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Jonathan Eisen
Jonathan Eisen

Talk by Jonathan Eisen on JGI-GEBA "Genomic encyclopedia of bacteria and archaea" project

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Seeking out the Dark Matter of the Biological Universe & The Need for a Phylogeny Driven Genomic Encyclopedia Jonathan A. Eisen August 11, 2009 CSB 2009 Tuesday, August 11, 2009
Tuesday, August 11, 2009
“Nothing in biology makes sense except in the light of evolution.” T. Dobzhansky (1973) Tuesday, August 11, 2009
Image removed because it comes from a non open access journal Fleischmann et al. 1995 Tuesday, August 11, 2009
From http://genomesonline.org Tuesday, August 11, 2009
rRNA Tree of Life Tuesday, August 11, 2009
The Tree is not Happy Tuesday, August 11, 2009
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, August 11, 2009
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, August 11, 2009
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, August 11, 2009
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 Archaea Thermotogae OP1 Based on OP11 Hugenholtz, 2002 Tuesday, August 11, 2009
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 Tuesday, August 11, 2009
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 Eisen, Ward, Chloroflexi Badger, Wu, TM7 Deinococcus-Thermus • Solution I: Dictyoglomus Wu, et al. Aquificae Thermudesulfobacteria sequence more Thermotogae OP1 phyla OP11 Tuesday, August 11, 2009
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, August 11, 2009
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 Tuesday, August 11, 2009
T. roseum genome Tuesday, August 11, 2009
The Tree of Life is Still Angry Tuesday, August 11, 2009
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 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, August 11, 2009
Proteobacteria TM6 OS-K • At least 100 phyla of Acidobacteria Termite Group OP8 bacteria Nitrospira Bacteroides Chlorobi • Genome sequences are Fibrobacteres Marine GroupA mostly from three phyla WS3 Gemmimonas Firmicutes • Most phyla with cultured Fusobacteria Actinobacteria species are sparsely OP9 Cyanobacteria Synergistes sampled Deferribacteres Chrysiogenetes NKB19 • Lineages with no cultured Verrucomicrobia Chlamydia OP3 taxa even more poorly Planctomycetes Spriochaetes sampled Coprothmermobacter OP10 Thermomicrobia Chloroflexi • Solution - use tree to really TM7 Deinococcus-Thermus fill gaps Dictyoglomus Aquificae Well sampled phyla Thermudesulfobacteria Thermotogae OP1 OP11 Tuesday, August 11, 2009
http://www.jgi.doe.gov/programs/GEBA/pilot.html Tuesday, August 11, 2009
GEBA Pilot Project Overview • Identify major branches in rRNA tree for which no genomes are available • Identify which lineages have type species available in DSMZ • Grow > 200 of these and prep. DNA • Sequence and finish 100 • Annotate, analyze, release data • Assess benefits of tree guided sequencing Tuesday, August 11, 2009
GEBA Pilot Project: Components • Project overview (Phil Hugenholtz, Nikos Kyrpides, Jonathan Eisen) • Project management (David Bruce, Lynne Goodwin et al) • Culture collection and DNA prep (DSMZ, Hans-Peter Klenk) • Libraries and DNA (Eileen Dalin et al) • Sequencing and closure (Susan Lucas, Alla Lapidus et al.) • Annotation and data release (Nikos Kyrpides) • Analysis (Dongying Wu, Kostas Mavrommatis, Martin Wu, Jenna Morgan, Victor Kunin, Marcel Huntemann, Neil Rawlings, Ian Paulsen, Patrick Chain, Patrik D’Haeseleer, Sean Hooper, Iain Anderson) • Adopt a microbe education project (Cheryl Kerfeld) • Outreach (David Gilbert) • $$$ (DOE, Eddy Rubin, Jim Bristow) Tuesday, August 11, 2009
GEBA Pilot: Selecting Targets Tuesday, August 11, 2009
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B: Ac tin ob ac te B: ria # of Genomes Am (H in igh 10 15 20 25 30 35 0 5 an G Tuesday, August 11, 2009 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
GEBA Current Status • >100 in progress • GEBA 56 (focus of first paper) – 34 finished genomes – Released to IMG-GEBA page, JGI-FTP site, and Genbank • All data is completely Open for anyone to use Tuesday, August 11, 2009
Tuesday, August 11, 2009
Assess Benefits of GEBA • All genomes have some value • But what, if any, is the benefit of tree- guided sequencing over other selection methods Tuesday, August 11, 2009
Why Increase Taxonomic Coverage? • Gene discovery • Annotation, functional prediction • Metagenomic analysis • Mechanisms of diversification • Species phylogeny and classification Tuesday, August 11, 2009
GEBA Lesson 1 rRNA Tree of Life is a Useful Guide for Genome Core Phylogenetic Diversity Tuesday, August 11, 2009
rRNA Tree of Life Tuesday, August 11, 2009
16s Says Hyphomonas is in Rhodobacteriales Badger et al. 2005 Tuesday, August 11, 2009
WGT Says Its Related to Caulobacterales Badger et al. 2005 Tuesday, August 11, 2009
Tuesday, August 11, 2009
Tuesday, August 11, 2009
Tuesday, August 11, 2009
GEBA Lesson 2 Phylogenetically Guided Selection Can Help Many Aspects of Genome Analysis Tuesday, August 11, 2009
Annotation Improves • Conversion of hypothetical into conserved hypotheticals • Linking distantly related members of protein families • Non-homology functional prediction methods Tuesday, August 11, 2009
Improved Rosetta Stone Predictions Tuesday, August 11, 2009
Al ph ap ro Be te ta o ba G 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 pr ct am ot er m eo ia ap ba ro ct D te er el ob ia ta pr ac Ep Tuesday, August 11, 2009 ot te U si lo eo ria nc ba la np ct ss ro er ifi te ia ed ob 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 iro es ch ae Fi te rm s ic ut Ch es lo ro U Phylogenetic Metagenomics nc fle la xi ss Ch ifi lo ed ro bi Ba ct er ia 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
16s Says Hyphomonas is in Rhodobacteriales Badger et al. 2005 Tuesday, August 11, 2009
WGT Says Its Related to Caulobacterales Badger et al. 2005 Tuesday, August 11, 2009
Tuesday, August 11, 2009
GEBA Lesson 3 We have still only scratched the surface of microbial diversity Tuesday, August 11, 2009
Protein Family Rarefaction Curves • Take data set of multiple complete genomes • Identify all protein families using MCL • Plot # of genomes vs. # of protein families Tuesday, August 11, 2009
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Tuesday, August 11, 2009
Tuesday, August 11, 2009
Tuesday, August 11, 2009
Tuesday, August 11, 2009
350000 300000 Number of proteins 250000 Total Gene Number 200000 150000 100000 S. agalactiae Enterobacteriaceae 50000 Actinobacteria Bacteria from GEBA project 0 0 10 20 30 40 50 60 70 80 Genome Number Tuesday, August 11, 2009
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, August 11, 2009
Within Family Novelty Example: Transporter Profiles inorganic ions amino acids, nitro compounds and peptides drugs/ toxins sugars carboxylates nucleosides/ tides, bases siderophores other 700 600 500 Number of transporters 400 300 200 100 0 halut pedhe thete denac kanko acife aliac chipi desa7 sanke capoc catac celfl dyafe sacvi sphth spili stana sulde theac tsupa xylce detpe haloc plali thecu atopa crycu kytse jonde slahe eggle halmu desr5 anapr strro halbo sebte lepbu actmi beuca brafa conwo nakmu krifl meisi desba geoob thebi gorbr meiru rhoma bramu Sebaldella termitidis ATCC 33386 has 2x number of sugar PTS transporters of any genome Tuesday, August 11, 2009
Phylogenetic Distribution Novelty: 1st Bacterial Actin Related Protein Haliangium ochraceum DSM 14365 Tuesday, August 11, 2009
Phylogenetic Diversity: Sequenced Bacteria & Archaea Tuesday, August 11, 2009
Phylogenetic Diversity with GEBA Tuesday, August 11, 2009
Phylogenetic Diversity: Isolates Tuesday, August 11, 2009
Phylogenetic Diversity: All Tuesday, August 11, 2009
Proteobacteria TM6 OS-K • At least 40 phyla of Acidobacteria Termite Group OP8 bacteria Nitrospira Bacteroides Chlorobi • Genome sequences are Fibrobacteres Marine GroupA mostly from three phyla WS3 Gemmimonas Firmicutes • Most phyla with cultured Fusobacteria Actinobacteria species are sparsely OP9 Cyanobacteria Synergistes sampled Deferribacteres Chrysiogenetes NKB19 • Lineages with no cultured Verrucomicrobia Chlamydia OP3 taxa even more poorly Planctomycetes Spriochaetes sampled Coprothmermobacter OP10 Thermomicrobia Chloroflexi TM7 Deinococcus-Thermus Dictyoglomus Aquificae Well sampled phyla Thermudesulfobacteria Thermotogae Poorly sampled OP1 OP11 No cultured taxa Tuesday, August 11, 2009
Uncultured Lineages: Technical Approaches • Get into culture • Enrichment cultures • If abundant in low diversity ecosystems • Flow sorting • Microbeads • Microfluidic sorting • Single cell amplification Tuesday, August 11, 2009
GEBA Lesson 4 Need Experiments from Across the Tree of Life too Tuesday, August 11, 2009
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, August 11, 2009
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, August 11, 2009
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, August 11, 2009
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 Coprothmermobacter OP10 Thermomicrobia Chloroflexi TM7 Deinococcus-Thermus Dictyoglomus Aquificae Thermudesulfobacteria Thermotogae OP1 OP11 Tuesday, August 11, 2009
Adopt a Microbe Tuesday, August 11, 2009
GEBA Lesson 5 The Importance of Culture (Collections that is) Tuesday, August 11, 2009
GEBA Biggest Challenge: Getting DNA • Getting quality DNA is biggest bottleneck • Sharing strains is also a bottleneck • Solution: Beg Borrow and Steal • DSMZ offered to do for free • ATCC is doing a small number for a fee • In discussions with other PCC and other collections Tuesday, August 11, 2009
Tuesday, August 11, 2009
Quantification gel of the genomic DNA isolated from Microorganisms Conexibacter woesei (DSM 14684T) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Lane 1: c(λ-Marker)= 15 ng Lane 9: DSM 18081, Patulibacter minatonensis Lane 2: c(λ-Marker)= 30 ng Lane 10: DSM 14684, Conexibacter woesei Lane 3: c(λ-Marker)= 50 ng Lane 11: DSM 11002, Dethiosulfovibrio peptidovorans Lane 4: DNA Molecular Weight Marker II (Roche Lane 12: DSM 11551, Halogeometricum borinquense 236250) Lane 13: DNA Molecular Weight Marker II (Roche Lane 5: DSM 13279, Collinsella stercoris 236250) Lane 6: DSM 43043, Intrasporangium calvum Lane 14: c(λ-Marker)= 125 ng Lane 7: DSM 18053, Dyadobacter fermentans Lane 15: c(λ-Marker)= 250 ng Lane 8: DSM 20476, Slackia heliotrinireducens Lane 16: c(λ-Marker)= 500 ng Conexibacter woesei (DSM 14684T) was taken from the German Collection of Microorganisms and Cell Cultures (DSMZ). The genomic DNA was isolated using the Qiagen Genomic 500 DNA Kit (Qiagen 10262). The genomic DNA was 10-250 kb in size as determined by Pulsed Field Gel Electrophoresis (PFGE). The bulk of DNA had a size of 50-250 kb (see attached PFGE image). The DNA concentration is 500 ng/µl as estimated from the gel. Spectrophotometric measurements yielded a DNA concentration of 450 µg/ml; 300 µl of genomic DNA are shipped (150 µg). Tuesday, August 11, 2009
Related Lesson 1 METADATA ROCKS Tuesday, August 11, 2009
SIGS • The Genomic Standards Consortium • The GSC is an open-membership working body which formed in September 2005. • The goal of this international community is to promote mechanisms that standardize the description of genomes and the exchange and integration of genomic data. • See http://gensc.org/gc_wiki/index.php/Main_Page Tuesday, August 11, 2009
Tuesday, August 11, 2009
Additional Lessons • Completeness matters • Computational methods need to be more automated • Need to limit analyses to subsets of all available data • Need for people to help interpret and study data is increasing not decreasing • Sequence is just the beginning • Need to train more students Tuesday, August 11, 2009
Tuesday, August 11, 2009
MICROBES Tuesday, August 11, 2009
A Happy Tree of Life Tuesday, August 11, 2009

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Eisen.Csb2009

  • 1. Seeking out the Dark Matter of the Biological Universe & The Need for a Phylogeny Driven Genomic Encyclopedia Jonathan A. Eisen August 11, 2009 CSB 2009 Tuesday, August 11, 2009
  • 3. “Nothing in biology makes sense except in the light of evolution.” T. Dobzhansky (1973) Tuesday, August 11, 2009
  • 4. Image removed because it comes from a non open access journal Fleischmann et al. 1995 Tuesday, August 11, 2009
  • 6. rRNA Tree of Life Tuesday, August 11, 2009
  • 7. The Tree is not Happy Tuesday, August 11, 2009
  • 8. 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, August 11, 2009
  • 9. 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, August 11, 2009
  • 10. 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, August 11, 2009
  • 11. 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 Archaea Thermotogae OP1 Based on OP11 Hugenholtz, 2002 Tuesday, August 11, 2009
  • 12. 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 Tuesday, August 11, 2009
  • 13. 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 Eisen, Ward, Chloroflexi Badger, Wu, TM7 Deinococcus-Thermus • Solution I: Dictyoglomus Wu, et al. Aquificae Thermudesulfobacteria sequence more Thermotogae OP1 phyla OP11 Tuesday, August 11, 2009
  • 14. 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, August 11, 2009
  • 15. 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 Tuesday, August 11, 2009
  • 16. T. roseum genome Tuesday, August 11, 2009
  • 17. The Tree of Life is Still Angry Tuesday, August 11, 2009
  • 18. 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 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, August 11, 2009
  • 19. Proteobacteria TM6 OS-K • At least 100 phyla of Acidobacteria Termite Group OP8 bacteria Nitrospira Bacteroides Chlorobi • Genome sequences are Fibrobacteres Marine GroupA mostly from three phyla WS3 Gemmimonas Firmicutes • Most phyla with cultured Fusobacteria Actinobacteria species are sparsely OP9 Cyanobacteria Synergistes sampled Deferribacteres Chrysiogenetes NKB19 • Lineages with no cultured Verrucomicrobia Chlamydia OP3 taxa even more poorly Planctomycetes Spriochaetes sampled Coprothmermobacter OP10 Thermomicrobia Chloroflexi • Solution - use tree to really TM7 Deinococcus-Thermus fill gaps Dictyoglomus Aquificae Well sampled phyla Thermudesulfobacteria Thermotogae OP1 OP11 Tuesday, August 11, 2009
  • 21. GEBA Pilot Project Overview • Identify major branches in rRNA tree for which no genomes are available • Identify which lineages have type species available in DSMZ • Grow > 200 of these and prep. DNA • Sequence and finish 100 • Annotate, analyze, release data • Assess benefits of tree guided sequencing Tuesday, August 11, 2009
  • 22. GEBA Pilot Project: Components • Project overview (Phil Hugenholtz, Nikos Kyrpides, Jonathan Eisen) • Project management (David Bruce, Lynne Goodwin et al) • Culture collection and DNA prep (DSMZ, Hans-Peter Klenk) • Libraries and DNA (Eileen Dalin et al) • Sequencing and closure (Susan Lucas, Alla Lapidus et al.) • Annotation and data release (Nikos Kyrpides) • Analysis (Dongying Wu, Kostas Mavrommatis, Martin Wu, Jenna Morgan, Victor Kunin, Marcel Huntemann, Neil Rawlings, Ian Paulsen, Patrick Chain, Patrik D’Haeseleer, Sean Hooper, Iain Anderson) • Adopt a microbe education project (Cheryl Kerfeld) • Outreach (David Gilbert) • $$$ (DOE, Eddy Rubin, Jim Bristow) Tuesday, August 11, 2009
  • 23. GEBA Pilot: Selecting Targets Tuesday, August 11, 2009
  • 28. B: Ac tin ob ac te B: ria # of Genomes Am (H in igh 10 15 20 25 30 35 0 5 an G Tuesday, August 11, 2009 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
  • 29. GEBA Current Status • >100 in progress • GEBA 56 (focus of first paper) – 34 finished genomes – Released to IMG-GEBA page, JGI-FTP site, and Genbank • All data is completely Open for anyone to use Tuesday, August 11, 2009
  • 31. Assess Benefits of GEBA • All genomes have some value • But what, if any, is the benefit of tree- guided sequencing over other selection methods Tuesday, August 11, 2009
  • 32. Why Increase Taxonomic Coverage? • Gene discovery • Annotation, functional prediction • Metagenomic analysis • Mechanisms of diversification • Species phylogeny and classification Tuesday, August 11, 2009
  • 33. GEBA Lesson 1 rRNA Tree of Life is a Useful Guide for Genome Core Phylogenetic Diversity Tuesday, August 11, 2009
  • 34. rRNA Tree of Life Tuesday, August 11, 2009
  • 35. 16s Says Hyphomonas is in Rhodobacteriales Badger et al. 2005 Tuesday, August 11, 2009
  • 36. WGT Says Its Related to Caulobacterales Badger et al. 2005 Tuesday, August 11, 2009
  • 40. GEBA Lesson 2 Phylogenetically Guided Selection Can Help Many Aspects of Genome Analysis Tuesday, August 11, 2009
  • 41. Annotation Improves • Conversion of hypothetical into conserved hypotheticals • Linking distantly related members of protein families • Non-homology functional prediction methods Tuesday, August 11, 2009
  • 42. Improved Rosetta Stone Predictions Tuesday, August 11, 2009
  • 43. Al ph ap ro Be te ta o ba G 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 pr ct am ot er m eo ia ap ba ro ct D te er el ob ia ta pr ac Ep Tuesday, August 11, 2009 ot te U si lo eo ria nc ba la np ct ss ro er ifi te ia ed ob 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 iro es ch ae Fi te rm s ic ut Ch es lo ro U Phylogenetic Metagenomics nc fle la xi ss Ch ifi lo ed ro bi Ba ct er ia 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
  • 44. 16s Says Hyphomonas is in Rhodobacteriales Badger et al. 2005 Tuesday, August 11, 2009
  • 45. WGT Says Its Related to Caulobacterales Badger et al. 2005 Tuesday, August 11, 2009
  • 47. GEBA Lesson 3 We have still only scratched the surface of microbial diversity Tuesday, August 11, 2009
  • 48. Protein Family Rarefaction Curves • Take data set of multiple complete genomes • Identify all protein families using MCL • Plot # of genomes vs. # of protein families Tuesday, August 11, 2009
  • 54. 350000 300000 Number of proteins 250000 Total Gene Number 200000 150000 100000 S. agalactiae Enterobacteriaceae 50000 Actinobacteria Bacteria from GEBA project 0 0 10 20 30 40 50 60 70 80 Genome Number Tuesday, August 11, 2009
  • 55. 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, August 11, 2009
  • 56. Within Family Novelty Example: Transporter Profiles inorganic ions amino acids, nitro compounds and peptides drugs/ toxins sugars carboxylates nucleosides/ tides, bases siderophores other 700 600 500 Number of transporters 400 300 200 100 0 halut pedhe thete denac kanko acife aliac chipi desa7 sanke capoc catac celfl dyafe sacvi sphth spili stana sulde theac tsupa xylce detpe haloc plali thecu atopa crycu kytse jonde slahe eggle halmu desr5 anapr strro halbo sebte lepbu actmi beuca brafa conwo nakmu krifl meisi desba geoob thebi gorbr meiru rhoma bramu Sebaldella termitidis ATCC 33386 has 2x number of sugar PTS transporters of any genome Tuesday, August 11, 2009
  • 57. Phylogenetic Distribution Novelty: 1st Bacterial Actin Related Protein Haliangium ochraceum DSM 14365 Tuesday, August 11, 2009
  • 58. Phylogenetic Diversity: Sequenced Bacteria & Archaea Tuesday, August 11, 2009
  • 59. Phylogenetic Diversity with GEBA Tuesday, August 11, 2009
  • 62. Proteobacteria TM6 OS-K • At least 40 phyla of Acidobacteria Termite Group OP8 bacteria Nitrospira Bacteroides Chlorobi • Genome sequences are Fibrobacteres Marine GroupA mostly from three phyla WS3 Gemmimonas Firmicutes • Most phyla with cultured Fusobacteria Actinobacteria species are sparsely OP9 Cyanobacteria Synergistes sampled Deferribacteres Chrysiogenetes NKB19 • Lineages with no cultured Verrucomicrobia Chlamydia OP3 taxa even more poorly Planctomycetes Spriochaetes sampled Coprothmermobacter OP10 Thermomicrobia Chloroflexi TM7 Deinococcus-Thermus Dictyoglomus Aquificae Well sampled phyla Thermudesulfobacteria Thermotogae Poorly sampled OP1 OP11 No cultured taxa Tuesday, August 11, 2009
  • 63. Uncultured Lineages: Technical Approaches • Get into culture • Enrichment cultures • If abundant in low diversity ecosystems • Flow sorting • Microbeads • Microfluidic sorting • Single cell amplification Tuesday, August 11, 2009
  • 64. GEBA Lesson 4 Need Experiments from Across the Tree of Life too Tuesday, August 11, 2009
  • 65. 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, August 11, 2009
  • 66. 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, August 11, 2009
  • 67. 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, August 11, 2009
  • 68. 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 Coprothmermobacter OP10 Thermomicrobia Chloroflexi TM7 Deinococcus-Thermus Dictyoglomus Aquificae Thermudesulfobacteria Thermotogae OP1 OP11 Tuesday, August 11, 2009
  • 69. Adopt a Microbe Tuesday, August 11, 2009
  • 70. GEBA Lesson 5 The Importance of Culture (Collections that is) Tuesday, August 11, 2009
  • 71. GEBA Biggest Challenge: Getting DNA • Getting quality DNA is biggest bottleneck • Sharing strains is also a bottleneck • Solution: Beg Borrow and Steal • DSMZ offered to do for free • ATCC is doing a small number for a fee • In discussions with other PCC and other collections Tuesday, August 11, 2009
  • 73. Quantification gel of the genomic DNA isolated from Microorganisms Conexibacter woesei (DSM 14684T) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Lane 1: c(λ-Marker)= 15 ng Lane 9: DSM 18081, Patulibacter minatonensis Lane 2: c(λ-Marker)= 30 ng Lane 10: DSM 14684, Conexibacter woesei Lane 3: c(λ-Marker)= 50 ng Lane 11: DSM 11002, Dethiosulfovibrio peptidovorans Lane 4: DNA Molecular Weight Marker II (Roche Lane 12: DSM 11551, Halogeometricum borinquense 236250) Lane 13: DNA Molecular Weight Marker II (Roche Lane 5: DSM 13279, Collinsella stercoris 236250) Lane 6: DSM 43043, Intrasporangium calvum Lane 14: c(λ-Marker)= 125 ng Lane 7: DSM 18053, Dyadobacter fermentans Lane 15: c(λ-Marker)= 250 ng Lane 8: DSM 20476, Slackia heliotrinireducens Lane 16: c(λ-Marker)= 500 ng Conexibacter woesei (DSM 14684T) was taken from the German Collection of Microorganisms and Cell Cultures (DSMZ). The genomic DNA was isolated using the Qiagen Genomic 500 DNA Kit (Qiagen 10262). The genomic DNA was 10-250 kb in size as determined by Pulsed Field Gel Electrophoresis (PFGE). The bulk of DNA had a size of 50-250 kb (see attached PFGE image). The DNA concentration is 500 ng/µl as estimated from the gel. Spectrophotometric measurements yielded a DNA concentration of 450 µg/ml; 300 µl of genomic DNA are shipped (150 µg). Tuesday, August 11, 2009
  • 74. Related Lesson 1 METADATA ROCKS Tuesday, August 11, 2009
  • 75. SIGS • The Genomic Standards Consortium • The GSC is an open-membership working body which formed in September 2005. • The goal of this international community is to promote mechanisms that standardize the description of genomes and the exchange and integration of genomic data. • See http://gensc.org/gc_wiki/index.php/Main_Page Tuesday, August 11, 2009
  • 77. Additional Lessons • Completeness matters • Computational methods need to be more automated • Need to limit analyses to subsets of all available data • Need for people to help interpret and study data is increasing not decreasing • Sequence is just the beginning • Need to train more students Tuesday, August 11, 2009
  • 80. A Happy Tree of Life Tuesday, August 11, 2009