OBJECTIVE: To confirm the HLA genotypes of the samples including 4 cases of magnetic bead probe HLA genotyping result pattern abnormality and 3 cases of ambiguous result detected by PCR sequence-specific oligonudeotide probe (SSOP) method. METHODS: All samples derived from HLA high-resolution typing laboratory were detected by PCR-SSOP. A total of 4 samples of magnetic bead probe HLA genotyping result pattern abnormality and 3 samples of ambiguous result were further confirmed by PCR sequence-based typing (SBT) technology and next-generation sequencing (NGS) technology. RESULTS: A total of 4 samples of magnetic bead probe HLA genotyping result pattern abnormality were detected by PCR-SSOP method. The results of SBT and NGS showed that the HLA-A genotype of sample 1 did not match any known genotypes. NGS analysis revealed that the novel allele was different from the closest matching allele A*31:01:02:01at position 154 with G>A in exon 2, which resulting in one amino acid substitution at codon 28 from Valine to Methionine (p.Val28Met). The HLA-C genotype of sample 2 was C*03:119, 06:02, sample 3 was C*03:03, 07:137, and sample 4 was B*55:02, 55:12. A total of 3 samples with ambiguous result were initially detected by PCR-SSOP method. The re-examination results of SBT and NGS showed that the HLA-B genotype of sample 5 was B*15:58, 38:02, sample 6 was DRB1*04:05, 14:101, and sample 7 was DQB1*03:34, 05:02. Among them, alleles C*03:119, C*07:137 and DRB1*14:101 were not included in the Common and Well-documented Alleles (CWD) v2.4 of the Chinese Hematopoietic Stem Cell Donor Database. CONCLUSION The abnormal pattern of HLA genotyping results of magnetic probe by PCR-SSOP method suggests that it may be a rare allele or a novel allele, which needs to be verified by sequencing.
Humans ; Alleles ; Polymerase Chain Reaction ; Genotype ; High-Throughput Nucleotide Sequencing ; Histocompatibility Testing/methods* ; Technology

As central players in cellular structure and function, proteins have long been central themes in life science research. Analyzing the impact of protein sequence variation on its structure and function is one of the important means to study proteins. In recent years, a technology called deep mutational scanning (DMS) has been widely used in the field of protein research. It introduces thousands of mutations in parallel in specific regions of proteins through high-abundance DNA libraries. After screening, high-throughput sequencing is employed to score each mutation, revealing sequence-function correlations. Due to its high-throughput, fast and easy, and labor-saving features, DMS has become an important method for protein function research and protein engineering. This review briefly summarizes the principle of DMS technology, highlighting its applications in mammalian cells. Moreover, this review analyzes the current technical bottlenecks, aiming to facilitate relevant research.
Animals ; Mutation ; Proteins/chemistry* ; Protein Engineering ; High-Throughput Nucleotide Sequencing/methods* ; Mammals/genetics*

OBJECTIVE: To assess the value of genetic screening by high-throughput sequencing (HTS) for the early diagnosis of neonatal diseases. METHODS: A total of 2 060 neonates born at Ningbo Women and Children's Hospital from March to September 2021 were selected as the study subjects. All neonates had undergone conventional tandem mass spectrometry metabolite analysis and fluorescent immunoassay analysis. HTS was carried out to detect the definite pathogenic variant sites with high-frequency of 135 disease-related genes. Candidate variants were verified by Sanger sequencing or multiplex ligation-dependent probe amplification (MLPA). RESULTS: Among the 2 060 newborns, 31 were diagnosed with genetic diseases, 557 were found to be carriers, and 1 472 were negative. Among the 31 neonates, 5 had G6PD, 19 had hereditary non-syndromic deafness due to variants of GJB2, GJB3 and MT-RNR1 genes, 2 had PAH gene variants, 1 had GAA gene variants, 1 had SMN1 gene variants, 2 had MTTL1 gene variants, and 1 had GH1 gene variants. Clinically, 1 child had Spinal muscular atrophy (SMA), 1 had Glycogen storage disease II, 2 had congenital deafness, and 5 had G6PD deficiency. One mother was diagnosed with SMA. No patient was detected by conventional tandem mass spectrometry. Conventional fluorescence immunoassay had revealed 5 cases of G6PD deficiency (all positive by genetic screening) and 2 cases of hypothyroidism (identified as carriers). The most common variants identified in this region have involved DUOX2 (3.93%), ATP7B (2.48%), SLC26A4 (2.38%), GJB2 (2.33%), PAH (2.09%) and SLC22A5 genes (2.09%). CONCLUSION Neonatal genetic screening has a wide range of detection and high detection rate, which can significantly improve the efficacy of newborn screening when combined with conventional screening and facilitate secondary prevention for the affected children, diagnosis of family members and genetic counseling for the carriers.
Child ; Infant, Newborn ; Humans ; Female ; Prospective Studies ; Connexins/genetics* ; Connexin 26/genetics* ; Glucosephosphate Dehydrogenase Deficiency ; Mutation ; Sulfate Transporters/genetics* ; DNA Mutational Analysis ; Genetic Testing/methods* ; Deafness/genetics* ; Neonatal Screening/methods* ; Hearing Loss, Sensorineural/genetics* ; High-Throughput Nucleotide Sequencing ; Solute Carrier Family 22 Member 5/genetics*

B cell receptor (BCR) is a key molecule involved in B cell specific recognition and the binding of antigens to produce adaptive humoral immune response. Gene rearrangement and high frequency mutation during B cell differentiation are the main mechanisms of BCR diversification. The enormous diversity and unique molecular structure of BCR determine the diversity and specificity of antigen recognition, shaping complex B cell repertoire with extensive collections of antigen specificities. Therefore, BCR antigen-specific information is vital to understanding the adaptive immune characteristics of different diseases. Our ability to connect BCR repertoire and antigen specificity has been enhanced with the development of B cell related research technologies, such as single cell sorting techniques, high-throughput sequencing (HTS), linking B cell receptor to antigen specificity through sequencing (LIBRA-seq). It could help researchers to better understand humoral immune responses, identify disease pathogenesis, monitor disease progression, design vaccines, and develop therapeutic antibodies and drugs. We summarizes recent studies on antigen-specific BCR of infections, vaccinations, autoimmune diseases and cancer. By analyzing autoantibody sequences of SLE as a case, the identification of autoantigens has become potentially possible due to this characterization.
Receptors, Antigen, B-Cell/metabolism* ; B-Lymphocytes/metabolism* ; Lymphocyte Activation ; High-Throughput Nucleotide Sequencing/methods*

RNA-based next-generation sequencing (NGS) has been recommended as a method for detecting fusion genes in non-small cell lung cancer (NSCLC) according to clinical practice guidelines and expert consensus. The primary targetable alterations in NSCLC consist of gene mutations and fusions, making the detection of gene mutations and fusions indispensable for assessing the feasibility of targeted therapies. Currently, the integration of DNA-based NGS and RNA-based NGS allows for simultaneous detection of gene mutations and fusions and has been partially implemented in clinical practice. However, standardized guidelines and criteria for the significance, application scenarios, and quality control of RNA-based NGS in fusion gene detection are still lacking in China. This consensus aims to provide further clarity on the practical significance, application scenarios, and quality control measures of RNA-based NGS in fusion gene detection. Additionally, it offers guiding recommendations to facilitate the clinical implementation of RNA-based NGS in the diagnosis and treatment of NSCLC, ultimately maximizing the benefits for patients from fusion gene detection.
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Humans ; Carcinoma, Non-Small-Cell Lung/drug therapy* ; Lung Neoplasms/drug therapy* ; RNA ; Consensus ; High-Throughput Nucleotide Sequencing/methods*

Objective: To explore the value of metagenomic next-generation sequencing (mNGS) in the etiology diagnosis of bacterial meningitis in children. Methods: The etiological results of 189 children diagnosed with "bacterial meningitis" or "purulent meningitis" or "central nervous system infection" in the Children's Hospital of Fudan University from 1st January 2019 to 31st December 2020 were analyzed retrospectively. The cerebrospinal fluid (CFS) of the children with bacterial meningitis was detected by culture and mNGS respectively, and the difference of pathogen detection rate between the 2 methods was analyzed. According to the age at the time of visit, the children were divided into neonatal group (≤28 days of age) and non-neonatal group (>28 days of age), and χ² test was used to compare the positive rate between the 2 groups. Taking CFS culture as the gold standard, the sensitivity and specificity of mNGS in the diagnosing of bacterial meningitis in children were analyzed. Results: Among these 189 children with bacterial meningitis, 116 were males and 73 were females. A total of 76 strains of pathogens were detected in blood and (or) CSF cultures, of which 50 strains (65.8%) were Gram-positive bacteria; among those, 18 strains (23.7%) of Streptococcus agalactiae, 17 strains (19.7%) of Escherichia coli and 15 strains (19.7%) of Streptococcus pneumoniae were detected with higher detection rate. The infection rate of Gram-positive bacteria in the non-neonatal group was higher than that in the neonatal group (76.0% (38/50) vs. 50.0% (13/26), χ²=5.24, P=0.020).The same CSF samples of 48 cases were tested by mNGS and culture at the same time, and the detection rate of mNGS was higher than that of CSF culture (20 cases (41.7%) vs. 12 cases (25.0%), χ²=16.45, P<0.001). The consistency of mNGS and culture results was 79.2% (38/48), and the same pathogen was detected in 11 children with both positive mNGS and CSF culture. Taking the results of CSF culture as the gold standard, the sensitivity of mNGS in the diagnosing of bacterial meningitis was 91.7%, and the specificity was 75.0%. Conclusions: The mNGS technology can improve the pathogen detection rate of bacterial meningitis in children, and has a high consistency with CSF culture. In suspected cases where the pathogen cannot be identified by traditional methods, CSF mNGS should be considered timely.
Child ; Escherichia coli ; Fatigue Syndrome, Chronic ; Female ; Gram-Positive Bacteria ; High-Throughput Nucleotide Sequencing/methods* ; Humans ; Male ; Meningitis, Bacterial/microbiology* ; Metagenomics/methods* ; Retrospective Studies ; Sensitivity and Specificity

Adulteration in meat products is a widespread issue that could lead to serious threats to public health and religious violations. Technology that offers rapid, sensitive, accurate and reliable detection of meat species is the key to an effectual monitoring and control against meat adulteration. In recent years, high-throughput sequencing-based DNA metabarcoding technology has developed rapidly. With the characteristics of being high-throughput, highly precise and high-speed, this technology can simultaneously identify multiple species in complex samples, thus offering pronounced advantages in the surveillance of adulteration in meat and meat products. Starting with an introduction of the major developments in the high-throughput sequencing technology in the past two decades, this review provides an overview of the technical characteristics and research methods of DNA metabarcoding, summarizes the application of DNA metabarcoding technology in meat adulteration detection over the last few years, discusses the challenges of using DNA metabarcoding technology in the detection of meat adulteration, and provides future prospects on the development of this technology.
DNA ; Food Contamination/analysis* ; High-Throughput Nucleotide Sequencing/methods* ; Meat/analysis* ; Meat Products ; Technology

Studies of cellular dynamic processes have shown that cells undergo state changes during dynamic processes, controlled mainly by the expression of genes within the cell. With the development of high-throughput sequencing technologies, the availability of large amounts of gene expression data enables the acquisition of true gene expression information of cells at the single-cell level. However, most existing research methods require the use of information beyond gene expression, thus introducing additional complexity and uncertainty. In addition, the prevalence of dropout events hampers the study of cellular dynamics. To this end, we propose an approach named gene interaction network entropy (GINE) to quantify the state of cell differentiation as a means of studying cellular dynamics. Specifically, by constructing a cell-specific network based on the association between genes through the stability of the network, and defining the GINE, the unstable gene expression data is converted into a relatively stable GINE. This method has no additional complexity or uncertainty, and at the same time circumvents the effects of dropout events to a certain extent, allowing for a more reliable characterization of biological processes such as cell fate. This method was applied to study two single-cell RNA-seq datasets, head and neck squamous cell carcinoma and chronic myeloid leukaemia. The GINE method not only effectively distinguishes malignant cells from benign cells and differentiates between different periods of differentiation, but also effectively reflects the disease efficacy process, demonstrating the potential of using GINE to study cellular dynamics. The method aims to explore the dynamic information at the level of single cell disorganization and thus to study the dynamics of biological system processes. The results of this study may provide scientific recommendations for research on cell differentiation, tracking cancer development, and the process of disease response to drugs.
Cell Differentiation/genetics* ; Entropy ; Gene Regulatory Networks ; High-Throughput Nucleotide Sequencing ; Single-Cell Analysis/methods*

Infectious diseases are commonly seen in clinical practice, and pathogen diagnosis is the key link in diagnosis and treatment; however, conventional pathogen detection methods cannot meet clinical needs due to time-consuming operation and low positive rate. As a new pathogen detection method, metagenomic next-generation sequencing (mNGS) has a wide detection range and can detect bacteria, viruses, fungi, parasites, rare pathogens, and even unknown pathogens. The technique of mNGS is unbiased and can rapidly, efficiently, and accurately obtain all nucleic acid information in test samples, analyze pathogens, and guide clinical diagnosis and treatment, thereby playing an important role in complicated infectious diseases. This article reviews the diagnostic advantages and clinical value of mNGS in bacterial, fungal, viral, and parasitic infections.
Bacteria ; Communicable Diseases/diagnosis* ; High-Throughput Nucleotide Sequencing/methods* ; Humans ; Metagenomics/methods* ; Sensitivity and Specificity

OBJECTIVES: To study the application value of metagenomic next-generation sequencing (mNGS) in children with severe infectious diseases. METHODS: An analysis was performed on the clinical data and laboratory test results of 29 children with severe infection who were admitted to the Second Affiliated Hospital of Wenzhou Medical University from June 2018 to December 2020. Conventional pathogen culture was performed for the 29 specimens (27 peripheral blood specimens and 2 pleural effusion specimens) from the 29 children, and mNGS pathogen detection was performed at the same time. RESULTS: Among the 29 children, 2 tested positive by conventional pathogen culture with 2 strains of pathogen, and the detection rate was 7% (2/29); however, 20 children tested positive by mNGS with 38 strains of pathogen, and the detection rate was 69% (20/29). The pathogen detection rate of mNGS was significantly higher than that of conventional pathogen culture (P<0.05), and mNGS could detect the viruses, fungi, and other special pathogens that conventional pathogen culture failed to detect, such as Orientia tsutsugamushi. The univariate analysis showed that gender, routine blood test results, C-reactive protein, procalcitonin, D-dimer, radiological findings, and whether antibiotics were used before admission did not affect the results of mNGS (P>0.05). CONCLUSIONS Compared with conventional pathogen culture, mNGS is more sensitive for pathogen detection, with fewer interference factors. Therefore, it is a better pathogenic diagnosis method for severe infectious diseases in children.
Anti-Bacterial Agents ; Child ; Communicable Diseases ; High-Throughput Nucleotide Sequencing/methods* ; Humans ; Metagenomics/methods* ; Sensitivity and Specificity