Single-cell genome-wide concurrent haplotyping and copy-number profiling through genotyping-by-sequencing in bovine embryos

Single-cell whole-genome haplotyping allows simultaneous detection of haplotypes associated with monogenic diseases, chromosome copy-numbering and subsequently, has revealed mosaicism in embryos and embryonic stem cells. Methods, such as karyomapping and haplarithmisis, were deployed as a generic and genome-wide approach for preimplantation genetic testing (PGT) and are replacing traditional PGT methods. While current methods primarily rely on SNP array, we envision sequencing-based methods to become more accessible and cost-efficient. Here, we developed a novel sequencing-based methodology to haplotype and copy-number profile single cells. Following DNA amplification, genomic size and complexity is reduced through restriction enzyme digestion and DNA is genotyped through sequencing. This single-cell genotyping-by-sequencing (scGBS) is the input for haplarithmisis, an algorithm we previously developed for SNP array-based single-cell haplotyping. We established technical parameters and developed an analysis pipeline enabling accurate concurrent haplotyping and copy-number profiling of single cells. We demonstrate its value in human blastomere and trophectoderm samples as application for PGT for monogenic disorders. Furthermore, we demonstrate the method to work in other species through analyzing blastomeres of bovine embryos. Our scGBS method opens up the path for single-cell haplotyping of any species with diploid genomes and could make its way into the clinic as a PGT application. A total of nine samples belonging to two bovine pedigrees were used to compare a sequencing-based with the SNP array-based approach for concurrent haplotyping and copy-number profiling. The semen from the same bull was used for embryo production in both pedigrees. Ovarian tissue samples from the donor cows (mothers) and semen from the bull (father) were used to extract bulk DNA (DNeasy Blood and Tissue kit, Qiagen, Germany). Ovaries were collected separately and retrieval of the oocytes was followed by routine in vitro embryo production. The embryos were treated with pronase to dissolve zona pellucida and single blastomeres were retrieved from zona free embryos and whole-genome amplified (WGA) using commercial multiple displacement amplification kit (Repli-G Single Cell kit, Qiagen). WGA products and bulk parental and sibling DNA were then SNP genotyped on BovineHD BeadChip arrays using Infinium HD whole-genome genotyping assay (Illumina Inc., USA). Subsequently, the SNP logR and SNP B-allele frequency (BAF) values, as well as the discrete SNP genotype calls were obtained for each single cell and applied in further single-cell analysis to obtain genome-wide haplarithm profiles using a modified version of the siCHILD algorithm (Zamani Esteki et al. 2015).