Single Cell Genome Analysis Platform Development in Blood Sample

Abstract

In this thesis, I introduce the development of single cell genome analyzing platform for blood sample. By using this platform, I observed abnormality of down syndrome by analyzing genome of 5 cells from real patient blood. The number of cells required for genome analysis is important because less number implies high possibility of detecting unique markers. To develop this platform, I improved targeted single cell isolating method and optimized whole genome amplification method. First, to isolate targeted cell, we need to observe the cell through microscope and isolate them right away. Conventional method burns surrounding of the targeted cells laid on polyethylene naphthalate film. I used indium tin oxide as a sacrificial layer and infrared pulse laser for intact cell isolation. At the same time, since this sacrificial layer has high physical and chemical durability similar to slide glass, modification of general protocol to observe cells are not required. Optimization of whole genome amplification improved uniformity of amplification of low input genome. Amplifying whole genome with low amplification bias is critical for high quality of genome analysis. Conventional protocol of whole genome amplification using isolated cells were not enough for high quality genome analysis. By optimal cell retrieving, cell lysis and pre-denaturation step improved the amplification efficiency. As the result, we could obtain uniform amplification product. In order to confirm this platform is applicable for various usage, I demonstrated with 2 cell lines and 2 blood sample from hereditary disorder patients. Since staining and storing decrease quality of genome which result in low uniformity of amplification, we had to find minimum cell number for reliable amplification product. We measured initial amplification rate, PCR success rate and positive rate of target sequencing and directly analyzed genome coverage by low depth whole genome sequencing. We found out 25 cells were minimum cell number required for reliable genome analysis using giemsa stained cells. I believe this novel platform is ready to use for wide usage for discovering biologically significant points, which was unrevealed by high noise from heterogeneous cell population.