Figure 2: Principles of family-specific haplotyping and embryo analysis based on SNP genotyping of Sanger sequencing.

Family-specific haplotype analysis based on SNP genotyping of the parents and paternal grandmother in five candidate SNPs. “P” represents “paternal grandmother”, “F” represents “father”, “M” represents “mother”, “F1” represents “haplotype 1 of the father”, “F2” represents “haplotype 2 of the father”, “M1” represents “haplotype 1 of the mother” and “M2” represents “haplotype 2 of the mother”. The five candidate SNPs were selected from the SNPs of the targeted NGS that were also confirmed to have stable and quality results in the direct detection of embryos by Sanger sequencing. The five SNP genotypes of the father were heterozygous, and both the paternal grandmother and mother were homozygous but had different SNP genotypes. By Mendelian analysis of the SNP genotypes of the parents and the paternal grandmother, it is possible to identify the four parental haplotypes. The SNP loci colored in red and green represent the informative SNPs that could determine the unique haplotype. The informative SNPs colored in red were from haplotype 1 of the father, and the green SNPs were from haplotype 2 of the father (A). “E4” represents “embryo 4”, and “E6” represents “embryo 6”. The two embryos illustrate the principle of embryo haplotype analysis. The results from the embryo SNP genotypes were obtained from Sanger sequencing. The SNP loci coding embryos 1 and 5 are represented by two letters because they were heterozygous. The SNP loci coding embryos 2, 3, and 4 are represented by one letter, likely due to ADO. Embryo 4 was determined to have an abnormality that resulted from inheritance of mutation haplotype 1 from the father. Embryo 6 was normal (B).