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 discussion about morning topics 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

Chasing the genetic basis of a QTL in chicken DJ de Koning

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

The observed trait is sum of many genes and environmental factors Complex Traits

Qq Detection of QTL using exotic crosses QQ qq QQ Qqqq 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.

Intermezzo: tool for QTL analysis

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 genomics Output QTL: Animal QTLdb Chicken 3162 QTL from 158 papers Pigs 6818 QTL from 290 papers Cattle 5920 QTL from 330 papers From QTL to QTN Pigs IGF2 Cattle DGAT1, 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 X

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

Very important meat quality trait Related to activity on the slaughter line Here measured 15 minutes post mortem PH in chicken meat

F. Ricard, 1975 Nadaf et al 2007 Chicken High growth Line, Low growth Line

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

700 F2 24 F2 RNA 12 Microarray chips (Agilent 44k) Genetic information Genomic information 12 QQ 12 qq Targeted genetical genomic approach

Enriched signals at the QTL position

Closer look at the QTL area QTL appears to act on a region < 1Mb

Top 10 ProbeNameGeneNametP.Valueadj.P.Val Alternative Gene name A_87_P016951CR E E-06 ZFY A_87_P RCJMB04_23c E E-06 ACOT9 A_87_P030344BU E PRDX4 A_87_P034725BU E A_87_P014256CR E KLHL15 A_87_P011383CR E KLHL15 A_87_P032384BU E PRDX4 A_87_P006189TC E MSL3L1 A_87_P025536BU E APOO A_87_P034683BU E PRDX4

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 SNPs analysis in non- coding regions SNPs analysis in coding regions CpG islands UTR regions Missense in exons Get gene data from Ensembl Candidate genes with candidate SNPs List of top rank SNPs Splicing sites

Non-synonymous mutations

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

ENSGALT

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