Every cell in our body has a genome that carries the blueprint of our lives. Our genome is dynamical, i.e., changing with time. Genomic instability gives rise to genetic variations among cells originating from the same lineage, particularly cancer cells. However, we have not yet been able to study such dynamics of genomes because tools are not available, despite the tremendous advances in the next generation of genome sequencing in the past few years. Single cell whole genome amplification and sequencing is highly desirable for characterizing such heterogeneity among cells. However, existing amplification methods, such as PCR or multiple displacement amplification (MDA), are severely limited by strong bias and artifacts such as chimeras. We have developed several strategies that can significantly reduce the bias and allow single cell quantification of genome and transcriptome. We have developed a new whole genome amplification method: Multiple Annealing and Looping Based Amplification Cycle (MALBAC), which greatly circumvents the above difficulties. It allows us to read out digitized copy number variations and identify unique single nucleotide polymorphisms with overall ~80% efficiency of a single cell. We were able to call SNVs with extremely low false positive rates and directly measure the genome-wide mutation rate for the first time. We have also developed a method for digital RNAseq, which will allow determination of a single cell transcriptome with single copy sensitivity and no amplification bias. Cancer is a genetic disease. There have been many theoretical models about the genesis of cancer that have been difficult to test experimentally. Single cell genome sequencing is the ultimate experiment. We propose to characterize the copy number and single nucleotide variations of one hundred individual cells from cancer tissues, from which we will be able to extract information regarding how genetic variations occur in real time in a solid tumor. We also plan to simultaneously determine the genome and the transcriptome of the same cell using the techniques described above. The implication of the proposed research on dynamics of the genome goes beyond cancer research and may have other broad implications to biology and medicine.
We propose to use the latest single cell DNA sequencing technologies developed in our lab to study how our genomes change with time, which is important not only to fundamental biology, but also to understanding of cancer.
