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Talk for UC Davis Applied Phylogenetics Course at Bodega Bay
The document discusses whole genome shotgun sequencing. It explains that shotgun sequencing involves breaking the genome into random fragments, sequencing the fragments, and then assembling the sequences back into the original genome. It notes that this approach was pioneered by The Institute for Genomic Research and has revolutionized genome sequencing by allowing rapid determination of entire microbial genomes.
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Talk for UC Davis Applied Phylogenetics Course at Bodega Bay
- 1. Phylogenomics Jonathan A. Eisen UC Davis Bodega Applied Phylogenetics Workshop March 7, 2011 Tuesday, March 8, 2011
- 2. Fleischmann et al. 1995 Science 269:496-512 Tuesday, March 8, 2011
- 3. Whole Genome ShotgunSequencing Tuesday, March 8, 2011
- 4. Whole Genome ShotgunSequencing Tuesday, March 8, 2011
- 5. Whole Genome ShotgunSequencing Warner Brothers, Inc. Tuesday, March 8, 2011
- 6. Whole Genome ShotgunSequencing shotgun Warner Brothers, Inc. Tuesday, March 8, 2011
- 7. Whole Genome ShotgunSequencing shotgun Warner Brothers, Inc. Tuesday, March 8, 2011
- 8. Whole Genome ShotgunSequencing shotgun Warner Brothers, Inc. sequence Tuesday, March 8, 2011
- 9. Whole Genome ShotgunSequencing shotgun Warner Brothers, Inc. sequence Tuesday, March 8, 2011
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- 11. Assemble Fragments sequencer output Tuesday, March 8, 2011
- 12. Assemble Fragments sequencer output Tuesday, March 8, 2011
- 13. Assemble Fragments sequencer output assemble fragments Tuesday, March 8, 2011
- 14. Assemble Fragments sequencer output assemble fragments Closure & Annotation Tuesday, March 8, 2011
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- 20. 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, March 8, 2011
- 21. General Steps inAnalysis of Complete Genomes • Identification/prediction of genes • Characterization of gene features • Characterization of genome features • Prediction of gene function • Prediction of pathways • Integration with known biological data • Comparative genomics Tuesday, March 8, 2011
- 22. Genome Size Tuesday, March8, 2011
- 23. Genome Structure: More Variable than Once Thought Tuesday, March 8, 2011
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- 25. Why Completeness is • Improves characterization of genome features – Gene order, replication origins • Better comparative genomics – Genome duplications, inversions • Presence and absence of particular genes can be very important • Missing sequence might be important (e.g., centromere) • Allows researchers to focus on biology not sequencing Tuesday, March 8, 2011
- 26. Vibrio cholerae Metabolism Tuesday,March 8, 2011
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- 29. 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 Tuesday, March 8, 2011
- 30. Outline • Phylogenomic Tales – Selecting genomes for sequencing – Species evolution – Predicting functions of genes – Uncultured microbes – Searching for novel organisms and genes Tuesday, March 8, 2011
- 31. Outline • Phylogenomic Tales – Selecting genomes for sequencing – Species evolution – Predicting functions of genes – Uncultured microbes – Searching for novel organisms and genes • All of these going to be told in context of a recent project “A Genomic Encyclopedia of Bacteria and Archaea” (aka GEBA) Tuesday, March 8, 2011
- 32. GEBA Introduction Knowing What We Don’t Know Tuesday, March 8, 2011
- 33. Major Microbial Sequencing Efforts • Coordinated, top-down efforts – Fungal Genome Initiative (Broad/Whitehead) – Gordon and Betty Moore Foundation Marine Microbial Genome Sequencing Project – Sanger Center Pathogen Sequencing Unit – NHGRI Human Gut Microbiome Project – NIH Human Microbiome Program • White paper or grant systems – NIAID Microbial Sequencing Centers – DOE/JGI Community Sequencing Program – DOE/JGI BER Sequencing Program – NSF/USDA Microbial Genome Sequencing • Covers lots of ground and biological diversity Tuesday, March 8, 2011
- 34. As of 2002 Tuesday,March 8, 2011
- 35. 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 Tuesday, March 8, 2011
- 36. 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 Tuesday, March 8, 2011
- 37. 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 Tuesday, March 8, 2011
- 38. 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 Tuesday, March 8, 2011
- 39. Need for TreeGuidance 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 Tuesday, March 8, 2011
- 40. 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 Eisen, Ward, Aquificae Thermudesulfobacteria sequence more Robb, Nelson, et Thermotogae phyla OP1 al OP11 Tuesday, March 8, 2011
- 41. 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) Tuesday, March 8, 2011
- 42. 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 Aquificae Eisen & Ward, PIs Thermudesulfobacteria Thermotogae OP1 OP11 Tuesday, March 8, 2011
- 43. Major Lineages ofActinobacteria 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 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 Rubrobacteraceae Tuesday, March 8, 2011
- 44. 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 Aquificae Eisen & Ward, PIs Thermudesulfobacteria Thermotogae OP1 OP11 Tuesday, March 8, 2011
- 45. 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 Aquificae Eisen & Ward, PIs Thermudesulfobacteria Thermotogae OP1 OP11 Tuesday, March 8, 2011
- 46. 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 Aquificae Eisen & Ward, PIs Thermudesulfobacteria Thermotogae OP1 OP11 Tuesday, March 8, 2011
- 47. 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 Thermudesulfobacteria Eisen & Ward, PIs Thermotogae OP1 OP11 Tuesday, March 8, 2011
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- 49. 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, March 8, 2011
- 50. rRNA Tree ofLife FIgure from Barton, Eisen et al. “Evolution”, CSHL Press. Based on tree from Pace NR, 2003. Tuesday, March 8, 2011
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- 54. B: Ac in t ob ac te B: ria # of Genomes Am (H Tuesday, March 8, 2011 in igh 10 15 20 25 30 35 0 5 an G a C B: B: er ) Ba Aq ob ct uif ia B: ero ica B: e D Ch ide B: e ef lo te r s D rri ofl ef ba e B: e c xi B: De B rrib ter Ep lta : D act es si Pr ei er lo o n es n te oc Pr ob oc ot a ci B: e ct G B: oba eri am B F ct a : ir e B: m Fu mi ria a G P so cut em ro ba e t c s B: ma eo te ba ri H tim c a a t B: loa ona eri a B: Pl nae de an r te Th c o s Phyla er B: to bia m S m le y s B: od piro ce es c te T u h B: he lfo ae s rm b te GEBA Pilot Target List Th o a s er de cte m s ri u a A: ove lfo H n bi A: alo abu a A: A b la M rc ac e A: et ha te M han eo ria et g ha ob lob ac i A: no te m r A: The icr ia Th rm obi er oc a m oc op ci ro te i
- 55. GEBA Pilot ProjectOverview • 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 200+ • Annotate, analyze, release data • Assess benefits of tree guided sequencing • 1st paper Wu et al in Nature Dec 2009 Tuesday, March 8, 2011
- 56. GEBA Phylogenomic Lesson1 The rRNA Tree of Life is a Useful Tool for Identifying Phylogenetically Novel Genomes Tuesday, March 8, 2011
- 57. rRNA Tree ofLife Bacteria Archaea Eukaryotes Figure from Barton, Eisen et al. “Evolution”, CSHL Press. 2007. Based on tree from Pace 1997 Science 276:734-740 Tuesday, March 8, 2011
- 58. The Core GetsSmall ... Tuesday, March 8, 2011
- 59. The Pangenome Tuesday, March8, 2011
- 60. Islands Among Synteny Tuesday,March 8, 2011
- 61. The Pangenome Tuesday, March8, 2011
- 62. Network of Life Bacteria Archaea Eukaryotes Figure from Barton, Eisen et al. “Evolution”, CSHL Press. Based on tree from Pace NR, 2003. Tuesday, March 8, 2011
- 63. Using the Core Tuesday,March 8, 2011
- 64. Wh Wholegenome tree built using AMPHORA by Martin Wu and Dongying Wu Tuesday, March 8, 2011
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- 66. Four Models forRooting TOL from Lake et al. doi: 10.1098/rstb.2009.0035 Tuesday, March 8, 2011
- 67. GEBA Phylogenomic Lesson2 rRNA Tree is good but not perfect and better genomic sampling improves phylogenetic inference Tuesday, March 8, 2011
- 68. 16s Says Hyphomonasis in Rhodobacteriales Badger et al. 2005 Tuesday, March 8, 2011
- 69. WGT and individualgene trees: Its Related to Caulobacterales Badger et al. 2005 Tuesday, March 8, 2011
- 70. 16s WGT, 23S Badger et al. 2005 Int J System Evol Microbiol 55: 1021-1026. Tuesday, March 8, 2011
- 71. Caveats: ignoring LGTand using concatenated alignments Tuesday, March 8, 2011
- 72. Concatenated Alignment MLTree Tuesday, March 8, 2011
- 73. Green Non SulfurBacteria Tuesday, March 8, 2011
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- 76. Zimmer. New YorkTimes. 2009 Tuesday, March 8, 2011
- 77. GEBA Phylogenomic Lesson3 Phylogenetics guided genome selection (and phylogenetics in general) improves genome annotation Tuesday, March 8, 2011
- 78. 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 Tuesday, March 8, 2011
- 79. From Eisen et al. 1997 Nature Medicine 3: 1076-1078. Tuesday, March 8, 2011
- 80. Blast Search ofH. pylori “MutS” • Blast search pulls up Syn. sp MutS#2 with much higher p value than other MutS homologs • Based on this TIGR predicted this species had mismatch repair Based on Eisen • Assumes functional constancy et al. 1997 Nature Medicine 3: 1076-1078. Tuesday, March 8, 2011
- 81. Predicting Function • Identification of motifs – Short regions of sequence similarity that are indicative of general activity – e.g., ATP binding • Homology/similarity based methods – Gene sequence is searched against a databases of other sequences – If significant similar genes are found, their functional information is used • Problem – Genes frequently have similarity to hundreds of motifs and multiple genes, not all with the same function Tuesday, March 8, 2011
- 82. MutL?? From http://asajj.roswellpark.org/huberman/dna_repair/mmr.html Tuesday, March 8, 2011
- 83. Phylogenetic Tree ofMutS Family Aquae Strpy Bacsu Synsp Deira Helpy Yeast Human Borbu Metth Celeg mSaco Yeast Human Yeast Mouse Arath Celeg Human Arath Human Mouse Spombe Fly Yeast Xenla Rat Mouse Yeast Human Spombe Yeast Neucr Arath Aquae Trepa Chltr DeiraTheaq Thema BacsuBorbu Based on Eisen, SynspStrpy 1998 Nucl Acids Ecoli Neigo Res 26: 4291-4300. Tuesday, March 8, 2011
- 84. MutS Subfamilies MSH5 MutS2 Aquae Strpy Bacsu Synsp Deira Helpy Yeast Human Borbu Metth Celeg mSaco MSH6 Yeast Human Mouse Arath Yeast MSH4 Celeg Human Arath Human MSH3 Mouse Fly Spombe Yeast Xenla Rat Mouse Yeast MSH1 Spombe Human Yeast MSH2 Neucr Arath Aquae Trepa Chltr Deira Theaq BacsuBorbu Thema SynspStrpy Ecoli Neigo Based on Eisen, 1998 Nucl Acids MutS1 Res 26: 4291-4300. Tuesday, March 8, 2011
- 85. Overlaying Functions ontoTree MutS2 MSH5 Aquae Strpy Bacsu Synsp Deira Helpy Yeast Human Borbu Metth Celeg MSH6 mSaco Yeast Human Mouse Arath YeastMSH4 Celeg Human Arath Human MSH3 Mouse Fly Spombe Yeast Xenla Rat Mouse Yeast Human MSH1 Spombe Yeast MSH2 Neucr Arath Aquae Trepa Chltr DeiraTheaq BacsuBorbu Thema SynspStrpy Based on Eisen, Ecoli Neigo 1998 Nucl Acids MutS1 Res 26: 4291-4300. Tuesday, March 8, 2011
- 86. Functional Prediction UsingTree MSH5 - Meiotic Crossing Over MutS2 - Unknown Functions Aquae Strpy Bacsu Synsp Deira Helpy Yeast Human Borbu Metth Celeg MSH6 - Nuclear mSaco Repair Yeast Of Mismatches Human MSH4 - Meiotic Crossing Mouse Yeast Over Arath Celeg Human Arath MSH3 - Nuclear Human Mouse RepairOf Loops Spombe Fly Yeast Xenla Rat Mouse MSH2 - Eukaryotic Nuclear Yeast Human Mismatch and Loop Repair MSH1 Spombe Yeast Neucr Mitochondrial Arath Repair Aquae Trepa Chltr DeiraTheaq BacsuBorbu Thema SynspStrpy Ecoli Based on Eisen, Neigo 1998 Nucl Acids MutS1 - Bacterial Mismatch and Loop Repair Res 26: 4291-4300. Tuesday, March 8, 2011
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- 88. PHYLOGENENETIC PREDICTION OFGENE FUNCTION EXAMPLE A METHOD EXAMPLE B 2A CHOOSE GENE(S) OF INTEREST 5 3A 1 3 4 2B 2 IDENTIFY HOMOLOGS 5 1A 2A 1B 3B 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 Duplication? 2A 3A 1B 2B 3B 1 2 3 4 5 6 1A INFER LIKELY FUNCTION OF GENE(S) OF INTEREST 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) Based on Eisen, 1998 Genome Duplication Res 8: 163-167. Tuesday, March 8, 2011
- 89. Phylogenetic Prediction of • Termed phylogenomics (Eisen, et al 1997) • Greatly improves accuracy of functional predictions compared to similarity based methods (e.g., blast) • Automated methods now available – Sean Eddy, Steven Brenner, Kimmen Sjölander, etc. • But … Tuesday, March 8, 2011
- 90. Example 2: RecentChanges • Phylogenomic functional prediction NJ * ** V.cholerae0512 VC V.cholerae VCA1034 V.cholerae VC V.cholerae VC V.cholerae VC A0974 A0068 V.cholerae VC 0825 0282 may not work well for very newly V.cholerae VCA0906 V.cholerae VC A0979 V.cholerae VCA1056 V.cholerae VC1643 V.cholerae VC2161 ** V.cholerae VCA0923 ** V.cholerae VC0514 V.cholerae VC 1868 V.cholerae VC A0773 V.cholerae VC1313 evolved functions V.cholerae VC 1859 V.cholerae VC1413 V.cholerae VCA0268 ** V.cholerae VC A0658 V.cholerae VC 1405 * V.cholerae VC1298 V.cholerae VC1248 V.cholerae VCA0864 V.cholerae VCA0176 ** V.cholerae VCA0220 V.cholerae VC 1289 ** V.cholerae VC1069 A V.cholerae VC2439 • Can use understanding of origin of V.cholerae VC967 1 V.cholerae VC A0031 V.cholerae VC1898 V.cholerae VC A0663 V.cholerae VC0988 A V.cholerae VC0216 * V.cholerae VC0449 V.cholerae VCA0008 V.cholerae VC1406 V.cholerae VC 1535 novelty to better interpret these cases? V.cholerae VC0840 B.subtilis gi2633766 Synechocystis sp. gi1001299 * Synechocystis sp.gi1001300 * Synechocystis sp. gi1652276 * Synechocystis sp. gi1652103 H.pylori gi2313716 ** **H.pylori 99 gi4155097 C.jejuni Cj1190c C.jejuni Cj1110c A.fulgidus gi2649560 A.fulgidus gi2649548 ** B.subtilis gi2634254 • Screen genomes for genes that have B.subtilis gi2632630 B.subtilis gi2635607 B.subtilis gi2635608 ** B.subtilis gi2635609 ** ** B.subtilisgi2635882 gi2635610 B.subtilis E.coligi1788195 E.coli gi2367378 * ** E.coligi1788194 E.coli A1092 gi1787690 V.cholerae VC changed recently V.cholerae VC 0098 E.coli gi1789453 H.pylori gi2313186 H.pylori 99 gi4154603 ** C.jejuni Cj0144 C.jejuni Cj1564 **C.jejuni C.jejuni Cj0262c Cj1506c ** H.pylori gi2313163 * ** H.pylori 99 gi4154575 ** H.pylori gi2313179 H.pylori 99 gi4154599 – Pseudogenes and gene loss ** C.jejuni Cj0019c C.jejuni Cj0951c C.jejuni Cj0246c B.subtilis gi2633374 T.maritima TM0014 V.cholerae VC1403 V.cholerae VCA1088 T.pallidum gi3322777 ** T.pallidum gi3322939 ** T.pallidum gi3322938 B.burgdorferi gi2688522 – Contingency Loci T.pallidum gi3322296 B.burgdorferi gi2688521 * T.maritima TM0429 **T.maritima TM0918 * **T.maritima T.maritima TM0023 TM1428 T.maritima TM1143 T.maritima TM1146 P.abyssi PAB1308 P.horikoshii gi3256846 ** P.abyssiPAB1336 – Acquisition (e.g., LGT) ** P.horikoshii gi3256896 ** **P.abyssi PAB2066 ** P.horikoshii ** P.abyssi gi3258290 * PAB1026 ** P.horikoshii DRA00354 gi3256884 D.radiodurans D.radiodurans ** D.radioduransDRA0353 ** DRA0352 ** V.cholerae VC 1394 P.abyssi PAB1189 P.horikoshii gi3258414 – Unusual dS/dN ratios ** B.burgdorferi gi2688621 M.tuberculosis gi1666149 V.cholerae VC 0622 – Rapid evolutionary rates – Recent duplications Tuesday, March 8, 2011
- 91. Example 3: Nonhomology methods • Many genes have homologs in other species but no homologs have ever been studied experimentally • Non-homology methods can make functional predictions for these • Example: phylogenetic profiling Tuesday, March 8, 2011
- 92. Phylogenetic profiling basis • Microbial genes are lost rapidly when not maintained by selection • Genes can be acquired by lateral transfer • Frequently gain and loss occurs for entire pathways/processes • Thus might be able to use correlated presence/ absence information to identify genes with similar functions Tuesday, March 8, 2011
- 93. Non-Homology Predictions: Phylogenetic Profiling • Step 1: Search all genes in organisms of interest against all other genomes • Ask: Yes or No, is each gene found in each other species • Cluster genes by distribution patterns (profiles) Tuesday, March 8, 2011
- 94. Carboxydothermus hydrogenoformans • Isolated from a Russian hotspring • Thermophile (grows at 80°C) • Anaerobic • Grows very efficiently on CO (Carbon Monoxide) • Produces hydrogen gas • Low GC Gram positive (Firmicute) • Genome Determined (Wu et al. 2005 PLoS Genetics 1: e65. ) Tuesday, March 8, 2011
- 95. Homologs of SporulationGenes Wu et al. 2005 PLoS Genetics 1: e65. Tuesday, March 8, 2011
- 96. Carboxydothermus sporulates Wu et al. 2005 PLoS Genetics 1: e65. Tuesday, March 8, 2011
- 97. Wu et al.2005 PLoS Genetics 1: e65. Tuesday, March 8, 2011
- 98. PG Profiling WorksBetter Using Orthology Tuesday, March 8, 2011
- 99. GEBA Lesson 3: Phylogeny driven genome selection (and phylogenetics) improves genome annotation • 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 Tuesday, March 8, 2011
- 100. GEBA Lesson 4: Metadata Important Tuesday, March 8, 2011
- 101. GEBA Phylogenomic Lesson5 Phylogeny-driven genome selection helps discover new genetic diversity Tuesday, March 8, 2011
- 102. Network of Life Bacteria Archaea Eukaryotes FIgure from Barton, Eisen et al. “Evolution”, CSHL Press. Based on tree from Pace NR, 2003. Tuesday, March 8, 2011
- 103. Protein Family Rarefaction • Take data set of multiple complete genomes • Identify all protein families using MCL • Plot # of genomes vs. # of protein families Tuesday, March 8, 2011
- 104. Wu et al.2009 Nature 462, 1056-1060 Tuesday, March 8, 2011
- 105. Wu et al.2009 Nature 462, 1056-1060 Tuesday, March 8, 2011
- 106. Wu et al.2009 Nature 462, 1056-1060 Tuesday, March 8, 2011
- 107. Wu et al.2009 Nature 462, 1056-1060 Tuesday, March 8, 2011
- 108. Wu et al.2009 Nature 462, 1056-1060 Tuesday, March 8, 2011
- 109. Synapomorphies exist Wu etal. 2009 Nature 462, 1056-1060 Tuesday, March 8, 2011
- 110. Families/PD not uniform +,%-./&#(%)"* !"#$%"&'(%)"* ! ! Tuesday, March 8, 2011
- 111. Structural Novelty • Of the 17000 protein families in the GEBA56, 1800 are novel in sequence (Wu) • Structural modeling suggests many are structurally novel too (D'haeseleer) • 372 being crystallized by the PSI (Kerfeld) Tuesday, March 8, 2011
- 112. GEBA Phylogenomic Lesson6 Improves analysis of genome data from uncultured organisms Tuesday, March 8, 2011
- 113. Great Plate CountAnomaly Culturing Microscope Count Count Tuesday, March 8, 2011
- 114. Great Plate CountAnomaly Culturing Microscope Count <<<< Count Tuesday, March 8, 2011
- 115. Environmental DNA Analysis DNA Culturing Microscope Count <<<< Count Tuesday, March 8, 2011
- 116. 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 Tuesday, March 8, 2011
- 117. rRNA PCR The Hidden Majority Richness estimates Hugenholtz 2002 Bohannan and Hughes 2003 Tuesday, March 8, 2011
- 118.
- 119. rRNA data increasingexponentially too Tuesday, March 8, 2011
- 120. rRNA phylotyping issues • Massive amounts of data – 1 x 10^6 new partial sequences with new 454 – 2 x 10^6 full length sequences in DB • Alignments of new sequences not always straightforward • Solutions: – Reliance on similarity scores (bad) – High throughput automated phylogenetic tools • STAP • WATERs Tuesday, March 8, 2011
- 121. Perna et al.2003 Tuesday, March 8, 2011
- 122.
- 123.
- 124.
- 125. Diversity of Proteorhodopsinsby PCR de la Torre et al 2003 Tuesday, March 8, 2011
- 126. Metagenomics shotgun sequence Tuesday, March 8, 2011
- 127. Massiuve Diversity ofProteorhodopsins Venter et al., 2004 Tuesday, March 8, 2011
- 128.
- 129.
- 130. Example I: FunctionalDiversity Tuesday, March 8, 2011
- 131. Functional Diversity ofProteorhodopsins? Venter et al., Science 304: 66. 2004 Tuesday, March 8, 2011
- 132. Example II: Phylotyping w/ many genes Tuesday, March 8, 2011
- 133. rRNA Phylotyping inSargasso Sea Venter et al., Science 304: 66. 2004 Tuesday, March 8, 2011
- 134. Shotgun Sequencing AllowsUse of Alternative Anchors (e.g., RecA) Venter et al., Science 304: 66. 2004 Tuesday, March 8, 2011
- 135. Weighted % ofClones 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 Tuesday, March 8, 2011 ac m t er ap ia ro 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 ter n ob ia ac t er 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 304: 66. 2004 Shotgun Sequencing Allows Use of Other Markers EFG Venter et al., Science EFTu rRNA RecA RpoB HSP70
- 136. Weighted % ofClones 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 Tuesday, March 8, 2011 ac m t er ap ia ro 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 ter n ob ia ac t er 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 EFG Venter et al., Science 304: 66-74. 2004 EFTu rRNA RecA RpoB HSP70
- 137. Weighted % ofClones 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 Tuesday, March 8, 2011 ac m t er ap ia ro 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 ter n ob ia ac t er 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 EFG Venter et al., Science 304: 66-74. 2004 EFTu rRNA RecA RpoB HSP70
- 138. Example III: Binning Tuesday, March 8, 2011
- 139.
- 140.
- 141. Binning challenge Best binning method: reference genomes Tuesday, March 8, 2011
- 142. Binning challenge Best binning method: reference genomes Tuesday, March 8, 2011
- 143. Binning challenge No reference genome? What do you do? Tuesday, March 8, 2011
- 144. CFB Phyla Tuesday, March8, 2011
- 145. Weighted % ofClones 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 Tuesday, March 8, 2011 ac m t er ap ia ro 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 ter n ob ia ac t er 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 Phylogenetic Binning 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 EFG Venter et al., Science 304: 66-74. 2004 EFTu rRNA RecA RpoB HSP70
- 146. Weighted % ofClones 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 Tuesday, March 8, 2011 ac m t er ap ia ro 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 ter n ob ia ac t er 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 EFG Venter et al., Science 304: 66-74. 2004 EFTu rRNA RecA RpoB HSP70
- 147. Weighted % ofClones 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 Tuesday, March 8, 2011 ac m t er ap ia ro 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 ter n ob ia ac t er Fi ia rm ic u te improves Ac s tin ob ac t er C ia hl or ob i C GEBA Project 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 metagenomic analysis 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 EFG Venter et al., Science 304: 66-74. 2004 EFTu rRNA RecA RpoB HSP70
- 148. GEBA Cyano Sequencing status (as of 01/14): Awaiting Material 11 Library 12 Production 22 Finishing 5 Grand Total 50 On-going/ Planed Activities: - Building Cyanobacterial Metadatabase (IMG-GOLD) - 10th Cyanobacterial Molecular Biology Workshop, Lake Arrowhead, CA (06/10) --> Cheryl will host: Workshop training as prep for virtual Jamboree 134 Tuesday, March 8, 2011
- 149. GEBA RNB Plan: Sequence multiple Root Nodule Bacteria (RNBs) across the planet. Pilot: 100 RNBs. Beta RNB Cupriavidis Goal: Burkholderia • Understand BioGeographical effects on species evolution Alpha RNB Azorhizobium and understand host-specificity. Allorhizobium Bradyrhizobium Mesorhizobium Rationale: Rhizobium Sinorhizobium • N2 fixation by legume pastures and crops provides 65% of the N Devosia Ochrobactrum currently utilized in agricultural production. Phyllobacterium Balneimonas-like • Contributes 25 to 90 million metric tones N pa. • Symbioses save $US 6-10 billion annually on N fertilizer. • Grain and animal production enhanced by fixed nitrogen supplied by the symbiosis. Nikos Kyrpides 135 Tuesday, March 8, 2011
- 150.
- 151. 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 not happy OP10 Thermomicrobia Chloroflexi TM7 Deinococcus-Thermus Dictyoglomus Aquificae Eisen & Ward, PIs Thermudesulfobacteria Thermotogae OP1 OP11 Tuesday, March 8, 2011
- 152. Shotgun Sequencing AllowsUse of Other Markers Sargasso Phylotypes 0.5000 0.3750 GEBA Project Weighted % of Clones 0.2500 improves EFG EFTu HSP70 0.1250 metagenomic analysis, RecA RpoB rRNA but only a little 0 ia ia ia ria ia ia s ia i FB xi s ia ch s a ta ob te te ar mu ot le er er er er er er er eo te C u ae or ae of t t t t t t ct ic r ac ac ac ac ac ac ac ha Eu The hl or ch ba rm b ob ob b b ob ob C rc hl iro eo eo eo so Fi s- na C e te n tin ry Sp Fu t ot t ot ya cu re ro ro ro Ac pr pr C C oc ap ap np ta lta oc ph m lo Be De si am in Al Ep De G Major Phylogenetic Group Venter et al., Science 304: 66-74. 2004 Tuesday, March 8, 2011
- 153. Phylogenomics Future 1 Need to adapt genomic and metagenomic methods to make better use of data Tuesday, March 8, 2011
- 154. Improving Metagenomic Analysis • Methods – More automation – Better phylogenetic methods for short reads – Improved tools for using distantly related genomes in metagenomic analysis • Data sets – Rebuild protein family models – New phylogenetic markers – Need better reference phylogenies, including HGT • More simulations Tuesday, March 8, 2011
- 155.
- 156. AMPHORA Guide tree Tuesday, March 8, 2011
- 157. 0 0.1750 0.3500 0.5250 0.7000 Al ph ap ro Be t eo ta ba pr ct G ot er i Tuesday, March 8, 2011 am eo m ba a ap ct er De rot ia lta eo pr bac Ep ot si eo teri lo ba a U np ct nc ro er la te ia ss ob ifi ac ed Pr te ria C ot yaeo noba bact e C cteria hl am ria Ac yd ia id e ob ac Ba te ct ria er oi Ac de tin te ob s ac Aq teri ui a Pl fic an ae ct om Sp yce iro te ch s a Fi ete rm s ic ut C es hl or of le C x U hl i or nc ob la i ss ifi ed Ba ct er ia frr tsf rpsI pgk rplL rplT rplF rplE rplS rplP rplA infC rplK rplB rplD rplN rplC rpsJ rplM rpsE rpsS rpsK rpsB rpsC rpoB pyrG nusA rpsM rpmA dnaG smpB
- 158. We have morethan 700 compete genome sequences: •Select 100 representatives •Build gene families •Identify families that present in all organisms with equal numbers •Hmm building and phylogenetic analysis to identify the true makers δ ε α β γ Proteobacteria Firmicutes Phylogenetic Tree of Bacteria (built from 31 concatenate marker align Tuesday, March 8, 2011
- 159. More Markers Tuesday, March8, 2011
- 160. AMPHORA 2 Comingw/ More Markers Phylogenetic group Genome Gene Maker Number Number Candidates Archaea 62 145415 106 Actinobacteria 63 267783 136 Alphaproteobacteria 94 347287 121 Betaproteobacteria 56 266362 311 Gammaproteobacteria 126 483632 118 Deltaproteobacteria 25 102115 206 Epislonproteobacteria 18 33416 455 Bacteriodes 25 71531 286 Chlamydae 13 13823 560 Chloroflexi 10 33577 323 Cyanobacteria 36 124080 590 Firmicutes 106 312309 87 Spirochaetes 18 38832 176 Thermi 5 14160 974 Thermotogae 9 17037 684 Tuesday, March 8, 2011
- 161. 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, March 8, 2011
- 162.
- 163. Phylogenetic challenge A single tree with everything Tuesday, March 8, 2011
- 164. PhylOTU: A High-ThroughputProcedure Quantifies Microbial Community Diversity and Resolves Novel Taxa from Metagenomic Data Thomas J. Sharpton1*, Samantha J. Riesenfeld1, Steven W. Kembel2, Joshua Ladau1, James P. O’Dwyer2,3, Jessica L. Green2, Jonathan A. Eisen4, Katherine S. Pollard1,5 1 The J. David Gladstone Institutes, University of California San Francisco, San Francisco, California, United States of America, 2 Center for Ecology and Evolutionary Biology, University of Oregon, Eugene, Oregon, United States of America, 3 Institute of Integrative and Comparative Biology, University of Leeds, Leeds, United Kingdom, 4 Department of Evolution and Ecology, University of California Davis, Davis, California, United States of America, 5 Institute for Human Genetics & Division of Biostatistics, Finding Metagenomic OTUs University of California San Francisco, San Francisco, California, United States of America Abstract Microbial diversity is typically characterized by clustering ribosomal RNA (SSU-rRNA) sequences into operational taxonomic units (OTUs). Targeted sequencing of environmental SSU-rRNA markers via PCR may fail to detect OTUs due to biases in priming and amplification. Analysis of shotgun sequenced environmental DNA, known as metagenomics, avoids amplification bias but generates fragmentary, non-overlapping sequence reads that cannot be clustered by existing OTU- finding methods. To circumvent these limitations, we developed PhylOTU, a computational workflow that identifies OTUs from metagenomic SSU-rRNA sequence data through the use of phylogenetic principles and probabilistic sequence profiles. Using simulated metagenomic data, we quantified the accuracy with which PhylOTU clusters reads into OTUs. Comparisons of PCR and shotgun sequenced SSU-rRNA markers derived from the global open ocean revealed that while PCR libraries identify more OTUs per sequenced residue, metagenomic libraries recover a greater taxonomic diversity of OTUs. In addition, we discover novel species, genera and families in the metagenomic libraries, including OTUs from phyla missed by analysis of PCR sequences. Taken together, these results suggest that PhylOTU enables characterization of part of the biosphere currently hidden from PCR-based surveys of diversity? Citation: Sharpton TJ, Riesenfeld SJ, Kembel SW, Ladau J, O’Dwyer JP, et al. (2011) PhylOTU: A High-Throughput Procedure Quantifies Microbial Community Diversity and Resolves Novel Taxa from Metagenomic Data. PLoS Comput Biol 7(1): e1001061. doi:10.1371/journal.pcbi.1001061 Editor: Oded Be ` , Technion-Israel Institute of Technology, Israel ´ja Received July 22, 2010; Accepted December 17, 2010; Published January 20, 2011 Copyright: ß 2011 Workflow. et al. This is an open-access article as squares and databases are represented as cylinders in this generalize Figure 1. PhylOTU Sharpton Computational processes are represented distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, 8, 2011 Results reproduction in any medium, provided the original author and source are credited. Tuesday, March distribution, and section for details. workflow of PhylOTU. See
- 165. • Build AMPHORA ALL reference tree with concatenated alignment • Align reads that match any of the HMMs to concatenated alignment • Place reads into reference tree one at a time Tuesday, March 8, 2011
- 166. Phylogenomics Future 2 We have still only scratched the surface of microbial diversity Tuesday, March 8, 2011
- 167. rRNA Tree ofLife Bacteria Archaea Eukaryotes Figure from Barton, Eisen et al. “Evolution”, CSHL Press. 2007. Based on tree from Pace 1997 Science 276:734-740 Tuesday, March 8, 2011
- 168. Phylogenetic Diversity: Genomes FromWu et al. 2009 Nature 462, 1056-1060 Tuesday, March 8, 2011
- 169. Phylogenetic Diversity withGEBA From Wu et al. 2009 Nature 462, 1056-1060 Tuesday, March 8, 2011
- 170. Phylogenetic Diversity: Isolates From Wu et al. 2009 Nature 462, 1056-1060 Tuesday, March 8, 2011
- 171. Phylogenetic Diversity: All From Wu et al. 2009 Nature 462, 1056-1060 Tuesday, March 8, 2011
- 172. Uncultured Lineages: • Get into culture • Enrichment cultures • If abundant in low diversity ecosystems • Flow sorting • Microbeads • Microfluidic sorting • Single cell amplification Tuesday, March 8, 2011
- 173. GEBA uncultured Number of SAGs from Candidate Phyla 406 1 OD1 OP1 OP3 SAR Site A: Hydrothermal vent 4 1 - - Site B: Gold Mine 6 13 2 - Site C: Tropical gyres (Mesopelagic) - - - 2 Site D: Tropical gyres (Photic zone) 1 - - - Sample collections at 4 additional sites are underway. Phil Hugenholtz 159 Tuesday, March 8, 2011
- 174.
- 175.
- 176.
- 177.
- 178. Phylogenomics Future 3 Need Experiments from Across the Tree of Life too Tuesday, March 8, 2011
- 179. 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 Tuesday, March 8, 2011
- 180. 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 Firmicutes studies 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 Tuesday, March 8, 2011
- 181. 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 Firmicutes studies are Fusobacteria Actinobacteria mostly from OP9 Cyanobacteria Synergistes three phyla Deferribacteres Chrysiogenetes NKB19 • Some studies Verrucomicrobia Chlamydia OP3 in other phyla Planctomycetes Spriochaetes Coprothmermobacter OP10 Thermomicrobia Chloroflexi TM7 Deinococcus-Thermus Dictyoglomus Aquificae Thermudesulfobacteria Thermotogae OP1 Based on OP11 Hugenholtz, 2002 Tuesday, March 8, 2011
- 182. 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 • Same trend in Dictyoglomus Aquificae Thermudesulfobacteria Eukaryotes Thermotogae OP1 Based on OP11 Hugenholtz, 2002 Tuesday, March 8, 2011
- 183. 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 • Same trend in Dictyoglomus Aquificae Thermudesulfobacteria Viruses Thermotogae OP1 Based on OP11 Hugenholtz, 2002 Tuesday, March 8, 2011
- 184. 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, March 8, 2011
- 185. Proteobacteria TM6 OS-K Adopt a Acidobacteria Termite Group OP8 Microbe Nitrospira Bacteroides Chlorobi Fibrobacteres Marine GroupA WS3 Gemmimonas Firmicutes 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, March 8, 2011
- 186. Conclusion • Phylogenetic sampling of genomes improves our understanding of microbial diversity in many ways • Still need – More biogeography – More phenotypic/experimental data – Deeper phylogenetic sampling Tuesday, March 8, 2011
- 187.
- 188.
- 189. A Happy Treeof Life Tuesday, March 8, 2011
- 190. Acknowledgements • GEBA: DOE-JGI, DSMZ • GWSS: Nancy Moran & lab, Dongying Wu • iSEEM: Katie Pollard, Jessica Green, Martin Wu • RecA: Dongying Wu, Craig Venter, Doug Rusch, et al. Tuesday, March 8, 2011