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

Abstract

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 single-nucleotide polymorphism (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.

Preimplantation genetic testing: A remarkable history of pioneering, technical challenges, innovations, and ethical considerations

“…NGS coupled to whole genome amplification emerged as a powerful new technology for PGT that could enable genome‐wide analysis of gene mutations using single cells or embryo biopsies, and often in combination with other methods. NGS can allow detection of single gene mutations, aneuploidies, structural rearrangements, and even distinction between normal embryos and those bearing balanced chromosomal translocations, potentially at lower cost than some other methods (He et al, 2022; Kuliev & Rechitsky, 2017; Masset et al, 2022; Niu et al, 2020; De Rycke & Berckmoes, 2020; Treff et al, 2013; Zhang et al, 2017). Low‐coverage NGS providing sequencing outputs for a limited set of markers across the genome has been employed for PGT‐SR (Zhai et al, 2022).…”

msCNVS: medium throughput single cell copy number variation sequencing with barcoded library construction free of preamplification toward clinical implementation

“…These efforts include degenerate oligonucleotide-primed PCR (DOP-PCR) 13 , multiple displacement amplification (MDA) 14 , multiple annealing and looping-based amplification cycles (MALBAC) 15 , and linear amplification via transposon insertion (LIANTI) 16 . These preWGA-based scCNV-seq methods enable the processing of the genome of single cells, after WGA, and have been applied widely for scCNV-seq with many fruitful results 17 . Some WGA methods are less biased and give faithful CNV calling, including examples eMDA 18 , MIDAS 19 , LIANTI 16 .…”

Genome-wide equine preimplantation genetic testing enabled by simultaneous haplotyping and copy number detection

“…However, PGT for horses remains underdeveloped as compared to humans or cattle with no publications on PGT-A, and few studies on PGT-M, limited to the interrogation of a maximum of 33 loci related to diseases or traits, including sex, ID markers, coat color, based on (multiplex) targeted PCR 3 , 55 – 58 . A generic method allowing for concurrent, genome-wide PGT-A, PGT-M and PGT-SR with the potential for PGT-P 59 – 61 , as successfully applied for humans 25 , 32 , 62 and cattle 27 , 31 , would increase the potential of PGT for both clinical and research-based equine applications.…”