Establishment And Applicability Verification Of A Novel Technology For Single-cell Copy Number Variation Sequencing

Background:With the development of single-cell sequencing technology and growing popularity of single-cell transcriptome,single-cell genomics has received increasing attention from researchers.Currently,most techniques for single-cell copy number variation are based on whole-genome amplification technology,PCR and amplified transposase library construction methods that require genome pre-amplification,microfluidic chips,and special sequencing programs.Such requirements often mean that these techniques are complicated,time consuming and less cost-efficient in terms of labor,machines,and sequencing costs.Among this,existing preimplantation genetic testing method for aneuploidy(including the detection of trace DNA copy number variation of a single cell or few cells on multiple single cells),is not only expensive and complex,but also plagued by lower detection rate of mosaicism and inaccurate interpretation of aneuploidy.Objective:While one of the single-cell sequencing technologies,single-cell copy number variation sequencing technology is a powerful tool in researching tumor evolution,reproductive health,drug screening and disease pathology,it comes with several limitations of being time-consuming,complicated,and expensive.This study aims at addressing this issue by establishing a novel single-cell copy number variation sequencing technology(Medium throughput Single Cell Copy Number Variation Sequencingv,MT-scCNV-seq)to meet growing demand for high-efficiency,high-throughput,and low-cost single-cell technology.In the application of preimplantation genetic testing(PGT),MT-scCNV-seq provides a feasible backup for the high-efficiency and low-cost detection of mosaicism and aneuploidy in third-generation IVF.Successful development of MT-scCNV-seq is expected to be followed by wide application in the field of scientific research and clinical treatment,especially in tumor heterogeneity,occurrence and development,as well as reproductive health.Method:This study uses traditional biochemistry and molecular biology methods combined with bioinformatics analysis.Nucleic acid,enzymology and PCR technologies are deployed to transform and optimize the technology of single-cell copy number variation second-generation sequencing library construction.At the same time,the data optimization,feasibility,and robustness of the MT-scCNV-seq are analyzed with bioinformatics for cell-lines.Finally,MT-scCNV-seq is used to detect preimplantation genetic testing for aneuploidy at single-cell level.Content and process:The research content of this study is divided into two parts.The first part covers the development of a single-cell copy number variation sequencing technology.The main process is to redesign the nucleic acid sequence(combined with the transposase)which carry a specific barcode segment.The segment is later inserted into sample fragments in the interrupting process for library establishment(as part of the second-generation sequencing procedure).The treated samples(single cells)can then be used for pooling in one-step hybrid amplification performed under the micro-reaction system to construct a library.Finally,coupled with the index sequences,MT-scCNV-seq is shown to be able to achieve meduim throughput.The second part of the study focuses on the application and robustness of this method.The main process here is to optimize the single-cell microdroplet selection method and to apply the technology developed to detecting the copy number variation of single cells in preimplantation embryos with known defects.Conclusions:The analysis of K562 proves the correlation between this single-cell technology and Bulk cells under 5K bins to be at least 0.8;the analysis of GM12878 shows the false-positive rate of this technology of 1.32%;the false-negative rate is 1.00%.The mixed single-cell analysis of cell lines and normal human peripheral blood mononuclear cells indicate that MT-scCNV-seq is robust in cell clustering and verification of heterogeneity,especially for cell lines with close relationships between Hela and Hela S3 to exhibit clustering effect.It proves the feasibility,robustness and reliability of the technology.In the detection of copy number variation of 6 scrap embryos before implantation,it is found that MT-scCNV-seq basically matched the detection of copy number variation at the 5K bins scale and can predict the specific situation of mosaicism.It is confirmed that there is severe aneuploidy and a great probability of mosaicism in embryos that trigger developmentally delayed or unsuitable for implantation.