR, 2003. Tuesday, June 29, 2010
- 65. “Whole Genome” Concatenation Tree w/ AMPHORA See Wu and Eisen, Genome Biology 2008 9: R151 http://bobcat.genomecenter.ucdavis.edu/AMPHORA/ Tuesday, June 29, 2010
- 66. Compare PD inTrees Tuesday, June 29, 2010
- 67. PD of rRNA,Genome Trees Similar From Wu et al. 2009 Nature 462, 1056-1060 Tuesday, June 29, 2010
- 68. GEBA Lesson 2 Phylogeny-driven genome selection helps discover new genetic diversity Tuesday, June 29, 2010
- 69. Network of Life Bacteria Archaea Eukaryotes FIgure from Barton, Eisen et al. “Evolution”, CSHL Press. Based on tree from Pace NR, 2003. Tuesday, June 29, 2010
- 70. Protein Family RarefactionCurves • Take data set of multiple complete genomes • Identify all protein families using MCL • Plot # of genomes vs. # of protein families Tuesday, June 29, 2010
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- 77. GEBA Lesson 3 Phylogeny-driven genome selection improves genome annotation Tuesday, June 29, 2010
- 78. Predicting Function • Key step in genome projects • More accurate predictions help guide experimental and computational analyses • Many diverse approaches • Comparative and evolutionary analysis greatly improves most predictions Tuesday, June 29, 2010
- 79. Phylogeny vs. Blast Many methods focus on “top EXAMPLE A METHOD EXAMPLE B blast hits” 2A CHOOSE GENE(S) OF INTEREST 5 1 3 4 3A 2 2B 5 1A 2A 1B 3B IDENTIFY HOMOLOGS 6 ALIGN SEQUENCES 1A 2A 3A 1B 2B 3B 1 2 3 4 5 6 CALCULATE GENE TREE Duplication? 1A 2A 3A 1B 2B 3B 1 2 3 4 5 6 OVERLAY KNOWN FUNCTIONS ONTO TREE But much better to build 1A 2A 3A 1B Duplication? 2B 3B 1 2 3 4 5 6 phylogenetic trees of genes and INFER LIKELY FUNCTION OF GENE(S) OF INTEREST compare to relatives Ambiguous Duplication? Species 1 Species 2 Species 3 1A 1B 2A 2B 3A 3B 1 2 3 4 5 6 ACTUAL EVOLUTION (ASSUMED TO BE UNKNOWN) Allows better integration of Duplication evolutionary history (e.g., orthologs and paralogs) Based on Eisen, 1998 Genome Res 8: 163-167. Tuesday, June 29, 2010
- 80. Wu et al.2005 PLoS Genetics 1: e65. Tuesday, June 29, 2010
- 81. Most/All Functional PredictionImproves w/ Better Phylogenetic Sampling • Conversion of hypothetical into conserved hypotheticals • Improved phylogenomics • Linking distantly related members of protein families • Improved non-homology prediction Tuesday, June 29, 2010
- 82. Known Unknowns 3: GEBA future Tuesday, June 29, 2010
- 83. GEBA Future 1 How much further should we go? Tuesday, June 29, 2010
- 84. rRNA Tree ofLife Bacteria Archaea Eukaryotes FIgure from Barton, Eisen et al. “Evolution”, CSHL Press. Based on tree from Pace NR, 2003. Tuesday, June 29, 2010
- 85. Phylogenetic Diversity: Sequenced Bacteria & Archaea From Wu et al. 2009 Tuesday, June 29, 2010
- 86. Phylogenetic Diversity withGEBA From Wu et al. 2009 Tuesday, June 29, 2010
- 87. Phylogenetic Diversity: Isolates From Wu et al. 2009 Tuesday, June 29, 2010
- 88. Phylogenetic Diversity: All From Wu et al. 2009 Tuesday, June 29, 2010
- 89. Proteobacteria TM6 OS-K • At least 40 phyla of bacteria Acidobacteria Termite Group OP8 • Genome sequences are mostly Nitrospira Bacteroides from three phyla Chlorobi Fibrobacteres Marine GroupA • Most phyla with cultured WS3 Gemmimonas Firmicutes species are sparsely sampled Fusobacteria Actinobacteria • Lineages with no cultured OP9 Cyanobacteria Synergistes taxa even more poorly Deferribacteres Chrysiogenetes NKB19 sampled Verrucomicrobia Chlamydia OP3 Planctomycetes Spriochaetes Coprothmermobacter OP10 Thermomicrobia Chloroflexi TM7 Deinococcus-Thermus Dictyoglomus Aquificae Well sampled phyla Thermudesulfobacteria Thermotogae Poorly sampled OP1 OP11 No cultured taxa Tuesday, June 29, 2010
- 90. Proteobacteria TM6 OS-K Acidobacteria Termite Group • At least 40 phyla of bacteria OP8 Nitrospira • Genome sequences are mostly Bacteroides Chlorobi Fibrobacteres from three phyla Marine GroupA WS3 • Most phyla with cultured Gemmimonas Firmicutes species are sparsely sampled Fusobacteria Actinobacteria OP9 • Lineages with no cultured taxa Cyanobacteria Synergistes even more poorly sampled Deferribacteres Chrysiogenetes NKB19 Verrucomicrobia Chlamydia OP3 Planctomycetes Spriochaetes Coprothmermobacter OP10 Thermomicrobia Chloroflexi TM7 Deinococcus-Thermus Dictyoglomus Aquificae Well sampled phyla Thermudesulfobacteria Thermotogae Poorly sampled OP1 OP11 No cultured taxa Tuesday, June 29, 2010
- 91. Uncultured Lineages: Technical Approaches • Get into culture • Enrichment cultures • If abundant in low diversity ecosystems • Flow sorting • Microbeads • Microfluidic sorting • Single cell amplification Tuesday, June 29, 2010
- 92. GEBA Future 2 How many gene families are there? Tuesday, June 29, 2010
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- 95. Compare PD inTrees Tuesday, June 29, 2010
- 96. Gene Families vsPD PD vs. Gene Families (per genome) 0.4 0.3 PD/Genome 0.2 0.1 0 0 275 550 825 1100 Gene families / genome Tuesday, June 29, 2010
- 97. How many proteinfamilies? GEBA Genomes PD/Genome ~0.1 PFAMs/Genome Text ~1000 PFAMs/PD ~10000 Total PFAMS ~10,000,000 From Wu et al. 2009 Tuesday, June 29, 2010
- 98. Caveats (of many) • Novel protein families per genome likely taxon specific • Parameters other than PD clearly important • Does not include viruses, eukaryotes Tuesday, June 29, 2010
- 99. GEBA Future 3 Need to better leverage improved phylogenetic sampling Tuesday, June 29, 2010
- 100. Example 1: ProteinFamily Space • Much less biased sampling of protein family space now available • Need to rebuild / reassess many protein family databases (e.g., HMMs) • Structural space Tuesday, June 29, 2010
- 101. Example 2: Experiments Tuesday,June 29, 2010
- 102. 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 Hugenholtz, OP11 2002 Tuesday, June 29, 2010
- 103. As of 2002 Proteobacteria TM6 OS-K • At least 40 Acidobacteria Termite Group OP8 phyla of Nitrospira Bacteroides bacteria Chlorobi Fibrobacteres Marine GroupA • Experimental WS3 Gemmimonas studies are Firmicutes Fusobacteria mostly from Actinobacteria OP9 Cyanobacteria three phyla Synergistes Deferribacteres Chrysiogenetes NKB19 Verrucomicrobia Chlamydia OP3 Planctomycetes Spriochaetes Coprothmermobacter OP10 Thermomicrobia Chloroflexi TM7 Deinococcus-Thermus Dictyoglomus Aquificae Thermudesulfobacteria Thermotogae OP1 Based on Hugenholtz, OP11 2002 Tuesday, June 29, 2010
- 104. As of 2002 Proteobacteria TM6 OS-K • At least 40 Acidobacteria Termite Group OP8 phyla of Nitrospira Bacteroides bacteria Chlorobi Fibrobacteres Marine GroupA • Experimental WS3 Gemmimonas studies are Firmicutes Fusobacteria mostly from Actinobacteria OP9 Cyanobacteria three phyla Synergistes Deferribacteres Chrysiogenetes • Some studies NKB19 Verrucomicrobia Chlamydia in other phyla OP3 Planctomycetes Spriochaetes Coprothmermobacter OP10 Thermomicrobia Chloroflexi TM7 Deinococcus-Thermus Dictyoglomus Aquificae Thermudesulfobacteria Thermotogae OP1 Based on Hugenholtz, OP11 2002 Tuesday, June 29, 2010
- 105. 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 sequences are Firmicutes Fusobacteria mostly from Actinobacteria OP9 Cyanobacteria three phyla Synergistes Deferribacteres Chrysiogenetes • Some other NKB19 Verrucomicrobia Chlamydia phyla are only OP3 Planctomycetes Spriochaetes sparsely Coprothmermobacter OP10 sampled Thermomicrobia Chloroflexi TM7 • Same trend in Deinococcus-Thermus Dictyoglomus Aquificae Eukaryotes Thermudesulfobacteria Thermotogae OP1 Based on Hugenholtz, OP11 2002 Tuesday, June 29, 2010
- 106. 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 sequences are Firmicutes Fusobacteria mostly from Actinobacteria OP9 Cyanobacteria three phyla Synergistes Deferribacteres Chrysiogenetes • Some other NKB19 Verrucomicrobia Chlamydia phyla are only OP3 Planctomycetes Spriochaetes sparsely Coprothmermobacter OP10 sampled Thermomicrobia Chloroflexi TM7 • Same trend in Deinococcus-Thermus Dictyoglomus Aquificae Viruses Thermudesulfobacteria Thermotogae OP1 Based on Hugenholtz, OP11 2002 Tuesday, June 29, 2010
- 107. Proteobacteria TM6 OS-K Need Acidobacteria Termite Group OP8 experimental Nitrospira Bacteroides Chlorobi studies from Fibrobacteres Marine GroupA WS3 across the tree Gemmimonas Firmicutes too Fusobacteria Actinobacteria OP9 Cyanobacteria Synergistes Deferribacteres Chrysiogenetes NKB19 Verrucomicrobia Chlamydia OP3 Planctomycetes Spriochaetes 0.1 Coprothmermobacter OP10 Thermomicrobia Chloroflexi TM7 Deinococcus-Thermus Dictyoglomus Aquificae Tree based on Thermudesulfobacteria Thermotogae Hugenholtz (2002) OP1 with some OP11 modifications. Tuesday, June 29, 2010
- 108. Example 3: Improvingthe tree • To make best use of GEBA data we need a better tree Tuesday, June 29, 2010
- 109. Wh Concatenated alignment “whole genome tree” built using AMPHORA Tuesday, June 29, 2010
- 110. Why Wh does the tree matter? Whole genome tree built using AMPHORA by Martin Wu and Dongying Wu Tuesday, June 29, 2010
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- 114. Many Alternatives toConcatenation • Gene presence/absence • Supertrees / consensus methods • Separate phylogeny of genes and then integration of results (e.g., networks) • Models that incorporate gain/loss as well as gene phylogeny Tuesday, June 29, 2010
- 115. Example 4: MetagenomicAnalysis Tuesday, June 29, 2010
- 116. Weighted % ofClones 0 0.1250 0.2500 0.3750 0.5000 Al ph ap ro t eo ba Be ct t er Tuesday, June 29, 2010 ap ia ro t eo G ba ct am er m ia ap ro t eo Ep ba si ct lo er np ia ro t eo De ba lta ct er pr ia ot eo ba ct C er ia ya no ba ct er ia Fi rm ic ut es Ac tin ob ac te ria C hl or ob i Major Phylogenetic Group C FB Sargasso Phylotypes C hl or of le xi Sp iro ch ae te s Fu so ba ct De er in ia oc oc cu s- Th er Eu m ry us ar ch Phylogeny for Typing and Binning ae ot C a re n ar ch ae ot a Venter et al., 2004 EFG EFTu rRNA RecA RpoB HSP70
- 117. Weighted % ofClones 0 0.1250 0.2500 0.3750 0.5000 Al ph ap ro t eo ba Be ct t er Tuesday, June 29, 2010 ap ia ro t eo G ba ct am er m ia ap ro t eo Ep ba si ct lo er np ia ro t eo De ba lta ct er pr ia ot eo ba ct C er ia ya no ba ct er ia Fi rm ic ut es Ac tin ob ac te ria C hl or ob i Major Phylogenetic Group C FB Sargasso Phylotypes C hl or of le xi Sp iro ch ae te s Fu so ba ct De er in ia oc Should improve with oc cu s- Th er Eu m better genomic sampling ry us ar ch Phylogeny for Typing and Binning ae ot C a re n ar ch ae ot a Venter et al., 2004 EFG EFTu rRNA RecA RpoB HSP70
- 118. Weighted % ofClones 0 0.1250 0.2500 0.3750 0.5000 Al ph ap ro t eo ba Be ct t er Tuesday, June 29, 2010 ap ia ro t eo G ba ct am er m ia ap ro t eo Ep ba si ct lo er np ia ro t eo De ba lta ct er pr ia ot eo ba ct C er ia ya no ba ct er ia Fi rm ic ut es Ac tin ob ac te ria C hl or ob i Major Phylogenetic Group C FB Sargasso Phylotypes C hl or of le xi Sp iro ch ae te s Fu so ba ct De er in ia oc oc cu Only improved a little s- Th er Eu m ry us ar ch Phylogeny for Typing and Binning ae ot C a re n ar ch ae ot a Venter et al., 2004 EFG EFTu rRNA RecA RpoB HSP70
- 119. How to improvephylogenetic analysis of metagenomic data • Better phylogenetic and OTU methods for fragmented data • Better assessment of which genes to use? • More automation of all methods Tuesday, June 29, 2010
- 120. Phylogenetic challenge How place all in one tree? How identify OTUs including all fragments? Can you analyze more than 1 gene family at a time? Tuesday, June 29, 2010
- 121. Approach 1: Place Reads on Reference Tree • Examples –AMPHORA (Wu and Eisen) –PPlacer (Erik Matsen) –FastTree (Morgan Price) • General approach –Precompute reference tree for full length sequences –Place individual reads on reference tree –Merge trees Tuesday, June 29, 2010
- 122. Variants • Use concatenated alignment of markers not just individual genes (Steven Kembel) • Apply to OTU identification not just classification (Thomas Sharpton) • CoBinning: look for linkage among fragments/genes (Aaron Darling) Tuesday, June 29, 2010
- 123. How to improvephylogenetic analysis of metagenomic data • Better phylogenetic and OTU methods for fragmented data • Better assessment of which genes to use? • More automation Tuesday, June 29, 2010
- 124. New “Marker Genes” • 100 representative genomes, including many GEBAs • MCL gene families • Identify gene families w/ –High universality –High uniformity of copy number –Phylogenetic tree similar to “whole genome tree” Tuesday, June 29, 2010
- 125. Distances between genetrees and the AMPHORA concatenated genome tree rpmA coaE coaE rpmA trmD rplL rpsS rpsQ radA rplR rplD rplQ tsf rpsH frr smpB ttf rpsO rplR rplP rplM rpsS rplI rplV rpsB rplT rpsO rplO mraW rpsP rpsH rpsK rplQ rplU rplL tsf rplT trmD rplE rplS rpsP ttf rplC rpsI rplV mraW rplS rpsL infC rpsG rpsM rplM rplO rplI rplU pyrH rpsL rpsM rpsQ ruvA guaA radA rpsG purA smpB rplK priA rplD rpsK infC rplK rplC serS rplE rplA rplA rplF frr ruvA rplF rpsC serS rplN rplN rplP guaA rpsE ruvB pyrH rpsB rpsI rpsJ secY rRNA16S rpsJ secY purA rplB rplB priA nusA rpsE ruvB rpsC rRNA16S nusA 0 1 2 3 4 5 6 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 NODAL distance SPLIT distance AMPHORA marker Ribosomal protein Transcription/translation related protein DNA repair protein Protein of other function Distance between the genome tree and 100 random trees (average ± standard deviation) Tuesday, June 29, 2010
- 126. Screen gene markersfor different taxonomic groups phylum Genome Gene Number Actinobacteria Number 63 267783 Alphaproteobac 94 347287 teria Betaproteobact 56 266362 eria Gammaproteob 126 483632 acteria Deltaproteobact 25 102115 eria Epislonproteoba 18 33416 cteria Bacteriodes 25 71531 Chlamydae 13 13823 Chloroflexi 10 33577 Cyanobacteria 36 124080 Firmicutes 106 312309 Spirochaetes 18 38832 Thermi 5 14160 Thermotogae 9 17037 Tuesday, June 29, 2010
- 127. Keep only thefamilies with: Universality * Evenness * monophyly >= 90*90*90 Phylogenetic group Genome Number Gene Number Maker Candidates Archaea 62 145415 102 Actinobacteria 63 267783 136 Alphaproteobacteria 94 347287 142 Betaproteobacteria 56 266362 294 Gammaproteobacteria 126 483632 141 Deltaproteobacteria 25 102115 44 Epislonproteobacteria 18 33416 446 Bacteriodes 25 71531 179 Chlamydae 13 13823 561 Chloroflexi 10 33577 140 Cyanobacteria 36 124080 532 Firmicutes 106 312309 80 Spirochaetes 18 38832 72 Thermi 5 14160 727 Thermotogae 9 17037 646 Tuesday, June 29, 2010
- 128. How to improvephylogenetic analysis of metagenomic data • Better phylogenetic and OTU methods for fragmented data • Better assessment of which genes to use? • More automation Tuesday, June 29, 2010
- 129. AMPHORA Guide tree Tuesday, June 29, 2010
- 130. Al ph ap ro Be te Tuesday, June 29, 2010 ta ob G 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 pr ac am ot te m eo ria ap b ac ro te D te ria el ob ta pr ac Ep ot te U si lo eo ria nc ba la np ss ro ct ifi te er ed ob ia Pr ac ot te eo ria ba Cy ct an er ob ia ac Ch te ria la m Ac yd id ia ob e Ba act ct er er ia Ac oi de tin te ob s ac te ria Aq Pl ui an fic ct om ae yc Sp et AMPHORA - each read on its own tree iro es ch ae Fi te rm s ic ut Ch es lo ro U fle nc xi la Ch ss lo ifi ro ed bi Ba ct er ia Phylogenetic Binning Using AMPHORA frr tsf pgk rplL rplF rplP rplT rplE infC rpsI rplS rplA rplB rplK rplC rpsJ rplN rplD rplM rpsE rpsS rpsB rpsK rpsC rpoB rpsM pyrG nusA dnaG rpmA smpB
- 131. Zorro • http://sourceforge.net/projects/probmask/ • ZORRO is a probabilistic masking program that assigns confidence scores to each column in a multiple sequence alignment. These scores can then be used to account for alignment accuracy in phylogenetic inference pipelines • Wu, Chatterji, Eisen submitted Tuesday, June 29, 2010
- 132. Conclusions • Phylogeny-driven sampling produces many benefits immediately • For the most benefits to come, we need to re- direct many informatics efforts to take advantage of less biased data • Still a long way away from full benefits • Note - most of the benefits can come from (aack) - unfinished genomes Tuesday, June 29, 2010
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- 135. A Happy Treeof Life Tuesday, June 29, 2010