| Background and objectiveRare cells have important biological significance,such as trophoblast cells and circulating tumor cells(CTCs),and play an important role in noninvasive prenatal screening and detection of tumor metastasis.Single-cell genome sequencing is dependent on whole genome amplification(WGA)due to the extremely low amount of genomic DNA in cells.With single-cell WGA,the complete genome information of the target cell can be obtained and detailed genetic analysis of the cell type can be performed.However,the current single-cell WGA technology has some amplification bias due to base bias and other problems,which causes the the expected coverage and uniformity not to be achieved.Multiple annealing and looping-based amplification cycles(MALBAC)are the most widely used single-cell WGA methods at present,considering the fidelity and uniformity of single-cell WGA.With the development of genetic engineering,the plasmid-cloning technique of transforming receptive cells has been widely used.At present,the application of plasmid-cloning technology in single-cell WGA products has not been reported.This study innovatively adopted plasmid linked cloning combined with MALBAC to evaluate the feasibility of this combined method to improve the coverage and quality of WGA products in rare cells.Methods1.To verify the transformation process of plasmid-cloning and evaluate the change of coverage before and after cloningIn this study,54 cases of WGA products from trophoblast cells and CTCs were investigated.Using plasmid-cloning technology,the WGA products of single cells were transferred into Escherichia coli DH5α competent cells by plasmid vector pMDTM19-T for prolifertion.Primers of 22 specific loci in 22 autosomes were designed for amplification,and the transformation of plasmid cloning was verified according to the amplification sites.The coverage of single-cell WGA products before and after cloning was calculated according to the positive rate of amplification sites.Then the paired t-test and scatter plot were used for statistical analysis.2.The quality changes of WGA products before and after plasmid-cloning were preliminarily evaluated2.1 Sanger sequencing was used to evaluate whether the samples before and after cloning had advantages in point mutation detection.2.2 Short Tandem Repeats(STR)were used to evaluate the changes in overall sample quality before and after cloning.2.3 Next generation resequencing was used to evaluate the changes in overall uniformity of samples before and after cloning,as well as the changes in CV values of common thalassemia gene rustle and common tumor sites.Results1.After cloning,the mean coverage of single-cell WGA products was improved(pre-clone 73%,post-clone 79%),especially for trophoblast cells(pre-clone 72%,post-clone 81%).2.Among the 12 randomly selected samples,the peak map quality and successs rate of Sanger sequencing of the cloned samples were better than those before cloning.3.In addition,the number of detected STR loci in the cloned samples was significantly higher than those before cloning in the two randomly selected samples.The number of STR loci in sample 23 was increased from 3 to 15 after cloning.4.In the randomly selected samples,the resequencing results of samples 38,40 and 41 after cloning showed that the amplification uniformity of the products was higher than that before cloning,and the CV values of sample 38 after cloning were lower in the common thalassemia and tumor locus regions.ConclusionCompared with traditional WGA methods,considering that rare cells cannot be wasted,plasmid-cloning combined with MALBAC not only maximized the utilization of cells and avoided waste,but also improved the coverage,quality and uniformity of single-cell WGA products to a certain extent,which will contribute to further exploring the biological significance and clinical value of rare cells. |
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