Genotype,haplotype Quantitative Study And Non-invasive Prenatal Diagnosis

As the Human Genome Project was completed and the costs of sequencing continuously reduced,genotype and haplotype analysis have become two basic technical means to solve crucial problems.On the basis of optimizing the accuracy of genotype and haplotype analysis,we focused on the application of genotyping and haplotyping in related fields,mainly including the field of non-invasive prenatal testing(NIPT)for quantitative analysis of fetal genotypes.In the first part,we used high-throughput sequencing and gene beadchips as tools to perform whole-genome sequencing and genotyping with an average depth of more than100× on 4 healthy human samples.We discussed the analysis method of sequencing data and established an accurate and effective genotyping pipeline.Subsequently,we adopted this strict genotyping pipeline to genotype the entire genomes of 6 individuals in 2pedigrees,and performed haplotype analysis through the sequencing results of 6individuals to establish a valid haplotyping pipeline;then we used haplotype quantification to verify the copy number variation detected in single cells,confirming that～1/100 of the X chromosome copy number variation in female individual single cells is a maternal or paternal haploid loss,which also verified the accuracy of our haplotype quantitative analysis.Last,after establishing the preliminary quantitative analysis methods of genotype and haplotype,we applied them to the optimization of the basic theoretical model of NIPT;we used haplotype results of 4 families to carry out the whole fetal genotype analysis.The genotype analysis achieved an accuracy of 99.99%.In the second part,which is the establishment of NIPT technology,we focused on the current principle application of NIPT in clinic: clinical testing of monogenic diseases.First,for pedigrees at high cardiovascular monogenic defect risk,we adopted the targeted enrichment method by probe capture,optimized its capture specificity and unbiased feature,improved the contamination-free character and effective performance of highdepth sequencing.We established targeted-seq suitable for low-fraction fetal genotype detection with high specificity,low bias and clean background,which solved the failure of conventional targeted-seq in low-fraction fetal detection.Second,we used titration samples to simulate the pregnant woman,calculated fetal fraction and fetal genotype likelihood ratio,verified with titration source sample genotypes,and constructed theoretical model of NIPT.The cutoff of clinical valid depth was set at 1000× for nonmaternal mutations and 3000× for maternal mutations by theoretical calculation of model sensitivity.Third,as for performance of clinical samples,836 samples from 303 pedigrees at high cardiovascular monogenic defect risk were determined with 86% detection rate of pathogenic locus among which 27 pedigrees had immediate NIPT requirements.We performed 100% accurate detection of fetal inheritance status in these 27 cases,including7 cases of maternal mutations,6 cases of paternal mutations and 14 cases of first-born mutations at risk of germ cell mosaicism.Based on our in-home database of normal samples and pathogenic gene annotation pipeline,a noise-free detection was performed in 6 de novo mutation cases;4 in 6 were effectively attributable to the pathogenic locus,and the left 2 cases had no pathogenic mutations either in NIPT or in the fetus.Through technical optimization,model establishment and clinical verification,a clinically friendly NIPT program for monogenic diseases was finally constructed.