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.  This Single Nucleotide Polymorphism (SNP) DNA chip contains more than SNPs (this can be seen as 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.

How to use GENOMICS to increase genetic gain ?

Option #1 (current situation): Mating of elite animals Transfers Calves genotyping to keep the best subjects

Option #2 (future…?): Mating of elite animals Embryo genotyping to transfer only the most promising embryos

Getting DNA for genotyping DNA Extraction DNA Extraction Bovine SNP50 Hybridization Bovine SNP50 Hybridization DNA Extraction DNA Extraction Bovine SNP50 Hybridization Bovine SNP50 Hybridization Impossible… cells in embryo biopsies times lower than requirements for Bovine SNP50 hybridization Impossible… cells in embryo biopsies times lower than requirements for Bovine SNP50 hybridization

Solution? Pre-amplification of genomic DNA recovered from biopsies > fold before SNP50 hybridization Pre-amplification of genomic DNA recovered from biopsies > fold before SNP50 hybridization GENTLE DNA « Liberation » GENTLE DNA « Liberation » Different methods: PCR based Isothermal amplification Plenty of commercial kits: Qiagen Nugen Sigma New England Biolabs GE healthcare Release DNA Decompact DNA Avoid DNA breakage Avoid loosing material

Be careful with commercial kits 10X 100X 1000X X EFFICIENT (Enough DNA for SNP50 hybridization) INNACURATE (Too much inconsistencies VS starting template) EFFICIENT (Enough DNA for SNP50 hybridization) INNACURATE (Too much inconsistencies VS starting template) Most of them were designed for bigger samples than embryo biopsies An EFFICIENT and ACCURATE method is absolutely required

Filling the holes…  Even with optimized conditions, some discrepancies between the end results and the starting templace are found following the amplification.  IMPUTATION is then a indispensable tool to fill the holes in the genome of the amplified biopsies generated by the amplification.  Imputation is done through FImpute V2.0, 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.

Efficiency of the method  We developped our own procedure for DNA extraction and amplification to get the most accurate coverage of the genome from embryo biopsies.  Phi-29 based isothermal amplification  So far:  681 out of 709 biopsies were successfully amplified (96% efficiency)  Call rate (% of SNPs that generated a signal on the SNP50 array): 91 ± 5%  583 samples were sent to USDA/CDN for genomic evaluation and 572 (98%) passed USDA/CDN quality controls and generated results

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).

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.

Evolution of DGVs during pregnancy  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.

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).

Importance of accurate genotype and imputation  SNPs analysed in the 25 Calf-Embryo pairs  Before imputation:  4909 ± 1190 missing SNPs (holes)  5286 ± 1439 missing and WRONGS SNPs  After imputation:  Basically, all missing SNPs (holes in the genome) are filled!  However, some errors persist: 266 ± 188 wrong calls.

Amplification accuracy and genotype errors  After amplification and hybridization, the % of called SNPs is an indicator of the quality of the overall genotype generated.  Indeed, the call rate decreases, the % of errors in the SNPs generated increases!

Impact of amplification quality on the accuracy of imputation  Errors generated in the amplification impacts the imputation:  When call rate decreases (amplification of lower quality), the # of divergent SNPs between amplified material and corresponding calf increases!

Genotype quality VS Genomic evaluation  Amplification quality impacts:  Exactitude of the SNPs generated  Accuracy of the imputation results  What are the consequences on the DGV?  Better the amplification is, more reliable is the DGV

Conclusion  A robust and accurate amplification procedure has been developped to generate high quantities of DNA from embryo biopsies.  The amplified DNA can be used for hybridization on the BovineSNP50 beadchip to generate genomic bovine evaluation from embryos.  Even if some changes happen during pregnancy, 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 amplification and imputation methods developed by our group.

Warnings…  Imputation « repairs » a lot of errors generated during the amplification.  However, its accuracy is impaired by poor quality genotype.  Therefore, biaised amplification impacts the final genomic evaluation.  Plenty of factors could impact the amplification:  Bad biopsy (too small, degraded material)  DNA extraction  Method of amplification  Commercial kits (none of them performed well enough to be used as it)

Opportunities…  Genomic at the embryonic level is now possible!  This technology can be combined to embryo freezing so breeders can accelerate their genetic gain by mutiplying crosses in a short period of time and only transfer embryos with higher potential.  Using multiple sires per session with good donors multiply the chances of getting very high profiles embryos.  Finally, this amplification procedure could be use for any diagnostic test involving DNA directly at the embryonic level.