<-

Genomics

<-

Transcriptomics

<-

Proteomics

Functional genomics to advance dairy cattle health

Sigbjørn Lien & Scott Fahrenkrug

Bio

info

rmat

ics

Func

tiona

l gen

omic

s

BOVINE GENOME SEQUENCINGBOVINE GENOME SEQUENCING

77--8X coverage8X coverage~~ 2X coverage ordered path of BAC clones + BAC end sequences2X coverage ordered path of BAC clones + BAC end sequences~~ 6X (2 kb), 16X (2 kb), 1--2X (10 kb) and 0.4X (50 kb) coverage shotgun library sequencing2X (10 kb) and 0.4X (50 kb) coverage shotgun library sequencing

Sequencing for genetic variation Sequencing for genetic variation --> > SNPsSNPs

Total costs: Total costs: ~US$51M~US$51M, where NHGRI pays ~50% (~US$25M), where NHGRI pays ~50% (~US$25M)

Norway participate with Norway participate with US$1MUS$1M

Sequencing at Baylor College of Medicine (Houston, Texas)Sequencing at Baylor College of Medicine (Houston, Texas)

Start Dec 2003 and Start Dec 2003 and complete 2006complete 2006

Step change in progress/cost of QTL/gene discoveryStep change in progress/cost of QTL/gene discovery

(up to 10X)(up to 10X)

Holstein, Jersey, Norwegian Red, Angus, Limousin, Brahman

Hereford

IdentifyIdentify putative putative SNPsSNPs from from shotgunshotgun sequencingsequencing PHASE IPHASE I

Fund Fund sequencingsequencing ofof 150.000 150.000 readsreads from from NorwegianNorwegian RedRed

>50.000 >50.000 SNPsSNPs from from NorwegianNorwegian Red Red -- Hereford Hereford comparisonscomparisons

ValidateValidate SNPsSNPs in in internationalinternational breedbreed panelpanelSNPSNP--panels and panels and technologytechnology for for highhigh--througputthrougput genotypinggenotyping

ValidateValidate >30.000 >30.000 SNPsSNPs

Genotype Norwegian resource population (biobank) PHASE IIPHASE II

Genotype >2.000 animals for 25.000 SNPs

Determine LD and haplotype structure in Norwegian Red cattle

Fine map QTL -> identify QTN (focus on health and fertility traits)

ParticipationParticipation in in thethe ’’BovineBovine HapMapHapMap projectproject’’

4378 4378 SekvensaSekvensa

NorwayNorway waswas thethe first first countrycountry to to establishestablish a a nationnation--widewide healthhealth cardcard recordingrecordingsystem in system in cattlecattleEachEach cowcow has an has an individualindividual healthhealth cardcard, drugs and , drugs and antibioticsantibiotics cancan onlyonly be be prescribedprescribed by by vetsvets --> > veryvery reliable reliable recordingrecording

10 traits

...........46 traits...........64 traits(1975) (1978) (1989)

10 traits

...........46 traits...........64 traits(1975) (1978) (1989)

••

AlsoAlso

intensive intensive recordingrecording

ofof

milkmilk, , beefbeef

and and reproductionreproduction

traitstraits••

90% 90% ofof

all all cowscows

have have beenbeen

registeredregistered

sincesince

1978 1978 --> > >>4 mill 4 mill cowscows

••

CompleteComplete

listing listing ofof

pedigreepedigree

structurestructure

--> > ~ 7,5 mill ~ 7,5 mill individualsindividuals••

ProgenyProgeny

testing testing ofof

250250--300 300 daughtersdaughters

per sireper sire

••

PaternityPaternity

testing testing ofof

bullsbulls••

SystematicSystematic

storagestorage

ofof

semensemen

from all bulls from all bulls sincesince

1982 1982 →→

DNADNA

••

SelectionSelection

groupsgroups: : lowlow

clinicalclinical

mastitismastitis

<<-->>

highhigh

protein protein yieldyield

NorwegianNorwegian BiobankBiobank

>250>250

ProgenyProgeny

testingtesting

......

QTLQTL--mappingmapping populationpopulation

Genotype Genotype ~25.000 ~25.000 SNPsSNPs⇓⇓

LD & LD & haplotypehaplotype

structurestructure⇓⇓

Fine Fine mapmap

QTL QTL affectingaffecting mastitismastitis

and and fertilityfertility

......

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

0 10 20 30 40 50 60 70 80 90

Map (cM)

Post

erio

r pro

babi

lity

(Olsen, Genetics, 2005)

CombinedCombined linkagelinkage and LD and LD analysisanalysis protein%protein%

Chr. 6

AAATCTTCCCCAAATCTTCCCC

TAAAGTTCCCGTAAAGTTCCCG

• ConstructConstruct

haplotypeshaplotypes

QQ

qq

++

÷÷

No. Freq.

AB

CG

2_49

AB

CG

2_25

6A

AFC

0214

4624

_757

84A

AFC

0214

4624

_031

29A

AFC

0214

4624

_031

28P

KD

2_74

6P

KD

2_11

75P

KD

2_14

51P

KD

2_13

49P

KD

2_65

0P

KD

2_35

3P

KD

2_61

1P

KD

2_61

0P

KD

2_34

9P

KD

2_38

3P

KD

2_90

1P

KD

2_37

7P

KD

2_44

7P

KD

2_12

41P

KD

2_22

56P

KD

2_27

59P

KD

2_36

10P

KD

2_39

09P

KD

2_97

141

PK

D2_

1013

PK

D2_

953

PK

D2_

597

OP

N_3

907

1 A G A G G G A G T C C G T G G A T A C T T T A G A C C T 0,2622 A G A G G G A A T C C G T G G A T A C T T T A G G C C T 0,1673 A G A A G G A A T T C G T G G A T A C T T T A G G C C T 0,0874 A G A G G G A A T C C G T G G A T A C T T T A A A C C D 0,0735 A G A A G G G A A C T G A T G G T A T T C C T G G C T T 0,0676 C G A G G G A A T C C G T G G A T A C T T T A G A C C D 0,0527 A A A A G G A A T C C G T G G A T A C T T T A G G C C T 0,0518 A A G A A A A A T C C G T G G A T A C T T T A G G C C T 0,0489 A A A A G G A A T C T G T G G A T A C T T T A G G C C T 0,047

10 A G G A A A A A T C T A T G G A A G C T T T A G G T C T 0,03411 A A G A A A A A T C T A T G G A A G C T T T A G G T C T 0,03112 A A G A A A A A T C C G T G G A T A C T T T A A A C C T 0,02513 A G A G G G A A T C T G T G A A T A C T T T A G A C C T 0,022

HAPLOTYPE

-8,00

-7,00

-6,00

-5,00

-4,00

-3,00

-2,00

-1,00

0,00

1,00

2,00

3,00

0 2 4 6 8 10 12 14

Haplotype

Effe

ct

New New projectproject in in NorwayNorway ((part part ofof UMB/UMN UMB/UMN collaborationcollaboration))

““A genome expression profiling strategy A genome expression profiling strategy towards better disease control and improved towards better disease control and improved animal welfareanimal welfare””

FundedFunded by by TheThe ResearchResearch CouncilCouncil ofof NorwayNorwayOneOne researcherresearcher (Siri (Siri KulbergKulberg))OneOne PhDPhD--studentstudent (to be (to be employedemployed))Total Total budgetbudget: 5 mill NOK (800.000 USD): 5 mill NOK (800.000 USD)FahrenkrugFahrenkrug lab to lab to provideprovide microarraysmicroarrays and and bioinformaticsbioinformatics

Siri has Siri has alreadyalready

visitedvisited

FahrenkrugFahrenkrug

lab and lab and willwill

returnreturn

in in OctoberOctober

NorwegianNorwegian

SelectionSelection

GroupsGroups

LCMHPY

1989-->

-

Cows

from nine

different

herds-

Progeny-tested

sires

(Q)(Q)(q)(q)

+PY ÷CM

UMB herd

• Identify

cows

for transcript

profiling

based

onhaplotype

structure

QQ

qq

TranscriptomicsMicroarraysMassARRAY (rcPCR)

Genomics

Transcriptomics

Proteomics

ReverseTranscription

Bovine Oligonucleotide Microarray Consortium (BOMC)

16,846 BOMC genes (which align to bovine genome assembly and have vertebrate protein homologs)

5943 3’ ESTs which align to bovine assembly at least 2 Kb away from BOMC genes

703 RefSeq predicted bovine genes

4 BoLa genes not included in previous sets

60 negative controls

360 mismatch controls

84 5’-3’ distance controls

_________________________________________________

Total of 24,000 BOMC oligo

probes

mammary glandSL CL

Data Processing and AnalysisData Processing and Analysis

Statistical Analysis

• Model: Y =X + a + b + …• F-test• T-test• Fold Change

• Results• Differential expressed

genes• Sample and gene expression

pattern cluster

• Data Quality• Dynamic Range• Signal-Noise Ratio• Signal Distribution of

Front/Background Channel• Sample Correlation

and Cluster

• Slide Quality• Spot Diameter• Spot Area• Footprint• Front Channel and Background

Channel Signal Uniformity

Biological Interpretation

Annotation

SequenceSequencer

Pipeline

Animal

Genotype

Markers

Arrays

LibrariesBiological Sample

Clones

SequenceAnalysis

Oligos

Minnesota Animal Genome and Ontology Database

Phenotype

Staphylococcal mastitis

Approximately 10% of the total US dairy farm annual milk sales (>$2 billion) is lost to mastitis

NorwegianNorwegian

SelectionSelection

GroupsGroups

LCMHPY

1989-->

-

Cows

from nine

different

herds-

Progeny-tested

sires

(Q)(Q)(q)(q)

((qqqq))((QqQq))

((QqQq))

HMY

1964

•2.5X more milk

•Low Reproductive Potential

•High Clinical Mastitis

1964

1964

2006

Minnesota Minnesota SelectionSelection GroupsGroups

New New projectproject in in NorwayNorway ((part part ofof UMB/UMN UMB/UMN collaborationcollaboration))

““A genome expression profiling strategy A genome expression profiling strategy towards better disease control and improved towards better disease control and improved animal welfareanimal welfare””

FundedFunded by by TheThe ResearchResearch CouncilCouncil ofof NorwayNorwayOneOne researcherresearcher (Siri (Siri KulbergKulberg))OneOne PhDPhD--studentstudent (to be (to be employedemployed))Total Total budgetbudget: 5 mill NOK (800.000 USD): 5 mill NOK (800.000 USD)FahrenkrugFahrenkrug lab to lab to provideprovide microarraysmicroarrays and and bioinformaticsbioinformatics

Siri has Siri has alreadyalready

visitedvisited

FahrenkrugFahrenkrug

lab and lab and willwill

returnreturn

in in OctoberOctober

““Identifying genes controlling milk production and Identifying genes controlling milk production and mastitis susceptibility using mastitis susceptibility using geneticalgenetical genomics genomics ””

Not Not yetyet fundedfundedOneOne PhDPhD--studentstudentTotal Total budgetbudget: $200,000: $200,000SeekingSeeking moneymoney from U from U ofof MN, USDAMN, USDANeedNeed to to ensureensure maintenancemaintenance ofof Control HerdControl Herd

Student to Student to visitvisit

ǺǺs to genotype Minnesota animals?s to genotype Minnesota animals?

New New projectproject in Minnesota?in Minnesota? ((part part ofof UMN/UMB UMN/UMB CollaborationCollaboration))