Identifying Multiple Genomic Abnormalities and Predicting Neoantigens from Single Tumor Cells [PRJNA1110294]

Tumors exhibit high heterogeneity due to distinct genomic aberrations in individual cells. Despite this, no methods currently exist to detect these genomic abnormalities at the single-cell level. To address this, we developed Uniform Chromosome Conformation Capture (Uni-C), an efficient method that precisely detects a variety of genomic anomalies in single cells, including single nucleotide polymorphisms (SNPs), insertions and deletions (INDELs), copy number variations (CNVs), structural variations (SVs), and focal amplifications such as extrachromosomal DNA (ecDNA) and homogeneously staining regions (HSRs). Utilizing Uni-C, we characterized varied structural variations and detailed the structure of ecDNA in circulating tumor cells (CTCs), highlighting their extensive heterogeneity. We also observed differences in chromatin conformation across CTCs in mitosis and interphase, potentially serving as markers for cell vitality. Additionally, by using genomic data from Uni-C, we detected driver gene mutations in CTCs and predicted neoantigens, significantly advancing early cancer detection and treatment strategies. To maintain the stability of chromatin spatial conformation throughout the experiment, we first employed EGS (ethylene glycol bis (succinimidyl succinate)) and formaldehyde for dual crosslinking of the cells, followed by lysis to release the nuclei. To prevent the aggregation of nuclei due to centrifugation, we excluded all centrifugation steps in later experimental stages. We used a 4-base cutter restriction endonuclease to enzymatically fragment the chromatin, conducting end-repair and proximity ligation within the same reaction setup. After proximity ligation, we transferred individual nuclei into an alkaline lysis buffer using a glass capillary, setting the stage for further single-nucleus amplification. For amplification of single nuclei, we utilized a heat-resistant phi29 DNA polymerase along with a mixture of dNTPs and Alpha Thiol-modified ddNTPs, and exonuclease-resistant random primers. During the extension process, the encounter with Alpha Thiol-modified ddNTPs terminates the extension reaction, thus controlling the size of the amplification products to be below 2 kb. The resulting shorter amplified products make it difficult for random primers to rebind, allowing only the original single-cell genome to serve as a template for further amplification. This prevents over-amplification of certain genomic regions and achieves uniform whole-genome amplification, significantly enhancing genome coverage. Moreover, the addition of the fast, heat-resistant phi29 polymerase has reduced the amplification time to approximately 2 hours. The amplified products from single nuclei were then subjected to size selection, followed by the preparation of libraries for high-throughput sequencing.