1. Wednesday from QTL to candidate genes Xidan Li Xiaodong Liu DJ de Koning

2. 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

3. Chasing the genetic basis of a QTL in chicken DJ de Koning dj.de-koning@slu.se

4. 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

5. From sequence to consequence Phenotype

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

7. Qq Detection of QTL using exotic crosses QQ qq QQ Qqqq Use of extreme crosses to unravel complex traits

8. 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

9. Quantitative trait locus (QTL) Region of the genome with a ’significant’ effect on our trait of interest. Large region with very many genes.

10. Intermezzo: tool for QTL analysis www.gridqtl.org.uk

11. 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

12. Challenge remains: What is the gene? Very large area Many candidate genes Very noisy signal Signal may not mark the gene

13. Livestock genomics Output QTL: Animal QTLdb http://www.animalgenome.org/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

14. 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.

15. QTL study AND gene expression study in Population. What are the gene expression effects of this QTL X

16. eQTL: Genome region that affects gene expression

17. Targeted eQTL Mapping Focus expression analysis on most informative individuals eQTL underlying functional QTL Increased power for target regions

18. Application to a chicken QTL

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

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

21. QTL affecting PH QTL Interval ~ 50 cM?

22. 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

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

25. Enriched signals at the QTL position

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

27. Top 10 ProbeNameGeneNametP.Valueadj.P.Val Alternative Gene name A_87_P016951CR385747-16.512.87E-107.19E-06 ZFY A_87_P014348 RCJMB04_23c 19-16.283.42E-107.19E-06 ACOT9 A_87_P030344BU299642-12.648.27E-090.0001 PRDX4 A_87_P034725BU10672911.034.38E-080.0005 A_87_P014256CR39028210.281.02E-070.0009 KLHL15 A_87_P011383CR5237639.253.57E-070.0025 KLHL15 A_87_P032384BU2309948.441.03E-060.0062 PRDX4 A_87_P006189TC2026597.225.78E-060.0304 MSL3L1 A_87_P025536BU4760937.047.56E-060.0353 APOO A_87_P034683BU1084636.899.55E-060.0401 PRDX4

29. 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.

30. 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

31. To be continued YOU will work with this NGS data today! The work up to the NGS has been published

32. Over to you! Then coffee

33. 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

34. Non-synonymous mutations

35. ACOT9

36. 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.”

37. F1NR19

38. ENSGALT0000026337

39. 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