A Tale of Two Worms:A Tale of Two Worms:Comparing the Genomes ofComparing the Genomes of

C. elegansC. elegans & C & C. briggsae. briggsae

Lincoln SteinCold Spring Harbor Laboratory

My LabMy Lab

International HapMap ProjectInternational HapMap Project

• Find common regions of genetic variation in human population to reduce cost of genetic association studies.

• Reduce cost of genetic association studies.

• 600,000 SNPs x 270 individuals

GrameneGramene

• Comparative genomics among monocots

• Rice as model system• Rice genome, maps, proteins,

mutants, QTLs, phenotypes• Map alignments to maize, wheat,

oats, barley &c.

Genome KnowledgeBaseGenome KnowledgeBase

• Biological pathways in human• Curated by experts in the field• Designed for

• Education• Data mining & discovery

• Open data/Open software

WormBaseWormBase

• Community database for C. elegans• C. elegans genome• C. briggsae genome• Genetic maps• Developmental anatomy• RNAi screens• Microarray screens• Evolutionary biology

Generic Model Organism Generic Model Organism DbDb• Reusable software for

building model organism databases

• Used by WormBase, FlyBase, Gramene, RatDB, SGD, MGD…

• Genome browsers, genetic maps, curation tools…

male (lateral)

male (ventral)

hermaphrodite

200 µm 200 µm

10 µm 10 µm

Sequencing Sequencing C. briggsaeC. briggsae

• Isolate DNA, make libraries (2 mo)• Map libraries (4 mo)• Shotgun sequence genome (1 wk)• Assemble genome (6 mo)• Analyze genome (9 mo)

The DraftThe Draft

Contig Type Count N50 (kb) Length (% genome)

Contigs 5341 41 105.6

Supercontigs

899 474 107.5

Scaffolds 142 1450 102.4 (98%)

BAC Map

Sequence Contigs

Supercontigs

Scaffolds

Jim Mullikin, Sanger Center; LaDeana Hillier, WUSTL

Calling Calling C. briggsae C. briggsae GenesGenes

briggsaebriggsae genes: “hybrid” genes: “hybrid” strategystrategy

Eleganspredictions

Briggsaepredictions

Avril Coghlan, University of Dublin

How accurate is it?How accurate is it?

• C. elegans gold standard• 2,257 genes entirely confirmed by mRNA

data

• Results on C. elegans set• 92% of time, hybrid method picked the

“gold standard gene” correctly• 32 genes incorrectly split into 2 or more

predictions (1.4%)• 49 genes incorrectly merged into 1

prediction (1%)

Gene Sets Very SimilarGene Sets Very SimilarTable 1: Comparison of the C. briggsae and C. elegans Protein-Coding Gene Sets

C. briggsae C. elegansWS77*

C. elegans Hybrid

Number of genes 19,507 18,808 20,621

Median gene length

1.90 kbp 1.91 kbp 1.83 kbp

Summed length of genes 55.7 Mbp 52.5 Mbp 55.6 Mbp

Average gene density

5.4 kbp per gene

5.3 kbp per gene

4.9 kbp per gene

Identifying OrthologsIdentifying Orthologs

Ce Cb ortholog pair

ortholog vs paralog?

best similarity match

best similarity match

Use colinearityto resolveambiguities

(Todd Harris, CSHL)

Comparing OrthologsComparing Orthologs

• 12,155 orthologs• 807 C. briggsae “orphans”• 1,061 C. elegans “orphans”• Divergence date: 80-110 Mya• All genes under various degrees of

purifying selection

(Todd Harris, Jason Stajich)

Orthologs Similar but Differ in Orthologs Similar but Differ in DetailDetail

Briggsae has 1 new intron every 5th gene.

Comparing Gene Families:TRIBE-MCL

Comparing Gene Families:TRIBE-MCL

Comparing Gene Families:TRIBE-MCL

Comparing Gene Families:TRIBE-MCL

Comparing Gene Families:TRIBE-MCL

Cluster 1

Cluster 2

Cluster 3

Cluster 4 Cluster 5

Cb/Ce protein clusters

(Jason Stajich, rotation student)

2169 clusters of >= 2 members24% of elegans single-copy genes28% of briggsae single-copy genes

Protein Family Clusters

Cluster Elegans Briggsae

Description

5 112 128 Zn-finger

3 128 150 Protein kinase

1 215 105 7TM receptor, subf 2

6 122 86 7TM receptor, subf 2

2 169 135 7TM receptor, subf 1

8 2 204 Unknown

17 116 22 DUF38>200 clusters unbalanced by more than 2-fold

A Rapidly Evolving Family:Olfactory Receptors

PFAM Class C. elegans C. briggsae

7tm_4 269 222

7tm_5 322 163

sra 37 18

srb 16 12

sre 55 51

srg 32 30

Total 718 476

Putative ortholog pair

elegans exclusive subtree

Sra Olfactory Receptor Family

Synteny: Aligning Synteny: Aligning C.b.C.b. to to C.e.C.e.

Type Intergen Upstr Downst CDS Intron 5' UTR 3'UTR Repeat TotalStrong 61,615 27,512 30,192 49,358 114,323 2,783 7,239 28,313 321,335Coding 41,817 11,600 15,571 152,086 49,135 855 1,557 12,095 284,716Weak 115,200 53,189 59,542 188,601 250,603 5,885 11,823 49,624 734,467

TOTAL 218,632 92,301105,305 390,045 414,061 9,523 20,619 90,032 1,340,518

(Todd Harris & Jason Stajich)

Synteny ReconstructionSynteny Reconstruction

raw aligned segments (WABA)

Merge overlaps

Merge adjacent

merged segments

Reconstruct interrupted segments

reconstructed segments

(Yours truly)

Reconstructing Reconstructing briggsaebriggsae

•4,837 reconstructed segments•~85% of genome•0.5-0.7 bkpts/Mb/My)

Rearrangement is LocalRearrangement is Local

I II III IV V X

I 335 17 22 31 32 16

II 256 28 21 30 9

III 289 42 28 6

IV 314 38 14

V 272 21

X 170

Junctions of elegans chromosomes onbriggsae contigs

Rearrangement is LocalRearrangement is Local

left arm center right arm

left arm 494 174 123

center 592 163

right arm

445

Junctions of elegans chromosome arms onbriggsae contigs

big mapbig mapSyntenic blocks

Genes & meiotic map

Orthologs

Orphans

Essential genes

Repetitive elements

KA/KS

KS

Improving Improving elegans: elegans: new new gene?gene?

Improving Improving elegans: elegans: bad bad exon?exon?

Corrections to Corrections to C. elegansC. elegansTable 11. Updating the C. elegans Gene Set Using C. briggsae Similarity

Gene Set WS77 WS103

New genes 1,275 985

New exons in existing genes 1,763 1,243

Exon extensions in existing genes

1,115 845

Exon deletions in existing genes

2,093 1,600

Exon truncations in existing genes

1,675 1,114

Recent Work:Recent Work:Chemosensory ReceptorsChemosensory Receptors

PFAM Class C. elegans C. briggsae7tm_4 269 222

7tm_5 322 163

sra 37 18

srb 16 12

sre 55 51

srg 32 30

• Third largest C. elegans protein family.• Subclass of GPCR 7TM receptors.

Questions Questions

• Are these differences real?• Mechanism of the differences?

• Amplification vs gene loss

• Why are some subfamilies unbalanced and not others?

• Phenotypic consequences of the differences?

Putative ortholog pair

elegans exclusive subtree

Sra Olfactory Receptor FamilySra Olfactory Receptor Family

Are the Differences Real?Are the Differences Real?

• Intensive search for missing sra family members.

C. elegans C. briggsae

srasra

srasrasra

sra sra

sra srasrasra

sra

elegans genome briggsae genome

(Jack Chen, Postdoc; Shraddha Pai, URP)

similaritysearching

newnew

newnew

newnew

ResultsResults

• Family size differences real (still roughly twice as manyelegans sra as briggsae sra)

• Differences due to species-specific tandem duplications, not due to conversion into pseudogenes.

• But…

Hitting non-sra Hitting non-sra eleganselegans genes?genes?

18 non-sra C. elegans genes17 non-sra C. briggsae genes

A New Nematode A New Nematode Chemosensory (Sub)family?Chemosensory (Sub)family?

srafamily

sra-likegenes

7TM Domain Structure7TM Domain Structure

C36C5.7

• Most candidates showed 7 transmembrane domain signatures characteristic of GPCR membrane receptors.

……UsuallyUsuallyGene Name Number of TMsC36C5.6 4C36C5.7 7C36C5.8 7C36C5.10 7C36C5.11 7C36C5.2 7C36C5.1 7T20D4.18 6T20D4.2 4T20D4.1 2C04F5.4 7C04F5.5 7C04F5.6 6C33G8.5 6C47A10.6 7T21H8.2 6T21H8.3 6T21H8.4 7

Gene Name Number of TMsCBG07353 6CBG07355 7CBG19062 6CBG19390 5CBG19391 7CBG13454 5CBG13479 5CBG06298 5CBG05677 7CBG18741 4CBG08673 6CBG08675 6CBG08677 5CBG21805 7CBG07352 7CBG21852 7

Repairing Incomplete Repairing Incomplete GenesGenes

missedexon

Before & AfterBefore & After

After repairing: 7 TMsBefore repairing: 6 TMs

After RepairAfter RepairGene Name Number of TMsC36C5.6 7C36C5.7 7C36C5.8 7C36C5.10 7C36C5.11 7C36C5.2 7C36C5.1 7T20D4.18 7T20D4.2 7T20D4.1 7C04F5.4 7C04F5.5 7C04F5.6 7C33G8.5 6C47A10.6 7T21H8.2 6T21H8.3 6T21H8.4 7

Gene Name Number of TMsCBG07353 7CBG07355 7CBG19062 6CBG19390 5 ψCBG19391 7CBG13454 5 ψCBG13479 5CBG06298 5CBG05677 7CBG18741 4 ψCBG08673 6CBG08675 6CBG08677 6 ψCBG21805 7CBG07352 7CBG21852 7

Expression Patterns Co-Expression Patterns Co-Cluster with sra Family Cluster with sra Family

GenesGenes

sra genes

sra-likegenes

Kim et al Science, 293: 2087-2092. 2001

Anatomic Expression Anatomic Expression PatternPattern

Promoter/GFP Fusion AnalysisPromoter/GFP Fusion Analysis(Collaboration w/ David Baillie)(Collaboration w/ David Baillie)

Gene Head TailT20D4.1 -

T21H8.4 -

C33G8.5

C36C5.6

C47A10.6

C05F5.4 -

T20D4.18 - -

Phasmid Neuron Phasmid Neuron ExpressionExpression

anus

PHA/PHB

T21H8.4-GFP

Amphid Neuron Amphid Neuron ExpressionExpression

axon

dendrite

ASx Cell body

C05F5.4

ConclusionConclusion

• Likely new olfactory receptor subfamily

• Closely related to sra subfamily• ~50% more members in elegans

than briggsae• Specific expression in both amphid

& phasmid sensory neurons

Next StepsNext Steps

• Continue refining families in the 2 species• More novel candidate (sub)families,

one with ~100 members.

• Characterize expression patterns

Longer TermLonger Term

• Deconvolute odorant combinatorial code.

chemotaxis aversion

odorant

chemosensory receptors

sensory neurons

interneurons

Resources to ApplyResources to Apply

• Neuronal wiring chart.• Receptor promoter::GFP fusions.• Calcium-flux sensitive GFP

constructs (“Cameleons”)• Phenotypic assays (elegans &

briggsae)• Transgenesis/rescue

And Coming SoonAnd Coming Soon

Who DunnitWho DunnitZhirong Bao Alan Coulson Shraddha Pai

Thomas Blumenthal Richard DurbinBob Plumb

Michael Brent Sam Griffith-Jones Jane Rogers

Jack Chen Todd Harris Mark Sohrmann

Laura Clarke LaDeana Hillier Jason Stajich

Chris Clee Patricia Kuwabara Robert Waterston

Avril Coghlan James Mullikin David WilleyCSHL, WUGSC, Trinity College Dublin, Wellcome Trust/Sanger Institute, NIH, Duke University, UW

Funding from: NIH & Wellcome Trust

Who DunnitWho DunnitZhirong Bao Alan Coulson Shraddha Pai

Thomas Blumenthal Richard DurbinBob Plumb

Michael Brent Sam Griffith-Jones Jane Rogers

Jack Chen Todd Harris Mark Sohrmann

Laura Clarke LaDeana Hillier Jason Stajich

Chris Clee Patricia Kuwabara Robert Waterston

Avril Coghlan James Mullikin David WilleyCSHL, WUGSC, Trinity College Dublin, Wellcome Trust/Sanger Institute, NIH, Duke University, UW

Funding from: NIH & Wellcome Trust

Who DunnitWho DunnitZhirong Bao Alan Coulson Shraddha Pai

Thomas Blumenthal Richard DurbinBob Plumb

Michael Brent Sam Griffith-Jones Jane Rogers

Jack Chen Todd Harris Mark Sohrmann

Laura Clarke LaDeana Hillier Jason Stajich

Chris Clee Patricia Kuwabara Robert Waterston

Avril Coghlan James Mullikin David Willey

David Baillie, University of British Columbia