Jonathan Eisen talk on 1$ Genome

The 1$ Bacterial Genome: Advances in Bioinformatics Jonathan A. Eisen U. C. Davis Genome Center

The 1$ Bacterial Genome: Oh $^#^ - We’re $&#$ Jonathan A. Eisen U. C. Davis Genome Center

The 1$ Bacterial Genome: Informatics, GEBA and me Jonathan A. Eisen U. C. Davis Genome Center

Outline • GEBA - The JGI Genomic Encyclopedia of Bacteria and Archaea • Insights into the 1$ genome from the GEBA project • Additional insights into the 1$ genome

GEBA: The Genomic Encyclopedia of Bacteria and Archaea Run by JGI $$ from DOE Work by many

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

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 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 OP11 Hugenholtz, 2002

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 three phyla Synergistes Deferribacteres Chrysiogenetes • Some other NKB19 Verrucomicrobia Chlamydia phyla are OP3 Planctomycetes Spriochaetes only sparsely Coprothmermobacter OP10 sampled Thermomicrobia Chloroﬂexi TM7 Deinococcus-Thermus Dictyoglomus Aquiﬁcae Thermudesulfobacteria Thermotogae OP1 Based on OP11 Hugenholtz, 2002

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 three phyla Synergistes Deferribacteres Chrysiogenetes • Some other NKB19 Verrucomicrobia Chlamydia phyla are OP3 Planctomycetes Spriochaetes only sparsely Coprothmermobacter OP10 sampled Thermomicrobia Chloroﬂexi TM7 • Same trend in Deinococcus-Thermus Dictyoglomus Aquiﬁcae Archaea Thermudesulfobacteria Thermotogae OP1 Based on OP11 Hugenholtz, 2002

Need for Tree Guidance Well Established • Common approach within some eukaryotic groups – NHGRI animal projects – FGI at Whitehead – Plant LSP • Phylogenetic gaps in bacterial and archaeal projects commonly lamented in literature, conversations, etc • Many small projects funded to ﬁll in some gaps – DOE/TIGR Sequencing – Multiple CSP projects – Multiple NSF/USDA projects – Private projects (e.g., Integrated Genomics, Diversa)

Proteobacteria • NSF-funded TM6 • At least 40 OS-K Tree of Life Acidobacteria Termite Group phyla of OP8 Project Nitrospira bacteria Bacteroides Chlorobi • A genome Fibrobacteres Marine GroupA • Genome from each of WS3 Gemmimonas sequences are Firmicutes eight phyla 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 Eisen, Ward, Chloroﬂexi • Solution I: TM7 Badger, Wu, Deinococcus-Thermus Wu, et al. Dictyoglomus Aquiﬁcae sequence more Thermudesulfobacteria Thermotogae OP1 phyla OP11

Proteobacteria TM6 OS-K • At least 100 phyla of Acidobacteria Termite Group bacteria OP8 Nitrospira Bacteroides • Genome sequences are Chlorobi 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 • Solution - use tree to really Chloroflexi TM7 Deinococcus-Thermus fill gaps Dictyoglomus Aquificae Well sampled phyla Thermudesulfobacteria Thermotogae OP1 OP11

http://www.jgi.doe.gov/programs/GEBA/pilot.html

GEBA Pilot Project Overview • Select 200 organisms using rRNA tree as a guide • Develop high throughput pipeline for strain growth and DNA preparation • Sequence and ﬁnish 100 genomes • Annotate, analyze, release data • Assess beneﬁts of tree guided sequencing

B: Ac tin ob ac te B: ria # of Genomes Am (H in igh 10 15 20 25 30 35 0 5 an G a C B: B: er ) Ba Aq ob ct uif ia e i B: B: ro cae D Ch ide B: efe 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 Pl na d a B: an er ete Th o B: cto bia s Phyla er 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 oba lob A: no cte i m r A: The icr ia Th rm obi er oc a m oc op ci ro te i

IMG/GEBA http://img.jgi.doe.gov/cgi-bin/geba/main.cgi

Why Increase Taxonomic Coverage? • Gene discovery • Annotation, functional prediction • Metagenomic analysis • Mechanisms of diversiﬁcation • Species phylogeny and classiﬁcation

Phylogenetic Metagenomics

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)

GEBA Lesson 1 Tree of Life is a Useful Guide

rRNA Tree of Life

GEBA Lesson 2 We have still only scratched the surface of microbial diversity

Phylogenetic Diversity: Sequenced Bacteria & Archaea

Phylogenetic Diversity with GEBA

Phylogenetic Diversity: GreenGenes

Viruses Too

First Bacterial Actin Related Protein - Haliangium ochraceum DSM 14365 First found by V. Kunin, Structure Analysis by Patrik D. et al

GEBA Lesson 3 Need Experiments from Across the Tree of Life too

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

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 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 OP11 Hugenholtz, 2002

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 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 OP11 Hugenholtz, 2002

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

GEBA Lesson 4 The Importance of Project Management

GEBA Project Flowchart Annotation Project Initiation Sequencing Draft IMG1 GEBA Sequencing Proposal and Assembly1 Shotgun Complete Scientific and Genome Genome Technical GenBank GenBank Review1 Submission1 Submission1 OK? OK? IMG – ER1 IMG – ER1 Finish Negotiate Sequencing Scope of and Draft Work Gene-QA1 Assembly2 Annotation3 Receive Starting Material1 Finish OK? Annotation3 1 PGF 2 LANL David Bruce, Lynne Goodwin et al 3 ORNL

GEBA Lessons 5 The Importance of Culture (Collections that is)

GEBA Biggest Challenge: Getting DNA • Getting quality DNA is biggest 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

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).

Related Lesson 1 METADATA ROCKS

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

Related Lesson 2 Completeness Matters

Completeness • Final quality of genome sequence inﬂuences what one can do with the data • Why completeness (closed, high quality) is important – Gene presence/absence – Gene order – Genome rearrangements – Identifying islands • See “The Value of Complete Microbial Genome Sequencing (You Get What You Pay For).” Fraser et al. J. Bact. 2002.

StrpB vs. StrpA 13623100 13622900 13622700 13622500 13622300 Series1 13622100 13621900 13621700 13621500 13621300 0 500 1000 1500 2000 2500

Mauve, Artemis

Additional Lessons • 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

MICROBES

Jonathan Eisen talk on 1$ Genome

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