通过牛津纳米孔（Oxford Nanopore）自适应测序结合新开发的生物信息学流程探索循环肿瘤细胞的基因突变

Circulating tumor cells (CTCs) represent an important and easily accessible biological material for monitoring treatment response and early detection of patients with cancer. In this study, we aimed to develop a precision workflow combining immunomagnetic bead- and negative selection-based enrichment of CTCs with the Nanopore adaptive sequencing technology to detect mutations in cancer-relevant genomic regions and to understand the intrinsic properties of CTCs. PowerMag system which we developed in our previous studies was used to deplete leukocytes and enrich CTCs from blood sample followed by whole genome amplification. The amplified DNA was subject to Nanopore adaptive sequencing using the Oxford Nanopore GridIon device and the BED file to define 76 common cancer gene mutation target regions. A bioinformatic analytical platform was also developed in-house for efficient extraction of high-confidence reads. The confidence for the presence of mutated nucleotides was determined by implementing a logistic calculation based on the quality score of the indicated nucleotides. The workflow has been validated by using human peripheral blood leukocytes as the biological source of DNA. In addition, preliminary analysis of OECM-1 oral cancer cells spiked into human peripheral blood also reveals that CTCs enrichment followed by adaptive sequencing and the use of analytical tools developed in-house are applicable to identify cancer-associated gene mutation even with a few numbers of reads spanning the gene mutation regions. Taken together, the combined platform represents a new tool for real-time, cost-effective genomic profiling of rare CTCs, with potential applications in the clinical management of cancer patients.