Gene expression labeling and conditional manipulation of gene function are important for elaborate dissection of gene function. However, contemporary generation of pairwise dual-function knockin alleles to achieve both conditional and geno-tagging effects with a single donor has not been reported. Here we first developed a strategy based on a flipping donor named FoRe to generate conditional knockout alleles coupled with fluorescent allele-labeling through NHEJ-mediated unidirectional targeted insertion in zebrafish facilitated by the CRISPR/Cas system. We demonstrated the feasibility of this strategy at sox10 and isl1 loci, and successfully achieved Cre-induced conditional knockout of target gene function and simultaneous switch of the fluorescent reporter, allowing generation of genetic mosaics for lineage tracing. We then improved the donor design enabling efficient one-step bidirectional knockin to generate paired positive and negative conditional alleles, both tagged with two different fluorescent reporters. By introducing Cre recombinase, these alleles could be used to achieve both conditional knockout and conditional gene restoration in parallel; furthermore, differential fluorescent labeling of the positive and negative alleles enables simple, early and efficient real-time discrimination of individual live embryos bearing different genotypes prior to the emergence of morphologically visible phenotypes. We named our improved donor as Bi-FoRe and demonstrated its feasibility at the sox10 locus. Furthermore, we eliminated the undesirable bacterial backbone in the donor using minicircle DNA technology. Our system could easily be expanded for other applications or to other organisms, and coupling fluorescent labeling of gene expression and conditional manipulation of gene function will provide unique opportunities to fully reveal the power of emerging single-cell sequencing technologies.
Alleles ; Animals ; CRISPR-Cas Systems ; DNA End-Joining Repair ; DNA, Circular/metabolism* ; Embryo, Nonmammalian ; Gene Editing/methods* ; Gene Knock-In Techniques ; Gene Knockout Techniques ; Genes, Reporter ; Genetic Loci ; Genotyping Techniques ; Green Fluorescent Proteins/metabolism* ; Integrases/metabolism* ; Luminescent Proteins/metabolism* ; Mutagenesis, Insertional ; Single-Cell Analysis ; Zebrafish/metabolism*

Identifying antimicrobial resistant (AMR) bacteria in metagenomics samples is essential for public health and food safety. Next-generation sequencing (NGS) technology has provided a powerful tool in identifying the genetic variation and constructing the correlations between genotype and phenotype in humans and other species. However, for complex bacterial samples, there lacks a powerful bioinformatic tool to identify genetic polymorphisms or copy number variations (CNVs) for given genes. Here we provide a Bayesian framework for genotype estimation for mixtures of multiple bacteria, named as Genetic Polymorphisms Assignments (GPA). Simulation results showed that GPA has reduced the false discovery rate (FDR) and mean absolute error (MAE) in CNV and single nucleotide variant (SNV) identification. This framework was validated by whole-genome sequencing and Pool-seq data from Klebsiella pneumoniae with multiple bacteria mixture models, and showed the high accuracy in the allele fraction detections of CNVs and SNVs in AMR genes between two populations. The quantitative study on the changes of AMR genes fraction between two samples showed a good consistency with the AMR pattern observed in the individual strains. Also, the framework together with the genome annotation and population comparison tools has been integrated into an application, which could provide a complete solution for AMR gene identification and quantification in unculturable clinical samples. The GPA package is available at https://github.com/IID-DTH/GPA-package.
Bacteria ; genetics ; Bayes Theorem ; DNA Copy Number Variations ; Genome, Bacterial ; Genotyping Techniques ; High-Throughput Nucleotide Sequencing ; Humans ; Klebsiella pneumoniae ; genetics ; Metagenomics ; methods ; Polymorphism, Genetic ; Software

OBJECTIVE: To investigate the molecular characteristics and intracellular growth ability of Legionella pneumophila (L. pneumophila) strains from 1989 to 2016 in Sichuan Province, China. METHODS: Seventy-nine isolates of L. pneumophila were collected from environmental and clinical sources, including cooling towers, hot springs, bath water, fountains, and patients, and identified with 16S rRNA gene analysis and serum agglutination assay. The isolates were then typed by Sequence-Based Typing (SBT), and Genotyping of forty-two LP1 strains were analyzed by means of multiple-locus VNTR analysis with 8 loci (MLVA-8). All strains were further analyzed for two virulence genes: Legionella vir homologue (lvh) and repeats in structural toxin (rtxA). The intracellular growth ability of 33 selected isolates was determined by examining their interaction with J774 cells. RESULTS: All isolates were identified to L. pneumophila including 11 serogroups, among which the main serogroup were LP1, accounting for 54.43%. Thirty-three different sequence types (STs) from five main clonal groups and five singletons were identified, along with 8 different MLVA patterns. Both the lvh and rtxA loci were found in all 79 strains. Thirty isolates showed high intracellular growth ability in J774 cells. CONCLUSION L. pneumophila is a potential threat to public health, and effective control and prevention strategies are urgently needed.
Bacterial Proteins ; genetics ; Bacterial Toxins ; genetics ; China ; Genotyping Techniques ; Humans ; Legionella pneumophila ; genetics ; growth & development ; isolation & purification ; RNA, Ribosomal, 16S ; genetics ; Water Microbiology

Deficiency Diseases ; blood ; diagnosis ; genetics ; Genotyping Techniques ; Humans ; Microfluidic Analytical Techniques ; Micronutrients ; deficiency ; Polymorphism, Single Nucleotide ; Precision Medicine ; methods ; Risk Assessment

Objective To evaluate the effectiveness of single nucleotide polymorphism （SNP） genoty-ping in combination with identity by state （IBS） strategy in full sibling testing. Methods Thirty-five blood samples were collected from a four-generation family. Ninety autosomal SNPs were genotyped using Precision ID Identity Panel. The distribution of IBS scores for full siblings and other relationships were calculated and compared. The relationships were determined using Fisher discriminant function and threshold method, respectively. Results Based on family members and previous research, 44, 30, 111, 71 and 1 000 pairs of full siblings （FS）, grandparent-grandchild （GG）, uncle/aunt-nephew/niece （UN）, first cousins （FC） and unrelated individuals （UI） were obtained, respectively. The average IBS scores were 148, 130, 132, 124 and 120, respectively. Except for the GG and UN pairs, the distribution differences among the other relationships had statistical significance （P<0.05）. The false rates of Fisher discriminant function to determine relationships were 1.3%, 22.3%, 17.0% and 38.7% for FS, GG, UN and FC, respectively. Based on the simulation data, the thresholds t₁=128 and t₂=141 were recommended to determine full sibling relationships （the false rate ≤0.05%）. Conclusion The 90 SNP genetic markers included in the Precision ID Identity Panel meet the testing requirements for full sibling relationships. The threshold method based on IBS has a relatively lower false rate and is more flexible.
Genotype ; Genotyping Techniques/methods* ; Humans ; Polymorphism, Single Nucleotide/genetics* ; Siblings

BACKGROUND: Endobronchial ultrasound guided transbronchial needle aspiration (EBUS-TBNA) has emerged as an innovative technique for diagnosis and staging of lung cancer. But whether the procedure can provide enough tissue for the detection of gene mutations is still to be defined. Here we evaluated the efficacy of lung cancer diagnosis and gene analysis using samples obtain via EBUS-TBNA. METHODS: Patients with suspected lung cancer and mediastinal lesions were referred for EBUS-TBNA. Diagnosis and sub-classifications were made by pathologists. Samples with non-squamous non small cell lung cancer sub type were tested for the EGFR and/or ALK mutations. RESULTS: A total of 377 patients were included in this study. The median needle passes were 2.07. Lung cancer was diagnosed in 213 patients. The diagnosis accuracy for malignancy was 92%. Epidermal growth factor receptor (EGFR) mutations, anaplasticlymphoma kinase (ALK) fusion genes and double genes analysis were successfully preformed in 84 (90%), 105 (95%) and 79 (90%) patients. The number of needle passes and the diameters of lymph node were not associated with the efficacy of gene testing in univariate analysis. However, samples of adenocarcinoma sub type showed a tendency associated with higher genotyping efficacy. CONCLUSIONS Tissue samples obtained through EBUS-TBNA are sufficient for pathological diagnosis and genetic analysis of lung cancer. The pathology type of sample affected genotyping efficacy.
Adult ; Carcinoma, Non-Small-Cell Lung ; diagnosis ; genetics ; pathology ; Endoscopic Ultrasound-Guided Fine Needle Aspiration ; Feasibility Studies ; Female ; Genotyping Techniques ; Humans ; Lung Neoplasms ; diagnosis ; genetics ; pathology ; Male

Tuberculosis (TB) has remained an ongoing concern in China. The national scale-up of the Directly Observed Treatment, Short Course (DOTS) program has accelerated the fight against TB in China. Nevertheless, many challenges still remain, including the spread of drug-resistant strains, high disease burden in rural areas, and enormous rural-to-urban migrations. Whether incident active TB represents recent transmission or endogenous reactivation has helped to prioritize the strategies for TB control. Evidence from molecular epidemiology studies has delineated the recent transmission of Mycobacterium tuberculosis (M. tuberculosis) strains in many settings. However, the transmission patterns of TB in most areas of China are still not clear. Studies carried out to date could not capture the real burden of recent transmission of the disease in China because of the retrospective study design, incomplete sampling, and use of low-resolution genotyping methods. We reviewed the implementations of molecular epidemiology of TB in China, the estimated disease burden due to recent transmission of M. tuberculosis strains, the primary transmission of drug-resistant TB, and the evaluation of a feasible genotyping method of M. tuberculosis strains in circulation.
China ; epidemiology ; Genotyping Techniques ; Humans ; Molecular Epidemiology ; Mycobacterium tuberculosis ; genetics ; Tuberculosis, Multidrug-Resistant ; epidemiology ; transmission ; Whole Genome Sequencing

OBJECTIVETo explore the association of KIR-HLA gene polymorphism with chronic myeloid leukemia (CML) among ethnic Hans from southern China.

METHODSA total of 172 adult CML patients and 480 unrelated healthy controls were screened for the presence of KIR with sequence-specific primers-PCR (PCR-SSP) and sequence-based typing (SBT) of HLA-A, -B and -C loci. Polymorphisms of the KIR-HLA system were analyzed at 4 levels, and the frequencies of KIR framework genes and KIR profiles, classⅠHLA ligands, matched KIR+HLA pairs and KIR-HLA compound profile were compared between the two groups. P values were calculated using SPSS 13.0 software.

RESULTSFor the CML group, the frequencies of HLA-C2 ligand, 2DL1+HLA-C2 pair and HLA-B Bw4-80I were significantly lower than those of the control group, suggesting a protective effect against CML (HLA-C2: OR=0.386, 95%CI:0.240-0.620, P<0.01; 2DL1+HLA-C2: OR=0.316, 95%CI:0.191-0.525, P<0.01; HLA-B Bw4-80I: OR=0.576, 95%CI:0.384-0.862, P<0.01). The frequencies of KIR2DL1 ligand (HLA-C2) and KIR3DL1 ligand (HLA-B Bw4-80I) in the CML group were significantly lower than that of the control group, suggesting that the HLA-C2 and HLA-B Bw4-80I expression is probably decreased in the CML patient group, which led to reduced inhibitory signal and enhanced activating signal of KIR2DL1and/or KIR3DL1NK cells. Notably, the frequency of KIR-HLA compound profiles ID2 (KIR AA1-HLA-C1/C1-Bw6/Bw6-A3/11) in CML patients significantly increased in the CML patient group compared with the control group, suggesting that the KIR-HLA compound profiles ID2 may be a risk factor for CML (OR=2.163, 95%CI 1.198-3.906, P<0.01).

CONCLUSIONAbove analysis has identified certain protective and risk factors for CML from the KIR-HLA system, which may provide a clue for the pathogenesis of leukemia and development of individualized immune therapy.

Asian Continental Ancestry Group ; genetics ; China ; Gene Frequency ; Genetic Predisposition to Disease ; ethnology ; genetics ; Genotyping Techniques ; HLA Antigens ; genetics ; HLA-A Antigens ; genetics ; HLA-B Antigens ; genetics ; HLA-C Antigens ; genetics ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; ethnology ; genetics ; Odds Ratio ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Protein Isoforms ; genetics ; Receptors, KIR ; genetics ; Risk Factors

OBJECTIVETo study genetic polymorphisms of the KIR2DS4 gene among ethnic Hans from southern China.

METHODSGenomic DNA was isolated from 306 unrelated individuals and amplified with KIR2DS4-specific PCR primers. KIR2DS4-positive samples were genotyped for the entire coding sequence by sequencing-based typing (SBT). Assignment of allelic genotypes was accomplished by using Assign 3.5 software. For samples with inconclusive SBT results, RT-PCR products covering the entire coding sequence of the KIR2DS4 gene were subjected to cloning and haplotype sequencing.

RESULTSAmong all tested samples, 297 were demonstrated to have carried the KIR2DS4 framework gene. For KIR2DS4-positive samples subjected to SBT for the entire coding sequences, no background was observed with the obtained sequences. Three of the seven identified alleles were of novel types, which were officially named by the KIR subcommittee of the World Health Organization Nomenclature Committee for Factors of HLA System. The observed frequencies for the 7 alleles were KIR2DS4*00101 (78.8%), *003 (10.5%), *004 (16.0%), *010 (23.2%), *017 (0.3%), *00105 (0.3%) and *018 (0.7%), respectively. Allele KIR2DS4*007 was not found. The overall frequency for normal cell-surface expression KIR2DS4 alleles including 2DS4*00101, *017 and *00105 was 79.4%, and that for non cell-surface expression alleles including 2DS4*003, *004, *010 and *018 was 50.4%. The ratio between the two was 1.6:1.

CONCLUSIONThe present study has elucidated the allelic diversity of KIR2DS4 among ethnic Hans from southern China, which may provide valuable data for transplantation as well as studies on KIR-associated disease and evolution.

Alleles ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; China ; Gene Frequency ; Genotype ; Genotyping Techniques ; methods ; Haplotypes ; Humans ; Polymorphism, Genetic ; Receptors, KIR ; genetics ; Sequence Analysis, DNA ; methods

OBJECTIVETo study the genetic polymorphisms of human leukocyte antigen (HLA)- A, B, C, DRB1, DQA1, DQB1, DPA1and DPB1among ethnic Hans from southern China.

METHODS481 randomly selected individuals were genotyped using a polymerase chain reaction (PCR) sequence-based typing (SBT) method for the above genes. Their allele frequencies were determined by direct counting.

RESULTSIn total, 28 HLA-A, 57 HLA-B, 28 HLA-C, 40 HLA-DRB1, 18 HLA-DQA1, 17 HLA-DQB1, 6 HLA-DPA1and 21 HLA-DPB1alleles were identified. Among these, common alleles (with allelic frequencies > 0.05) included A*1101, A*2402, A*0207, A*3303, A*0201, B*40:01, B*46:01, B*58:01, B*13:01, B*15:02, C*01:02, C*07:02, C*03:04, C*03:02, C*08:01, C*03:03, C*04:01, DRB1*09:01, DRB1*15:01, DRB1*12:02, DRB1*08:03, DRB1*03:01, DRB1*04:05, DRB1*11:01, DQA1*01:02, DQA1*03:02, DQA1*03:03, DQA1*06:01, DQA1*01:03, DQA1*05:05, DQA1*01:04, DQA1*03:01, DQA1*05:01, DQB1*03:01, DQB1*03:03, DQB1*06:01, DQB1*05:02, DQB1*03:02, DQB1*02:01, DQB1*03:02, DQB1*06:02, DPA1*02:02, DPA1*01:03, DPA1*02:01, DPB1*05:01, DPB1*02:01, DPB1*13:01, DPB1*04:01and DPB1*02:02.For each of the locus, the overall frequencies of common alleles were 75.57%, 52.81%, 78.28%, 62.16%, 86.70%, 77.23%, 95.32% and 81.59%, respectively.

CONCLUSIONThe allelic frequencies of the 8 selected HLA loci among ethnic Hans from southern China may served as a reference for anthropology, legal medicine, transplantation and disease association studies.

Alleles ; Asian Continental Ancestry Group ; genetics ; China ; Gene Frequency ; Genotype ; Genotyping Techniques ; methods ; HLA-A Antigens ; genetics ; HLA-B Antigens ; genetics ; HLA-C Antigens ; genetics ; HLA-DP Antigens ; genetics ; HLA-DQ alpha-Chains ; genetics ; HLA-DQ beta-Chains ; genetics ; HLA-DRB1 Chains ; genetics ; Histocompatibility Antigens Class I ; genetics ; Histocompatibility Antigens Class II ; genetics ; Humans ; Linkage Disequilibrium ; Polymerase Chain Reaction ; Polymorphism, Genetic