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Thursday from QTL to candidate genes

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

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Thursday from QTL to candidate genes

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  1. Thursdayfrom QTL tocandidate genes Xidan Li Xiaodong Liu DJ de Koning

  2. Overviewoftoday • Schedule for teachingday • MorningLectures • 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 targetedsequencingof QTL region – Xiaodong Liu • 10:45 – 11:00 leg stretcher • 11:00 – 11:30 Lecture: Identification and evaluationofcausativegenetic variants – Xidan Li • 11.30-12.00 discussionaboutmorningtopics • 12.00-13.00 lunch • Afternoonexercises • 13:00 – 14:00 NGS data aligning • 14:00 – 15:00 SNPs calling • 15:00 – 16:00 Identify and evaluatecausativegenetic variants • 16:00 – 17:00 evaluateresults 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 RecherchesAvicoles • Cécile Berri • Elizabeth Le Bihan-Duval

  5. From sequence to consequence Phenotype

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

  7. Detection of QTL using exotic crosses qq QQ Qq Useof extreme crossestounravelcomplextraits QQ Qqqq

  8. Detectionof ”QTL” Useheritable variation in the genome as DNA Markers Followinheritanceof DNA markers through population Compareinheritancepatternwithcharacterofinterest

  9. Quantitativetraitlocus (QTL) Region of the genomewith a ’significant’ effect on ourtraitofinterest. Large region withverymany genes.

  10. Intermezzo: tool for QTL analysiswww.gridqtl.org.uk

  11. Nowadays: Association studies Take a large (thousands), representative, sampleof the population Characterise for a verylargenumberof DNA variants Estimate a putative effectofevery DNA variant on the trait ofinterest

  12. Challenge remains: What is the gene? • Verylarge area • Manycandidate genes • Verynoisy signal • Signal may not mark the gene

  13. 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 … Livestock genomicsOutput 1000’s of QTL, very few QTN

  14. Next step up: Gene expression studies Measure the expression ofthousandsof genes simultaneously Snapshot ofwhat is happening in a given tissue at a given time.

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

  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. Applicationto a chicken QTL

  19. PH in chickenmeat Veryimportantmeatqualitytrait Relatedtoactivity on the slaughterline Heremeasured 15 minutes post mortem

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

  21. QTL affecting PH QTL Interval ~ 50 cM?

  22. Experimental design • Whatare the local and global effectsofthis QTL on gene expression? • Identify 12 birdswith QQ genotypes on the basis offlanking markers and 12 withqqgenotype • PerformmicroarrayanalyisusingmRNA from breastmuscle (P. Major) • Agilent 44k Array: 2-colour, dye-balanced

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

  24. Enriched signals at the QTL position

  25. Closer look at the QTL area QTL appearstoact on a region < 1Mb

  26. Top 10

  27. 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.

  28. Next Step: Re-Sequencing the QTL region • 5 birdsofeach QTL genotype • Selected DNA from 1 Mb around QTL with Agilent SureSelect Target Enrichment • Onelane on Illumina GA flow cell: 151 bppaired-end • 4.9 Gbaseofrawsequencingreads • ~200 x coverageofeachindividualchicken

  29. To be continued • YOU willworkwiththis NGS data today! • The workupto the NGS has beenpublished

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