Abstract: Objective To evaluate the potential application value of circulating tumor cells (CTCs) combined with tissue dual template sequencing method in the treatment monitoring and medication guidance of non-small cell lung cancer (NSCLC) patients. 
Methods This study applied a sequential sorting and enrichment system (Ep LMB/Vi LMB) modified with epithelial cell adhesion molecule (EpCAM, Ep) and vimentin (Vi) to achieve efficient capture of CTCs. Based on this technology platform, CTC Deoxyribonucleic acid (DNA) was integrated with tissue DNA for analysis, and a dual template sequencing system was constructed. CTC testing was performed on peripheral blood samples of 26 enrolled patients, with tissue samples additionally obtained from 5 patients and fixed in formalin and embedded in paraffin for next-generation sequencing (NGS). 
Results The CTC count of lung cancer patients was significantly higher than that of the healthy control group ［(34.6±5.2) vs. (3.8±1.4), P＜0.001］. The dual template sequencing technology was used to detect an average of 2.44 tumor specific gene variations per case, which could identify 28.7% more low-frequency mutations than single sample sequencing, and the difference was statistically significant (P＜0.05). Simultaneously, dual template sequencing not only detected all expected variations in tissue sequencing, but also other variations that were not detected in tissue sequencing. 
Conclusion The capture technology based on multi-target LMB can overcome the limitations of traditional EpCAM dependent methods and achieve effective enrichment of CTCs during epithelial mesenchymal transition. The dual source (tissue+CTC) sequencing workflow not only covers all gene variations detected by tissue biopsy, but also identifies clinically significant mutation sites. 

Key words: neoplastic cells, circulating, liposome magnetic beads, high throughput sequencing