Thursday from QTL to candidate genes Xidan Li Xiaodong Liu DJ de Koning.
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Transcript of Thursday 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
• 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
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
Detection of QTL using exotic crosses
QQ qq
QQ Qq qq
Use of extreme crosses to unravel complex traits
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.
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
• Many candidate genes
• Very noisy signal
• Signal may not mark the gene
Livestock genomicsOutput
•QTL: Animal QTLdbhttp://www.animalgenome.org/QTLdb/
•Chicken3162 QTL from 158 papers
•Pigs6818 QTL from 290 papers
•Cattle5920 QTL from 330 papers
• From QTL to QTN
• PigsIGF2
• CattleDGAT1, ABCG2
…
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.
• QTL study AND gene expression study in Population.
• What are the gene expression effects of this QTL
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Targeted eQTL Mapping
• Focus expression analysis on most informative individuals
• eQTL underlying functional QTL
• Increased power for target regions
• Very important meat quality trait
• Related to activity on the slaughter line
• Here measured 15 minutes post mortem
PH in chicken meat
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
700 F2
24 F2
RNARNA
12 Microarray chips (Agilent 44k)
Genetic information
Genomic information
12 QQ 12 qq
Targeted genetical genomic approach
Top 10
ProbeName GeneName t P.Value adj.P.ValAlternative Gene name
A_87_P016951 CR385747 -16.51 2.87E-10 7.19E-06 ZFY
A_87_P014348RCJMB04_23c
19 -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
Enriched signals at the QTL position
• 16 differentially expressed gene 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