functional genomics to advance dairy cattle health · functional genomics. bioinformatics. bovine...
TRANSCRIPT
<-
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
++
÷÷
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
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))