Let's start with the tumor. It is difficult to treat tumors thoroughly, not only because of the complexity of their genomes, but also because of the heterogeneity extended by their complexity. Heterogeneity is actually a phenotypic attribute caused by the complexity of tumor genome. Heterogeneity is usually discussed in two situations. The first case refers to the heterogeneity of tumor cells in the same patient. The gene mutation of tumor cells is different in different stages of tumorigenesis, resulting in many subgroups in each tumor cell population. When tumor cells undergo metastasis, tumor cells belonging to different subgroups will invade new places and form new tumors. It is necessary to introduce the concept of CTC (circulating tumor cells). As shown in the following figure 1, some people think that CTC is the main inducement of tumor metastasis caused by primary tumor cells. CTC cells have many characteristics that ordinary tumor cells do not have, such as being larger, more "dry", or easier to enter EMT (epithelial interstitial) pathway. Studies have shown that CTC has the following relationship with the development of tumor:1.The number of CTC can be used as a marker); Inferring the development of tumor; Second, a large amount of CTC in the blood will accelerate the progress of tumor and reduce the time of tumor recurrence; Thirdly, CTC can also be used as a clinical index to guide the treatment process. However, such important cells are difficult to study because they are difficult to obtain, because their content in blood is extremely small. For example, in patients with advanced breast cancer, only 1.43% patients have more than 500 CTC in every 7.5 ml of blood. This means that there are many obstacles to the research of CTC, because there are too few cells. The second situation is that, in addition to the heterogeneity of tumor caused by different tumor cells of the same patient, the heterogeneity of tumor is also reflected in the fact that different patients may have the same tumor, but the "similarity" is not necessarily the same-just the same phenotype does not mean the same genotype. Figure 2 below shows the response of tumor heterogeneity. Different colors represent different tumor subgroups, and different tumor subgroups invade different places to form new branches of tumor "evolution" (which should be understood as development), thus producing tumor heterogeneity; And there is heterogeneity among tumors obtained from different patients.
Before discussing why we should use single-cell genome sequencing to solve the problem, we need to sort out the problems we face again: 1. The tumor genome is too complex, with many mutations, different mutations in different periods and serious heterogeneity. If a large number of tumor cell genomes are still sequenced, the mixed results will often interfere with judgment. 2. The content of CTC, an important cell group related to tumor metastasis, is very small in blood, and there is heterogeneity among different CTCs-so on the one hand, it is difficult to obtain a large number of CTC cell genomes, on the other hand, even if relevant information is obtained, it still cannot explain the problem.
In order to solve these two problems and provide more accurate personalized treatment for cancer patients, the author found that single cell sequencing can really solve these problems (at least conceptually at present).
The earliest method was the research group led by Roger Lasken, which optimized and established the first generation of MDA (Multiplex Displacement Amplification) kit. This technology uses the patented Phi29 DNA polymerase from Yale University. The enzyme has the characteristics of multiple substitutions-in the reaction, the extension of the latter primer can exceed the previously bound DNA without being blocked by it; The enzyme also has strong template DNA binding ability, and can continuously synthesize products with the length from 10 kb to 50 kb, with the longest reaching 100 KB. At the same time, it has the exonuclease activity of 3'-5' nucleic acid and the ability of self-repairing errors, so it has high fidelity. However, due to the extremely small amount of DNA in the initial genome, some fragments (such as GC) will be directly amplified, so there will be a strong amplification bias, which will reduce the coverage of the genome, and MDA method can not solve this problem well. 20 12 academician Xie Xiaoliang, a tenured professor at Harvard university, developed a new single-cell genome sequencing method-malbac (multiple annealing and loop-based amplification cycle). It can greatly reduce the amplification preference. Let me briefly introduce its principle, as shown in the figure below.
The core step of MALBAC method: still use the enzyme in MDA method (which can be used to replace the original extended chain). But the primer used is designed in advance, and this primer is randomly combined and extended on the genome.