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What is genomics ?

Donor / Embryo Genomics Patrick Blondin L’Alliance Boviteq AABP Embryo Transfer Seminar Montréal, 2012 Sept 19 th. What is genomics ?. A consortium of Universities and North American artificial insemination centers developed the Illumina BovineSNP50 Beadchip .

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What is genomics ?

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  1. Donor / EmbryoGenomicsPatrick BlondinL’Alliance Boviteq AABP Embryo TransferSeminar Montréal, 2012 Sept 19th

  2. Whatisgenomics? • A consortium of Universities and North American artificial insemination centers developed the Illumina BovineSNP50 Beadchip. • This Single Nucleotide Polymorphism (SNP) DNA chip contains more than 43 000 SNPs (this can be seen as 43 000 potential mutation between different individuals). • The pattern of SNPs obtained following SNP50 hybridization have been correlated to production, fertility and health traits. • Genomic values are then generated and used to rank animals in terms of genetic potential.

  3. How to use GENOMICS to increasegenetic gain ?

  4. Option #1 (current situation): Mating of eliteanimals Transfers Calvesgenotyping to keep the best subjects

  5. Option #2 (future…?): Mating of eliteanimals Embryogenotyping to transferonly the mostpromisingembryos

  6. Getting DNA for genotyping Bovine SNP50 Hybridization DNA Extraction Bovine SNP50 Hybridization Impossible…. 10-12 cells in embryo biopsies 10 000 times lower thanrequirements for Bovine SNP50 hybridization DNA Extraction

  7. Solution? Pre-amplification of genomic DNA recoveredfrom biopsies > 10 000 foldbefore SNP50 hybridization AMPLIFICATION STEP • Differentmethods: • PCR based • Isothermal amplification • Plenty of commercial kits: • Qiagen • Nugen • Sigma • New EnglandBiolabs • GE healthcare GENTLEDNA « Liberation » • Release DNA • Decompact DNA • Avoid DNA breakage • Avoidloosingmaterial

  8. Be carefulwith commercial kits Most of themweredesigned for biggersamplesthanembryo biopsies • EFFICIENT (Enough DNA for SNP50 hybridization) • INNACURATE (Toomuchinconsistencies VS startingtemplate) 10X 10 000X 100X 1000X An EFFICIENT and ACCURATE methodisabsolutelyrequired

  9. Filling the holes… • Evenwithoptimized conditions, somediscrepanciesbetween the end results and the startingtemplace are foundfollowing the amplification. • IMPUTATION isthen a indispensable tool to fill the holes in the genome of the amplified biopsies generated by the amplification. • Imputation is done through FImputeV2.0, a software developped in house and optimized for embryo biopsies. This software performs a combined family and population imputation and reconstruct the genome using the data generated from the parents on the 50K.

  10. Efficiency of the method • Wedeveloppedourownprocedure for DNA extraction and amplification to get the mostaccuratecoverage of the genomefromembryo biopsies. • Phi-29 basedisothermal amplification • So far: • 681 out of 709 biopsies weresuccessfullyamplified (96%efficiency) • Call rate (% of SNPsthatgenerated a signal on the SNP50 array): 91 ± 5% • 583 sampleswere sent to USDA/CDN for genomicevaluation and 572 (98%) passed USDA/CDN qualitycontrols and generatedresults

  11. Variation intra embryo flushes • Some crosses generated very variable Direct Genomic Values (DGV) between different embryos (flushes #1 and #2) • For others, the DGV obtained were very similar (flush #3). • In some cases, very large divergences (1842 pts of DGV) were found between two embryos of a same flush (flush #4).

  12. Parentage possible • Multiple sires can be used for one embryo production in good donors since pedigree validation is part of the process and then, sire identification is executed. It is then possible to try many crosses in a shorter period of time while being able to know the pedigree of each embryo.

  13. Evolution of DGVsduringpregnancy • Parental average and genomic values may change over time. • What is the effect of those changes on the genomic on the embryos since the data are generated 9 months before birth… • Even if variations were observed between both groups, the overall pattern remained the same as the best embryos of the cohort were still the best embryos 9 months later.

  14. DGV differences between embryo and corresponding calf: • The first calves born from embryos genotyped were genotyped to measure the accuracy of our amplication method (Figure 4). • A mean divergence of 106 ± 68 pts of DGV was calculated for the first 25 samples (4.3 ± 3.6% from the DGV).

  15. Importance of accurate genotype and imputation • 42 503 SNPsanalysed in the 25 Calf-Embryo pairs • Before imputation: • 4909 ± 1190 missingSNPs (holes) • 5286 ± 1439 missing and WRONGS SNPs • After imputation: • Basically, all missingSNPs (holes in the genome) are filled! • However, someerrorspersist:266 ± 188 wrong calls.

  16. Amplification accuracy and genotypeerrors • After amplification and hybridization, the % of calledSNPsis an indicator of the quality of the overallgenotypegenerated. • Indeed, the call rate decreases, the % of errors in the SNPsgeneratedincreases!

  17. Impact of amplification quality on the accuracy of imputation • Errorsgenerated in the amplification impacts the imputation: • When call rate decreases (amplification of lowerquality), the # of divergent SNPsbetweenamplifiedmaterial and corresponding calf increases!

  18. Genotypequality VS Genomicevaluation • Amplification quality impacts: • Exactitude of the SNPsgenerated • Accuracy of the imputation results • What are the consequences on the DGV? • Better the amplification is, more reliableis the DGV

  19. Conclusion • A robust and accurate amplification procedure has been developped to generate high quantities of DNA fromembryo biopsies. • The amplified DNA canbeused for hybridization on the BovineSNP50 beadchip to generategenomic bovine evaluationfromembryos. • Even if some changes happenduringpregnancy, the best embryos of a flush remain the best of their cohort over time and thus, decisions made at the embryonic level are still valid at the time of calving. • Very small divergences were observed between the genomic evaluations predicted from the embryos and the ones obtained from the resulting calves. • This confirmed the accuracy of the amplificationand imputation methods developed by our group.

  20. Warnings… • Imputation « repairs » a lot of errorsgeneratedduring the amplification. • However, itsaccuracyisimpaired by poorqualitygenotype. • Therefore, biaised amplification impacts the final genomicevaluation. • Plenty of factorscould impact the amplification: • Bad biopsy (toosmall, degradedmaterial) • DNA extraction • Method of amplification • Commercial kits (none of themperformedwellenough to beused as it)

  21. Opportunities… • Genomic at the embryoniclevelisnow possible! • This technologycanbecombined to embryofreezingso breeders canacceleratetheirgenetic gain by mutiplying crosses in a short period of time and onlytransferembryoswithhigherpotential. • Using multiple sires per session with good donorsmultiply the chances of gettingvery high profiles embryos. • Finally, this amplification procedurecouldbe use for any diagnostic test involving DNA directly at the embryoniclevel.

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