The new technology for non-invasive early diagnosis of liver cancer refers to methylated CpG short tandem amplification and sequencing (MCTA-Seq). This technology is a breakthrough in cancer diagnosis methods by performing comprehensive sequencing analysis of abnormally hypermethylated CpG islands in patients’ plasma free DNA to achieve early diagnosis of liver cancer. DNA methylation is an epigenetic modification in which a methyl cytosine (C) is added to DNA to form a methyl cytosine, and occurs mainly in CpG dinucleotides, which are highly clustered in genomic regions called CpG islands, which are mainly located at the beginning of gene transcription and have important transcriptional regulatory functions. In tumor cells, a large number of CpG islands are aberrantly hypermethylated. aberrant CpG island hypermethylation is not only closely related to tumor development, but also a very promising tumor marker. Necrotic cancer cells release DNA into the blood as circulating free DNA. Can early cancer be detected by detecting free DNA carrying abnormally hypermethylated CpG islands in the blood? Although such attempts have been started as early as the 1990s, research has been slow to progress. One of the key bottlenecks is the lack of high-throughput technology that can detect a large number of CpG islands simultaneously. The free DNA released from early-stage tumors into peripheral blood is extremely small and highly fragmented, and the currently available DNA methylation group detection techniques are low in sensitivity and cannot meet the requirements for high-throughput detection. MCTA-Seq technology cleverly breaks this bottleneck. By selective amplification of methylated CpG short tandem CGCGCGG sequences and subsequent high-throughput sequencing analysis, MCTA-Seq can detect nearly nine thousand CpG islands simultaneously in one reaction; the lower limit of detection can be as low as 1-2 cells of genomic DNA. Liver cancer is one of the most prevalent malignant tumors in the world, with the third highest mortality rate. The incidence of hepatocellular carcinoma has been high in China due to the large number of chronic hepatitis B virus infected patients. Currently, serum alpha-fetoprotein (AFP) is the most commonly used clinical marker for early screening of liver cancer, but has a false-negative rate of about 40%, so there is an urgent need to develop new biomarkers for early screening of liver cancer. The investigators used MCTA-Seq technology to analyze a total of 151 clinical samples, including 57 cancer and paracancer tissue samples and 94 plasma samples from patients with hepatocellular carcinoma, patients with cirrhosis and normal individuals. In hepatocellular carcinoma tissues, they found that nearly nine hundred CpG islands were aberrantly hypermethylated. In contrast, nearly four hundred CpG islands with significantly increased methylation levels were identified in plasma samples from patients with small hepatocellular carcinoma (≤3 cm); further raising the screening criteria resulted in more than forty best-performing plasma CpG island markers. It was found that plasma CpG island markers in hepatocellular carcinoma patients could be divided into two categories, one of which was partially derived directly from hepatocellular carcinoma tissue, while the other was released by both hepatocellular carcinoma tissue and non-cancerous liver tissue. In the plasma of patients with small hepatocellular carcinoma (≤3 cm), the latter group of markers increased even more than the former group. After surgical resection of the tumor, both classes of markers decreased significantly, indicating that they are both closely associated with the tumor. The discovery of the latter group of novel plasma CpG island markers demonstrates the advantages of unbiased screening by MCTA-Seq technology. By combining the two types of plasma CpG island markers, the MCTA-Seq technique has a sensitivity of 94% and a specificity of 89% for the diagnosis of hepatocellular carcinoma. Of particular importance, MCTA-Seq successfully made the correct diagnosis in all 15 patients with hepatocellular carcinoma who presented false-negative AFP in this study. Given that abnormal hypermethylation of CpG islands occurs in most types of tumors, MCTA-Seq technology is also expected to be used for non-invasive early screening of other cancer types. In addition, since different types of tumors have unique methylation profiles, MCTA-Seq also has the potential to identify the origin of tumor tissue. the three-step library building reaction of MCTA-Seq can be completed in a single PCR tube with only superficial sequencing, making it a new technology for free DNA sequencing that is simple, economical and has promising applications.