Live births following Karyomapping of human blastocysts: experience from clinical application of the method  Michalis Konstantinidis, Renata Prates, N-Neka Goodall, Jill Fischer, Victoria Tecson, Tsion Lemma, Bo Chu, Amy Jordan, Erin Armenti, Dagan Wells, Santiago Munné  Reproductive BioMedicine Online  Volume 31, Issue 3, Pages 394-403 (September 2015) DOI: 10.1016/j.rbmo.2015.05.018 Copyright © 2015 Reproductive Healthcare Ltd. Terms and Conditions

Figure 1 Explanation of ‘key’ and ‘non-key’ informative single nucleotide polymorphism (SNP) loci. (A) Paternal informative SNP in region of interest. Each paternal haplotype in this example shows informative ‘key’ SNP and ‘non-key’ SNP. As shown in the diagram, each ‘key’ SNP locus has a unique allele linked with one of the paternal haplotypes, and can therefore clearly indicate which paternal haplotype was inherited by the embryo. In contrast, the genotype of ‘non-key’ SNP loci involves paternal alleles that have the same genotype as maternal alleles. As a result, such loci are more prone to erroneous results caused by phenomena such as allele dropout (ADO), e.g. in certain occasions a ‘key’ SNP might be incorrectly detected as ‘non-key’ due to ADO at the specific locus (instead of the original AB genotype [B being the unique paternal allele] only A alleles are detected). Even in situations in which erroneous results might be obtained for some of the SNP, however, the BlueFuse Multi software is able to provide accurate diagnosis by using the cumulative result of all the SNP at the region of interest and especially, the ‘key’ SNP. (B) Maternal informative SNP in region of interest. As with the paternal haplotypes, each maternal haplotype has its own ‘key’ and ‘non-key’ SNP used by the software to determine inheritance in assessed embryos. Reproductive BioMedicine Online 2015 31, 394-403DOI: (10.1016/j.rbmo.2015.05.018) Copyright © 2015 Reproductive Healthcare Ltd. Terms and Conditions

Figure 2 Conventional PCR test and Karyomapping test results obtained for an embryo tested for autosomal dominant polycystic kidney disease. (A) Karyomapping results for embryo 3. A detailed view of the PKD1 gene is shown along with 2Mb upstream and downstream of the gene. The top chromosome shown indicates paternal inheritance (always shown in blue and red colours). The chromosome below that indicates maternal inheritance (always shown in orange and green colours). The dots on top of each chromosome denote ‘key’ SNP, the dots below each chromosome indicate ‘non-key’ SNP and white dots in the middle of each chromosome indicate SNP that were not called. Owing to lack of additional family members' DNA, embryo 4 (diagnosed as unaffected by conventional polymerase chain reaction testing [PCR]) was used as reference. Embryo 4 in this case had inherited the normal copy of the gene from the affected female (orange colour). On the basis of this information, embryo 3 was diagnosed as unaffected since the immediate areas upstream and downstream of the gene indicated inheritance of the normal copy of the gene (orange colour). Owing to the high SNP coverage of the area, the recombination event that occurred could be clearly identified, and was found to be about 0.7Mb away from the gene. Contrary, when the conventional PCR method with short tandem repeats analysis was carried out (B), results were unclear and embryo was diagnosed as ‘recombinant’. Although recombination was detected, it was unclear where exactly it took place and therefore, it was not possible to give a diagnosis to the embryo. Although the mutation site indicated only inheritance of the normal allele, this could well be due to allele dropout of the mutant allele and consequently, no risk could be taken to provide any diagnosis to this embryo based on just the PCR test results. Reproductive BioMedicine Online 2015 31, 394-403DOI: (10.1016/j.rbmo.2015.05.018) Copyright © 2015 Reproductive Healthcare Ltd. Terms and Conditions