The Establishment And Evaluation Of High-throughput Sequencing Method Based On Single Cell Whole-genome Amplification To Simultaneously Detecting Point Mutation And Copy Number Variation

Objective:A single cell whole genome amplification was performed by biopsy of discarded blastomere embryos combined with isolation of peripheral blood mononuclear leukocytes.To study the effects of high-throughput sequencing library construction methods and different sequencing depths on DNA high-throughput sequencing’s sequencing coverage,sensitivity,specificity,etc.Establish a high-throughput sequencing method which can detect gene mutations and chromosomal copy number variations based on single-cell whole genome amplification.Optimize the processes of single-cell whole genome amplification products for high-throughput sequencing to diagnose the chromosomal diseases and single genetic diseases simultaneously in the same detection system.Provide a more comprehensive,economical and accurate method for preimplantation genetic diagnosis of chromosomal diseases and monogenic diseases.Methods:Peripheral blood single leukocytes and discarded blastomere embryos were isolated or biopsied under stereoscopic microscope.The peripheral blood was collected from β-thalassemia patients with known mutations.Subsequently,single cell whole genome amplification was performed,and amplificated the HBB gene target region using the single cell whole genome amplification product as PCR templates.Two methods were used to construct a high-throughput sequencing library:(1)Single-cell whole-genome amplification products and PCR products were mixed with the ratio of 1: 99;3: 97;5: 95 and 10: 90 respectively,and then high-throughput sequencing libraries was constructed;(2)Constructed highthroughput sequencing libraries with single-cell whole genome amplification products and PCR products separately,then mixed the two sequencing libraries with the ratio of 1: 99;3: 97;5: 95 and 10: 90 respectively.High-throughput sequencing was performed at 0.1× and 2× sequencing depth separately.By comparing the detection efficiency of target mutations,the coverage depth and coverage rate of chromosome,and the homogeneity of chromosome copy number variation detection in each detection scheme,establish and optimize the processes of single-cell whole genome amplification products for high-throughput sequencing to diagnose the chromosomal diseases and single genetic diseases.Finally,validated this method with five discarded blastomere embryos and five peripheral blood single leukocytes which were collected from β-thalassemia patients with known mutations.Results:1.The sensitivity and specificity of the detection of target mutations for all protocols were 100%.2.With an increase in the proportion of single-cell whole genome amplification products,the scatter plots of chromosome copy number variation detection were more centralized,and the average chromosome coverage depth and coverage rate could be increased to 10 times.3.The scatter plots of chromosome copy number variation detection of assays which used the second method of library construction were more centralized than that of method one,and the average coverage depth and coverage rate of chromosomes were 2.2 times higher on average.4.When high-throughput sequencing was performed at 2× sequencing depth,the scatter plots detected for chromosome copy number variation were more centralized than 0.1× sequencing depth,and the average chromosome coverage depth and coverage rate were 3 times higher on average.Conclusion:1.All the detection protocols constructed in this study can accurately detect the target mutations.2.With the increase of the proportion of single-cell whole genome amplification products,the average coverage depth and coverage rate of chromosomes were higher,and the homogeneity of chromosome copy number variation detection was better.3.The detection protocols adopted the second method for library construction,the average coverage depth and coverage rate of chromosomes as well as the homogeneity of detection of chromosome copy number variation were better than that of the first library construction method.4.When high-throughput sequencing was performed at 2× sequencing depth,the average coverage depth and coverage rate of chromosomes as well as the homogeneity of detection of chromosome copy number variation were better than that of 0.1× sequencing depth.In summary,the detection protocol constructed in this study that constructed high-throughput sequencing libraries with single-cell whole genome amplification products and PCR products separately,then mixed the two sequencing libraries with the ratio of 10: 90,performed high-throughput sequencing at 2× sequencing depth can detect the gene mutations and chromosomal copy number variations simultaneously in the same detection system.