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Wednesday
from QTL to candidate genes
Xidan Li
Xiaodong Liu
DJ de Koning
Overview of today
• Schedule for teaching day
• Morning Lectures
• 9:00 – 10:00 Lecture: Chasing the genetic basis of a QTL in chicken –
DJ de Koning
• 10:00 – 10:15 coffee break
• 10:15 – 10:45 Lecture: Bioinformatics pipeline for targeted sequencing
of QTL region – Xiaodong Liu
• 10:45 – 11:00 leg stretcher
• 11:00 – 11:30 Lecture: Identification and evaluation of causative genetic
variants – Xidan Li
• 11.30-12.00 discussion about morning topics
• 12.00-13.00 lunch
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•
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Afternoon exercises
13:00 – 14:00 NGS data aligning
14:00 – 15:00 SNPs calling
15:00 – 16:00 Identify and evaluate causative genetic variants
16:00 – 17:00 evaluate results and questions
Chasing the genetic basis of a
QTL in chicken
DJ de Koning [email protected]
Contributors
• Swedish University of Agricultural Sciences, Uppsala University
• Xidan Li
• Xiaodong Liu
• Roslin Institute, University of Edinburgh
•
•
•
•
Javad Nadaf
Ian Dunn
Chris Haley
Ark-Genomics: Alison Downing, Mark Fell, Frances Turner
• INRA, Unité de Recherches Avicoles
• Cécile Berri
• Elizabeth Le Bihan-Duval
From sequence
to consequence
Phenotype
Complex Traits
The observed trait is sum of many genes
and environmental factors
Detection
QTL using
exotic crosses
Use of of
extreme
crosses
to unravel complex traits
qq
QQ
Qq
QQ Qq qq
Detection of ”QTL”
• Use heritable variation in the
genome as DNA Markers
• Follow inheritance of DNA
markers through population
• Compare inheritance pattern
with character of interest
Quantitative trait locus (QTL)
• Region of the genome with a ’significant’ effect on
our trait of interest.
• Large region with very many genes.
Intermezzo: tool for QTL analysis
www.gridqtl.org.uk
Nowadays: Association studies
• Take a large (thousands), representative,
sample of the population
• Characterise for a very large number of DNA
variants
• Estimate a putative
effect of every DNA
variant on the trait
of interest
Challenge remains:
What is the gene?
• Very large area
• Very noisy signal
• Many candidate genes
• Signal may not mark
the gene
Livestock genomics
Output
•QTL: Animal QTLdb
http://www.animalgenome.org/QTLdb/
• Chicken
3162 QTL from 158 papers
• Pigs
• From QTL to QTN
• Pigs
IGF2
• Cattle
DGAT1, ABCG2
6818 QTL from 290 papers
…
• Cattle
5920 QTL from 330 papers
1000’s of QTL, very few QTN
Next step up:
Gene expression studies
• Measure the expression of thousands of genes
simultaneously
• Snapshot of what is happening in a given tissue
at a given time.
X
50
Test Statistic
40
30
20
10
0
0
50
100
150 cM
• QTL study AND gene expression
study in Population.
• What are the gene expression
effects of this QTL
50
Test Statistic
40
30
20
10
0
0
50
100
150 cM
eQTL: Genome region
that affects gene expression
Targeted eQTL
Mapping
• Focus expression analysis on most
informative individuals
• eQTL underlying functional QTL
• Increased power for target regions
Application to a chicken QTL
PH in chicken meat
• Very important meat
quality trait
• Related to activity on
the slaughter line
• Here measured 15
minutes post mortem
Chicken High growth Line, Low growth Line
F. Ricard, 1975
Nadaf et al 2007
QTL affecting PH
QTL Interval ~ 50 cM?
Experimental design
• What are the local and global effects of this QTL
on gene expression?
• Identify 12 birds with QQ genotypes on the basis
of flanking markers and 12 with qq genotype
• Perform microarray analyis using mRNA from
breast muscle (P. Major)
• Agilent 44k Array: 2-colour, dye-balanced
Targeted genetical genomic approach
700 F2
24 F2
12 QQ
12 qq
12 Microarray chips (Agilent 44k)
RNA
RNA
Enriched signals at the QTL
position
Closer look at the QTL area
QTL appears to act on a region < 1Mb
Top 10
t
P.Value
adj.P.Val
Alternative Gene
name
CR385747
RCJMB04_23c
A_87_P014348
19
-16.51
2.87E-10
7.19E-06
ZFY
-16.28
3.42E-10
7.19E-06
ACOT9
A_87_P030344
BU299642
-12.64
8.27E-09
0.0001
PRDX4
A_87_P034725
BU106729
11.03
4.38E-08
0.0005
A_87_P014256
CR390282
10.28
1.02E-07
0.0009
KLHL15
A_87_P011383
CR523763
9.25
3.57E-07
0.0025
KLHL15
A_87_P032384
BU230994
8.44
1.03E-06
0.0062
PRDX4
A_87_P006189
TC202659
7.22
5.78E-06
0.0304
MSL3L1
A_87_P025536
BU476093
7.04
7.56E-06
0.0353
APOO
A_87_P034683
BU108463
6.89
9.55E-06
0.0401
PRDX4
ProbeName
GeneName
A_87_P016951
Enriched signals at the QTL
position
• 16 differentially expressed probes in 1Mb region around
QTL
• QTL acting at chromatin or methylation level?
• PH simply one of the downstream effects.
Next Step: Re-Sequencing
the QTL region
•
5 birds of each QTL genotype
•
Selected DNA from 1 Mb around QTL with Agilent
SureSelect Target Enrichment
•
One lane on Illumina GA flow cell: 151 bp paired-end
•
4.9 Gbase of raw sequencing reads
•
~200 x coverage of each individual chicken
To be continued
• YOU will work with this NGS data today!
• The work up to the NGS has been published
Over to you!
• Then coffee 
Process for identifying
candidate SNPs
Re-sequencing with
200 X
SNPs calling
Get gene data from
Ensembl
SNPs analysis in noncoding regions
Splicing
sites
CpG
islands
Candidate genes
with candidate
SNPs
SNPs analysis in coding
regions
UTR regions
Missense in
exons
List of top rank
SNPs
Non-synonymous
mutations
ACOT9
Description: acyl-Coenzyme A thioesterase 2, mitochondrial [Source:RefSeq
peptide;Acc:NP_001012841]
Mutations:
QQ
coordinate in chr
121615732
position in proteins
290
ref codon VS. Mutated
codon
[ TAC / TAT V / I ]
121615675
121615732
309
290
[ CGC / CGA A / S ]
[ TAC / TAT V / I ]
qq
Mutations predictions:
A - S (309)
V - I (290)
conservaton score
0.08
0.09
physico-chemical properties
0.54
0.201
final effect
0.3726
0.201
All scores are in range (0 - 1). The higher score, the more effect to protein function.
ACOT9
• Most significant gene from eQTL study
• Mitochondrial gene
• Function of this particular gene not clear.
• “Acyl-CoA thioesterases are a group of enzymes
that catalyze the hydrolysis of acyl-CoAs to the
free fatty acid and coenzyme A (CoASH),
providing the potential to regulate intracellular
levels of acyl-CoAs, free fatty acids and CoASH.”
F1NR19
Description: DNA polymerase Source: UniProtKB/TrEMBL F1NR19
Mutations:
QQ
coordinate in chr
121338134
121464309
121466769
121478463
position in proteins
1396
523
466
289
ref codon VS. Mutated
codon
[ CGT / CAT T / M ]
[ AGG / AAG P / L ]
[ CGC / CAC A / V ]
[ ATG / ACG H / R ]
121464309
121466769
523
466
[ AGG / AAG P / L ]
[ CGC / CAC A / V ]
qq
Mutations predictions:
H - R (289)
A - V (466)
P - L (523)
T-M
(1396)
conservaton score
0.04
0.05
0.11
physico-chemical properties
0.543
0.304
0.703
final effect
0.02172
0.0152
0.07733
0.03
0.412
0.01236
ENSGALT0000026337
PRDX4
• Peroxiredoxin 4
• Antioxidant enzyme, regulates NFĸB
• Highly differentially expressed but no candidate
SNPs
• 2 probes up, 1 down => Splicing?
• Still a strong functional candidate