Identification Of PKD2 Gene Mutation In Human Preimplantation Embryos In Vitro Using Targeted Next-generation Sequencing Combined With Targeted Haplotyping

Current and imminent technologies for PGD/PGS include single-cell PCR,fluorescent in sit hybridization（FISH）,array comparative genomic hybridization（arrayCGH）,single nucleotide polymorphism arrays（SNP arrays）and next-generation sequence（NGS）.The disadvantages of the former two methods are laborious,expensive and time consuming and usually need specific locus and pedigree.The latter three methods are the latest single-cell genomics methodologies with high sensitivity,throughput and speed,which would replace PCR and FISH.In recent years,NGS is developing rapidly and more and more applications in the field of medical detection.The technology has a wide range of detection,high throughput and good stability.And it will make the sequencing costs lower and lower.By combining NGS with the capture technology,it is possible to choose the detection area with freedom,further reducing the detection costs.To evaluate the applicability of a new method which combines targeted next-generation sequencing（NGS）with targeted haplotyping in identification of PKD2 gene mutation in human preimplantation embryos in vitro,a proband family with a heterozygous deletion of c.（595₅95+14）delGGTAAGAGCGCGCGA in exon 1 of PKD2 gene was studied.An array-based gene chip to capture all of the exons of 21 genes（including PKD2 gene）,approximately 2,000 SNP（Minor Allele Frequency,MAF> 0.3）loci regions around each gene and X,and Y chromosome specific regions were designed.10 samples including 7 embryos from a PKD2 proband family were tested by targeted next-generation sequencing combined with haplotyping analysis.And Sanger sequencing combined with targeted haplotyping was performed to evaluate the feasibility of the new method.7.09 G data was obtained from 10 samples by targeted NGS.The total number of informative SNPs was 24,142 and 287 of each gene haplotype on average.Haplotyping analysis through some selected informative SNPs of PKD2 showed that three embryos（number3,5,6）were normal with no inheriting mutation haplotypes of PKD2 gene,and the other four embryos（number1,2,4,7）were abnormal with inheriting mutation haplotypes of PKD2 gene.The results was consistent with the Sanger method,and the accuracy was 100%.The sex identification results of targeted NGS also showed that the number 6 embryo probably lost X chromosome,which was further confirmed by array-CGH.From our study,we can infer that targeted next-generation sequencing combined with targeted haplotyping can be used to identification of PKD2 gene mutation in human preimplantation embryos in vitro with high sensitivity,fidelity,throughput and speed.