Application of single cell sequencing technology in liver regeneration / 中华消化外科杂志

The liver is a highly proliferative organ. As the liver injured, the hepatocytes can quickly enter the cell cycle to restore the volume and function of liver. Liver regeneration involves complex processes that depend on the interaction of many different cell types. As limited by the average cell change level in tissues, traditional sequencing methods can only acquire the average genetic information reflecting dominant cell subpopulations, but ignore the secondary cell subpopu-lations, which leads to the loss of cellular heterogeneity information. Single-cell sequencing tech-nology can analyze the biological behavior of single cell, which helps to better understand the distri-bution, interaction and cell heterogeneity of different cells during liver regeneration. The authors review the application of single cell sequencing technology in liver regeneration.

Progress of single-cell sequencing in lymphoma / 白血病·淋巴瘤

Single-cell sequencing (SCS) sequences the genetic information of a single cell to better understand the differences amongst cells and reveal the unique changes of each cell type. The specific analysis of cell subsets at the single-cell level can accurately evaluate tumor cells and microenvironment cells to reveal the complexity of molecular components and the difference from the corresponding components in non-malignant tissues. Lymphoma is highly heterogeneous, some have unknown pathological types, etiology and poor prognosis. SCS is helpful to clarify the molecular mechanisms of lymphomagenesis and pathological staging, and guide clinical practice. This article reviews SCS and its application in lymphoma.

Visual Analysis of Research on Lung Cancer Immunotherapy by Using CiteSpace / 肿瘤防治研究

Objective To understand the current status of research on lung cancer immunotherapy to provide a reference for further investigation and future topic selection in this field. Methods CiteSpace visualization analysis software was used to analyze 400 Chinese studies in CNKI and 5 001 English studies in the Web of Science database from 2005 to 2021, with "lung cancer" and "immunotherapy" as keywords. Keyword co-occurrence analysis was performed on 17 English studies of "Lung Cancer" "Immunotherapy" and "Single cell sequencing" in the Web of Science database. Results "Non-small cell lung cancer" "immunosuppressants" "PD-L1" "dendritic cells" and "cytokine-induced killer cells" are current research hotspots in lung cancer immunotherapy. Monoclonal antibody drugs including nivolumab, pembrolizumab, atezolizumab, and durvalumab are hotspot drugs. Immunotherapy combined with chemotherapy as well as PD-L1 expression have become the focus of continuous research. The majority of studies on lung cancer immunotherapy are conducted in the United States, followed by China. Conclusion Lung cancer immunotherapy has gradually become a research hot spot in China. In the future, in-depth research is needed to provide cutting-edge directions for lung cancer immunotherapy.

Multiplexed single-cell transcriptome analysis reveals molecular characteristics of monkey pluripotent stem cell lines / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Efforts have been made to establish various human pluripotent stem cell lines. However, such methods have not yet been duplicated in non-human primate cells. Here, we introduce a multiplexed single-cell sequencing technique to profile the molecular features of monkey pluripotent stem cells in published culture conditions. The results demonstrate suboptimized maintenance of pluripotency and show that the selected signaling pathways for resetting human stem cells can also be interpreted for establishing monkey cell lines. Overall, this work legitimates the translation of novel human cell line culture conditions to monkey cells and provides guidance for exploring chemical cocktails for monkey stem cell line derivation.

Comparison of pretreatment techniques for single-cell RNA sequencing of airway aspirate specimens / 中华微生物学和免疫学杂志

Objective:To investigate the appropriate pretreatment methods for single cell RNA sequencing of airway aspirate cells.Methods:Four fresh airway aspirate specimens were collected from four patients with acute respiratory tract infections. These specimens were digested with airway aspirate digester and prepared into single cell suspension. The cells were used for library construction directly (DE), or fixed with 10×Genomics Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit and then mixed to construct the library (DF), or cryopreserved, thawed, fixed (FF) before mixed to construct the library. All three methods were treated with oil emulsion using 10 4 cells and subjected to single-cell sequencing using the 10×Genomics platform. The number of obtained cells, data quality, annotated cell types and expression of marker genes were analyzed. Differences in the expression of highly variable genes (HVGs) of the same cell subsets obtained by the three pretreatment methods were compared using Pearson correlation. Expression of the differentially expressed genes in the same cell subpopulation obtained by different pretreatment methods was also compared. The correlation of the expression of differentially expressed genes between the same cell subsets obtained by the three pretreatment methods was analyzed by Pearson correlation. Results:The median numbers of single cells obtained using DE, FF and DF methods were 2 733, 1 140 and 5 897 ( P>0.05). The unique molecular identifiers were higher than 500. The median numbers of genes obtained using the three methods were 801, 887 and 1 259 ( P>0.05). The cells with novelty score over 0.8 accounted for 99%, 87% and 93%, respectively. There were nine cell subsets obtained by the three methods, including squamous cells, secretory cells, ciliated cells, T cells, B cells, macrophages, plasma cells and neutrophils. DF and FF methods could obtain more basal cells with specific high expression of keratin 5 than DE method. The differentially expressed and highly variable genes in the same cell subsets obtained by the three pretreatment methods showed high consistency in their expression with a significant correlation ( P<0.001). Conclusions:Under the same sequencing data volume, the quality of data obtained from fixed airway aspirate single-cell suspensions using the method of probe hybridization and transcriptome sequencing was comparable to that obtained directly from fresh cells. This method was more suitable for the pretreatment of clinical samples used for single-cell RNA sequencing.

Resolving the lineage relationship between malignant cells and vascular cells in glioblastomas

Glioblastoma multiforme (GBM), a highly malignant and heterogeneous brain tumor, contains various types of tumor and non-tumor cells. Whether GBM cells can trans-differentiate into non-neural cell types, including mural cells or endothelial cells (ECs), to support tumor growth and invasion remains controversial. Here we generated two genetic GBM models de novo in immunocompetent mouse brains, mimicking essential pathological and molecular features of human GBMs. Lineage-tracing and transplantation studies demonstrated that, although blood vessels in GBM brains underwent drastic remodeling, evidence of trans-differentiation of GBM cells into vascular cells was barely detected. Intriguingly, GBM cells could promiscuously express markers for mural cells during gliomagenesis. Furthermore, single-cell RNA sequencing showed that patterns of copy number variations (CNVs) of mural cells and ECs were distinct from those of GBM cells, indicating discrete origins of GBM cells and vascular components. Importantly, single-cell CNV analysis of human GBM specimens also suggested that GBM cells and vascular cells are likely separate lineages. Rather than expansion owing to trans-differentiation, vascular cell expanded by proliferation during tumorigenesis. Therefore, cross-lineage trans-differentiation of GBM cells is very unlikely to occur during gliomagenesis. Our findings advance understanding of cell lineage dynamics during gliomagenesis, and have implications for targeted treatment of GBMs.

Application progress of single cell sequencing technology in diagnosis of genetic and reproductive / 国际生物医学工程杂志

The technique of prenatal diagnosis before embryo implantation has been greatly developed in assisted reproduction, especially for the people with abnormal genetic material to provide the technical possibility of eugenics and eugenics. As an emerging sequencing technology, single-cell sequencing can analyze the genome and transcriptome of cells from the level of a single cell, and reflect the heterogeneity between cells, thus helping to reveal the mechanism of the occurrence and development of diseases. Through prenatal diagnosis before embryo implantation and high-throughput sequencing of single cells obtained from embryo biopsy, euploidy of embryonic chromosomes can be effectively detected, and SNPs and chromosomal copy number variation, insertion, deletion and other variations can also be better detected. It can conduct the comprehensive detection and research of genomic polymorphism and mutation of individual and other species. In this paper, the single-cell sequencing technology and relevant methods was introduced, and the application scenarios of single-cell sequencing in genetic reproduction diagnosis were summarized. The applications of the technology in the field of genetic reproduction were described, and the future directions of the technology were discussed.

Research progress of single cell sequencing technique in predicting the immunotherapeutic efficacy of lung cancer / 国际生物医学工程杂志

Globally, lung cancer is the leading cause of cancer death. Although survival rates for lung cancer patients have improved over the past few decades, the survival rates have not yet reached the levels of other common malignancies. In recent years, immune checkpoint inhibitors (ICIs) have shown great promise in clinical trials and have been rapidly incorporated into the standard of care for patients with advanced non-small cell lung cancer (NSCLC). However, many patients do not benefit from treatment. Our findings suggest that the heterogeneity of the tumor microenvironment (TME) is closely related to the efficacy of ICIs. Single-cell sequencing is a technology that can specifically analyze cell populations at the genome and transcriptome levels at the single-cell level. This article reviews the potential value of single-cell sequencing technology in predicting immune responses to lung cancer.

Aging weakens Th17 cell pathogenicity and ameliorates experimental autoimmune uveitis in mice

Aging-induced changes in the immune system are associated with a higher incidence of infection and vaccination failure. Lymph nodes, which filter the lymph to identify and fight infections, play a central role in this process. However, careful characterization of the impact of aging on lymph nodes and associated autoimmune diseases is lacking. We combined single-cell RNA sequencing (scRNA-seq) with flow cytometry to delineate the immune cell atlas of cervical draining lymph nodes (CDLNs) of both young and old mice with or without experimental autoimmune uveitis (EAU). We found extensive and complicated changes in the cellular constituents of CDLNs during aging. When confronted with autoimmune challenges, old mice developed milder EAU compared to young mice. Within this EAU process, we highlighted that the pathogenicity of T helper 17 cells (Th17) was dampened, as shown by reduced GM-CSF secretion in old mice. The mitigated secretion of GM-CSF contributed to alleviation of IL-23 secretion by antigen-presenting cells (APCs) and may, in turn, weaken APCs' effects on facilitating the pathogenicity of Th17 cells. Meanwhile, our study further unveiled that aging downregulated GM-CSF secretion through reducing both the transcript and protein levels of IL-23R in Th17 cells from CDLNs. Overall, aging altered immune cell responses, especially through toning down Th17 cells, counteracting EAU challenge in old mice.

Application of single cell sequencing technology in atherosclerosis research / 中华神经科杂志

Atherosclerosis is one of the most common cardiovascular and cerebrovascular diseases, and it is also an important cause of stroke. However, the research on the pathogenesis of atherosclerosis is still incomplete. Single cell technology, as an emerging technology in the study of differences in cell biology, has become a new tool and provides a new way of exploring the etiology of atherosclerosis. This article reviewed the research progress of single cell sequencing technology in atherosclerosis in recent years.

Bioinformatics based analysis of key genes underlying the development of nonalcoholic fatty liver disease / 中华内分泌代谢杂志

Objective:To identify key genes and their potential biological mechanisms in the progression of non-alcoholic fatty liver disease (NAFLD) using bioinformatics technology.Methods:Genes differentially expressed in simple non-alcoholic fatty liver disease (NAFL) and non-alcoholic steatohepatitis (NASH) were analyzed by integrating NAFLD-related sequencing datasets GSE135251 and GSE167523 from the Gene Expression Omnibus (GEO) datebase. Gene Ontology (GO) functional enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome signaling pathway analysis were performed. Key genes were identified by STRING database and Cytoscape3.7.2 software, and the expression of key genes under different fibrosis grades and activity scores was observed. In addition, the expression of key genes in different cell clusters was observed based on the single-cell RNA-seq dataset of NAFLD mice.Results:Bioinformatics methods were used to obtain 97 common differential genes in NAFLD from two datasets. GO functional enrichment analysis was mainly performed in Extracellular Matrix (ECM) tissues. The main signaling pathway is ECM-receptor interaction. Five key genes were identified based on PPI network and Cytoscape software: COL1A1, THBS2, CXCL8, THY1 and LOXL1. The expression of key genes was significantly positively correlated with fibrosis grade and activity score, indicating that they were closely related to the progression of NAFLD. These key genes are highly expressed in hepatic stellate cells (HSCs) and natural killer/T cells (NK/T cells).Conclusion:In this study, bioinformatics technology was used to identify five key genes that may be involved in the NAFL-NASH transformation, suggesting that the ECM-receptor interaction signaling pathway may be a key molecular mechanism of NAFLD disease progression.

Application of single cell sequencing technology in the cell analysis of cerebrospinal fluid / 中华检验医学杂志

The analysis of cerebrospinal fluid (CSF) facilitates the diagnosis and therapy of central nervous system (CNS) diseases. Compared to traditional methods, single-cell sequencing is conducive to analyze the cells composition and heterogeneity and discover scarce cells in CSF. Recent studies of single-cell sequencing in CSF has mainly focused on the neuroinfectious diseases, neurodegenerative and neuroinflammatory diseases, and leptomeningeal metastases (LM), reflecting the superiority and clinical value of single-cell sequencing in CNS diseases and providing new directions for the diagnosis and treatment of CNS diseases.

Single cell RNA sequencing of bone marrow mononuclear cells from elderly patients with hip fracture / 中华创伤骨科杂志

Objective:To determine whether subpopulations may exist which are related to regulation of immunization or inflammation in bone marrow mononuclear cells (BMMNCs) from 2 elderly patients with hip fracture and whether there might be any difference in the subpopulations between them.Methods:Two elderly patients with hip fracture were enrolled in this study. Their venous blood was harvested to determine subpopulations of complement (C)3, C4, interleukins (IL)-2, IL-6, IL-10, and lymphocytes. Single cell RNA sequencing (scRNA-Seq) was used to group their BMMNCs. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed of the top 20 differentially expressed genes for each subpopulation to judge the main function of each subpopulation. The subpopulations and the key genes related to regulation of immunization or inflammation were found out. The relationships were explored between subpopulations and prognosis of the patients.Results:By the venous blood indexes, IL-10 was slightly high in patient A; C3 below normal, C4 close to the lower normal limit, IL-2, IL-6 and IL-10 were significantly high, CD8 +T % was low, and CD4 +/CD8 + high in patient B. After scRNA-Seq and bioinformatics analyses, the BMMNCs in the 2 patients were divided into 5 subpopulations. GO and KEGG enrichment analyses showed that the functions of subpopulation 2 and subpopulation 4 were related to immunization or inflammation. CCL4, CCL5, LTB and CXCR4 in subpopulation 2 and C1QA, C1QB, CD14 and SPP1 in subpopulation 4 were related to the regulation of immunization or inflammation. The final prognosis of patient A was much better. The proportions of BMMNCs involved in subpopulation 2 and subpopulation 4 from patient A were higher than those from patient B [47.00% (1,431/3,045) versus 29.28% (882/3,012); 5.88% (179/3,045) versus 3.85% (116/3,012)]. Conclusions:The BMMNCs from elderly patients with hip fracture can be divided into subpopulations by scRNA-Seq. Some of the subpopulations may be related to regulation of immunization or inflammation, which may affect the post-injury immune inflammatory state and prognosis of the patients.

Clinical importance of screening differential gene set of monocytes based on single-cell sequencing and digital polymerase chain reaction technology for early diagnosis of sepsis / 中华危重病急救医学

Objective:To verify the specific differentiated subsets of monocytes in sepsis, and to screen and construct the differential gene set of monocytes used for early diagnosis of sepsis.Methods:Patients with sepsis admitted to Guangdong Provincial People's Hospital from June 2020 to March 2021 were enrolled, and peripheral blood mononuclear cells (PBMC) were extracted. Single-cell sequencing technology and pseudo-time analysis were used to verify the differential subsets of monocytes. Bioinformatics methods were used to analyze the expression of genes in differential subsets of monocytes and screen out differential genes for the preliminary construction of a candidate differential gene set. The digital polymerase chain reaction (PCR) technology was used to verify the candidate differential genes in PBMC of sepsis patients and sepsis human myeloid leukemia mononuclear cells (THP-1) models, and the Venn diagram was used to construct the final differential gene set of monocytes. Gene Expression Omnibus (GEO) database was used to validate the differential gene set of monocytes.Results:① The results of cell annotation and pseudo-time analysis showed that the differentiation of NEAT1 +CD163 + monocyte occurred in the early stage of sepsis was significantly different from other subsets, which validated that NEAT1 +CD163 + monocyte was the characteristic subset in the pathological process of sepsis. ② Twenty-two differential genes related to sepsis were screened out from the gene expression of NEAT1 +CD163 + monocyte. After further verification by digital PCR, basic leucine zipper ATF-like transcription factor (BATF), JUNB proto-oncogene, carcinoembryonic antigen-related cell adhesion molecule 4 (CEACAM4), chromosome 9 open reading frame 95 (C9orf95), G protein subunit alpha 15 (GNA15), complement C3a receptor 1 (C3AR1), transforming growth factor beta 1 (TGFB1) and mitochondrial carrier homolog 1 (MTCH1) were screened out to construct the final differential gene set of monocytes. ③ The external validation results showed that C9orf95 gene had no data in GSE154918 and GSE133822 from GEO, it was excluded during validation. In GSE154918, the expressions of BATF, JUNB, CEACAM4, GNA15, C3AR1, TGFB1, and MTCH1 in the sepsis group were significantly higher than those in the healthy control group (log 2expression level: BATF was 12.78±0.08 vs. 11.39±0.35, JUNB was 16.88±0.07 vs. 16.04±0.03, CEACAM4 was 14.73±0.08 vs. 13.77±0.05, GNA15 was 13.16±0.06 vs. 12.30±0.04, C3AR1 was 14.62±0.13 vs. 12.87±0.05, TGFB1 was 16.95±0.05 vs. 16.57±0.36, MTCH1 was 14.80±0.02 vs. 14.61±0.15, all P < 0.05). In GSE133822, the expressions of BATF, CEACAM4, GNA15, and C3AR1 in the sepsis group were significantly higher than those in the health control group (log 2expression level: BATF was 8.66±0.16 vs. 7.92±0.14, CEACAM4 was 9.20±0.16 vs. 8.36±0.20, GNA15 was 10.66±0.18 vs. 10.13±0.16, C3AR1 was 11.49±0.27 vs. 10.48±0.16, all P < 0.05), while the expressions of JUNB, TGFB1, and MTCH1 were not statistically different between two groups. The results of gene set variation analysis (GSVA) showed that the enrichment scores of monocytes differential gene set of sepsis group were significantly higher than those of the healthy control group in both GSE154918 (0.38±0.04 vs. -0.44±0.02) and GSE133822 (0.56±0.02 vs. 0.20±0.05, both P < 0.01). Receiver operator characteristic curve (ROC curve) analysis showed that the differential gene set of monocytes had a reliable diagnostic value for early sepsis with the area under ROC curve (AUC) of 0.993 [95% confidence interval (95% CI) was 0.980-1.000] in GSE154918 and 0.944 (95% CI was 0.873-1.000) in GSE133822. Conclusion:A differential gene set of monocytes (BATF, JUNB, CEACAM4, GNA15, C3AR1, TGFB1, and MTCH1) screened out by single-cell sequencing and digital PCR technology has a reliable diagnostic value for the early sepsis, and may provide a new idea for the early diagnosis of sepsis.

Research highlights of renal transplantation in 2020: voice from China / 器官移植

Renal transplantation is the optimal approach to improve the quality of life and restore normal life for patients with end-stage renal diseases.With the development of medical techniques and immunosuppressants, the shortterm survival of renal graft has been significantly prolonged, whereas the long-term survival remains to be urgently solved.Renal ischemia-reperfusion injury (IRI), acute rejection, chronic renal allograft dysfunction, renal fibrosis and other factors are still the major problems affecting the survival of renal graft.Relevant researches have always been hot spots in the field of renal transplantation.Meantime, 2020 is an extraordinary year.The novel coronavirus pneumonia (COVID-19) pandemic severely affects the development of all walks of life.Researches related to renal transplantation have also sprung up.In this article, the frontier hotspots of clinical and basic studies related to renal transplantation and the COVID-19 related researches in the field of renal transplantation in China were reviewed, aiming to provide novel therapeutic ideas and strategies.

Progress in Single-cell RNA Sequencing of Lung Adenocarcinoma / 中国肺癌杂志

Lung adenocarcinoma (LUAD) is the most common subtype of lung cancer and one of the main causes of cancer-related deaths. In the past decade, with the widespread use of computed tomography (CT) in routine screening for lung cancer, the incidence of LUAD presenting as small pulmonary nodules radiologically, has increased remarkably. The mechanisms of the occurrence and progression of LUADs are complex, and the prognoses of patients with LUAD vary significantly. Although significant progress has been made in targeted therapy and immunotherapy for LUADs in recent years, the drug resistance of tumor cells has not been effectively overcome, which limits the benefits of patients. With the accomplishment of the Human Genome Project, sequencing-based genomic and transcriptomics have come into the field of clinical and scientific researches. Single-cell sequencing, as a new type of sequencing method that has captured increasing attention recently, can perform specific analysis of cell populations at single-cell level, which can reveal the unique changes of each cell type. Single-cell sequencing can also provide accurate assessment on heterogeneous stromal cells and cancer cells, which is helpful to reveal the complexity of molecular compositions and differences between non- and malignant tissues. To sum up, it is an urgent need for clinicians and basic scientists to deeply understand the pathogenesis and development of LUAD, the heterogeneity of tumor microenvironment (TME) and the mechanism of drug resistance formation through single-cell sequencing, so as to discover new therapeutic targets. In this paper, we reviewed and summarized the application and progress in single-cell sequencing of LUADs.
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Research Progress of Single Cell Sequencing in Lung Cancer / 中国肺癌杂志

Lung cancer is the malignant tumor with the highest mortality rate in the world. Heterogeneity of lung cancer, usually studied by sequencing technology, is considered to have important clinical significance in current studies. However, general sequencing technology can only explain the differences between samples integrally and its resolution is not enough to describe the differences between the individual cells. Therefore, people urgently hope to understand the cell type, state, subgroup distribution in the tumor microenvironment and the communication behavior between cells in the single cell level. Single-cell sequencing technology solves this problem. Using this technique will contribute to further understanding the mechanism of the occurrence and development of lung cancer, discovering new diagnostic markers and therapeutic targets, and providing theoretical references for the precise treatment of lung cancer patients in the future. This article reviews the progress of single-cell sequencing technology and focuses on its research on lung cancer tumor heterogeneity, tumor microenvironment, invasion and metastasis, treatment response, and drug resistance.
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Application of single-cell sequencing technology in malignant tumor research / 中国肿瘤临床

Currently, malignant tumors are a major public health problem and pose a serious threat to human health. Because of the diversity and complexity and the high mortality rates, they have been a popular but complicated research topic for several years. Heterogeneity is one of the main characteristics of malignant tumors, and thus, this characteristic may affect many other aspects, such as tumorigenesis, development, metastasis, and therapeutic effects. With the completion of the Human Genome Project and the continuous advancement in sequencing technology, single-cell sequencing technology has been applied in many medical research fields; in particular, it is useful for analyzing tumor cell subpopulations, cell heterogeneity, tumor occurrence, evolution, and drug resistance. Therefore, this technology has provided us with a new tool for clinical diagnosis and treatment of malignant tumors and for estimating the prognosis at an early stage. In this article, we aimed to review the advances in single-cell sequencing technology and its application in malignant tumor research.

A human circulating immune cell landscape in aging and COVID-19

Age-associated changes in immune cells have been linked to an increased risk for infection. However, a global and detailed characterization of the changes that human circulating immune cells undergo with age is lacking. Here, we combined scRNA-seq, mass cytometry and scATAC-seq to compare immune cell types in peripheral blood collected from young and old subjects and patients with COVID-19. We found that the immune cell landscape was reprogrammed with age and was characterized by T cell polarization from naive and memory cells to effector, cytotoxic, exhausted and regulatory cells, along with increased late natural killer cells, age-associated B cells, inflammatory monocytes and age-associated dendritic cells. In addition, the expression of genes, which were implicated in coronavirus susceptibility, was upregulated in a cell subtype-specific manner with age. Notably, COVID-19 promoted age-induced immune cell polarization and gene expression related to inflammation and cellular senescence. Therefore, these findings suggest that a dysregulated immune system and increased gene expression associated with SARS-CoV-2 susceptibility may at least partially account for COVID-19 vulnerability in the elderly.

Development status and clinical applications of precision detection technology in the times of precision oncology / 中国肿瘤生物治疗杂志

@#Precision detection techniques have promoted the development of individualized diagnosis and treatment of tumors in the era of precision medicine. At the same time, clinical demands of precision treatments have further driven the development and application of precision detection techniques. In recent years, precision medical detection techniques realized rapid transformations from low-throughput to high-throughput genomic sequencing, from tissue biopsies to liquid biopsies, and from multicell promiscuous detection to single cell precision sequencing. All these changes have promoted the emergence and development of new technologies, new targets, and new drugs in the era of precision oncology medicine. In the future, multi-dimensional combined detection could help to improve the accuracy of precision medicine; ctDNA methylation detection analysis could broaden the research field of precision medicine; and the transformation of clinical trial design could also contribute to promote the in-depth development of precision medicine.