Digital-WGS: Automated, highly efficient whole-genome sequencing of single cells by digital microfluidics

单细胞全基因组测序(whole-genome sequencing, WGS)是表征细胞内DNA动态变化的关键手段，可为我们提供全面、深度的生物学信息，是生命科学领域研究的热点。然而，单细胞全基因组测序仍面临着诸如样品制备复杂、成本高、扩增偏置性强、误差大的挑战。杨朝勇教授课题组搭建了基于数字微流控的单细胞操控与纳升级全基因组测序平台Digital-WGS，可用于一体化、全自动的单细胞处理与分析。通过Digital-WGS方法制备的单细胞样本在不同测序深度下均展示出了高均一性、高覆盖率、高准确性的优势，解决了当前单细胞基因组测序操作复杂、成本高、均一性差、准确度低等问题，为单细胞基因组测序的广泛应用提供了新的思路。 本研究工作在化学化工学院杨朝勇教授的指导下完成，2015级iChEM直博生阮庆宇为第一作者。Single-cell whole-genome sequencing (WGS) is critical for characterizing dynamic intercellular changes in DNA. Current sample preparation technologies for single-cell WGS are complex, expensive, and suffer from high amplification bias and errors. Here, we describe Digital-WGS, a sample preparation platform that streamlines high-performance single-cell WGS with automatic processing based on digital microfluidics. Using the method, we provide high single-cell capture efficiency for any amount and types of cells by a wetted hydrodynamic structure. The digital control of droplets in a closed hydrophobic interface enables the complete removal of exogenous DNA, sufficient cell lysis, and lossless amplicon recovery, achieving the low coefficient of variation and high coverage at multiple scales. The single-cell genomic variations profiling performs the excellent detection of copy number variants with the smallest bin of 150 kb and single-nucleotide variants with allele dropout rate of 5.2%, holding great promise for broader applications of single-cell genomics.We thank the National Natural Science Foundation of China (21927806, 21735004, 21521004, and 21325522), the National Key R&D Program of China (2018YFC1602900, 2019YFA0905800), Innovative Research Team of High-Level Local Universities in Shanghai (SSMU-ZLCX20180701), and the Program for Changjiang Scholars and Innovative Research Team in University (IRT13036) for financial support. 研究工作得到了国家自然科学基金(21927806、21735004、21521004、21325522), 国家重点研发项目(2018YFC1602900、2019YFA0905800)和教育部“长江学者和创新团队发展计划” (IRT13036)的资助与支持

Identification and Functional Analysis of a Novel von Willebrand Factor Mutation in a Family with Type 2A von Willebrand Disease

von Willebrand factor (VWF) is essential for normal hemostasis. VWF gene mutations cause the hemorrhagic von Willebrand disease (VWD). In this study, a 9-year-old boy was diagnosed as type 2A VWD, based on a history of abnormal bleeding, low plasma VWF antigen and activity, low plasma factor VIII activity, and lack of plasma high-molecular-weight (HMW) VWF multimers. Sequencing analysis detected a 6-bp deletion in exon 28 of his VWF gene, which created a mutant lacking D1529V1530 residues in VWF A2 domain. This mutation also existed in his family members with abnormal bleedings but not in >60 normal controls. In transfected HEK293 cells, recombinant VWF ΔD1529V1530 protein had markedly reduced levels in the conditioned medium (42±4% of wild-type (WT) VWF, p<0.01). The mutant VWF in the medium had less HMW multimers. In contrast, the intracellular levels of the mutant VWF in the transfected cells were significantly higher than that of WT (174±29%, p<0.05), indicating intracellular retention of the mutant VWF. In co-transfection experiments, the mutant reduced WT VWF secretion from the cells. By immunofluorescence staining, the retention of the mutant VWF was identified within the endoplasmic reticulum (ER). Together, we identified a unique VWF mutation responsible for the bleeding phenotype in a patient family with type 2A VWD. The mutation impaired VWF trafficking through the ER, thereby preventing VWF secretion from the cells. Our results illustrate the diversity of VWF gene mutations, which contributes to the wide spectrum of VWD

Flow cytometric immunobead assay for quantitative detection of platelet autoantibodies in immune thrombocytopenia patients

Abstract Background Platelet autoantibody detection is critical for immune thrombocytopenia (ITP) diagnosis and prognosis. Therefore, we aimed to establish a quantitative flow cytometric immunobead assay (FCIA) for ITP platelet autoantibodies evaluation. Methods Capture microbeads coupled with anti-GPIX, -GPIb, -GPIIb, -GPIIIa and P-selectin antibodies were used to bind the platelet-bound autoantibodies complex generated from plasma samples of 250 ITP patients, 163 non-ITP patients and 243 healthy controls, a fluorescein isothiocyanate (FITC)-conjugated secondary antibody was the detector reagent and mean fluorescence intensity (MFI) signals were recorded by flow cytometry. Intra- and inter-assay variations of the quantitative FCIA assay were assessed. Comparisons of the specificity, sensitivity and accuracy between quantitative and qualitative FCIA or monoclonal antibody immobilization of platelet antigen (MAIPA) assay were performed. Finally, treatment process was monitored by our quantitative FCIA in 8 newly diagnosed ITPs. Results The coefficient of variations (CV) of the quantitative FCIA assay were respectively 9.4, 3.8, 5.4, 5.1 and 5.8% for anti-GPIX, -GPIb, -GPIIIa, -GPIIb and -P-selectin autoantibodies. Elevated levels of autoantibodies against platelet glycoproteins GPIX, GPIb, GPIIIa, GPIIb and P-selectin were detected by our quantitative FCIA in ITP patients compared to non-ITP patients or healthy controls. The sensitivity, specificity and accuracy of our quantitative assay were respectively 73.13, 81.98 and 78.65% when combining all 5 autoantibodies, while the sensitivity, specificity and accuracy of MAIPA assay were respectively 41.46, 90.41 and 72.81%. Conclusions A quantitative FCIA assay was established. Reduced levels of platelet autoantibodies could be confirmed by our quantitative FCIA in ITP patients after corticosteroid treatment. Our quantitative assay is not only good for ITP diagnosis but also for ITP treatment monitoring

Benzodiazepine-Receptor Agonist Utilization in Outpatients with Anxiety Disorder: A Retrospective Study Based on Electronic Healthcare Data from a Large General Tertiary Hospital

Benzodiazepine-receptor agonists (BZRAs), including benzodiazepines (BZDs) and drugs related to BZDs (Z-drugs), are commonly used for anxiety, but often have side effects. We retrospectively investigated the utilization and prescription characteristics of BZRAs for patients with anxiety disorders in a large tertiary care general hospital between 2018 and 2021, based on electronic healthcare records. We also examined the pattern of simultaneous consumption of multiple BZRA drugs, and the diseases coexisting with anxiety that are associated with this. The numbers of patients and BZRA prescriptions increased over the 4 years. Moreover, 7195 prescriptions from 694 patients contained two or more BZRAs, of which 78.08% contained both BZDs and Z-drugs, 19.78% contained multiple BZDs, and 2.14% contained multiple Z-drugs. For anxiety patients with concomitant Alzheimer’s disease or Parkinson’s disease, and dyslipidemia, they were more likely to consume multiple BZRAs simultaneously, whereas patients with concomitant insomnia, depression, hypertension, diabetes, or tumors were less likely to consume multiple BZRAs (all p < 0.05). Furthermore, older patients who consume multiple BZRAs simultaneously may have higher probabilities of long-term drug use. Better interventions supporting standardized BZD utilization may be needed to minimize the side effects of inappropriate BZRA administration

Highly Sensitive and Automated Surface Enhanced Raman Scattering-based Immunoassay for H5N1 Detection with Digital Microfluidics

Digital microfluidics (DMF) is a powerful platform for a broad range of applications, especially immunoassays having multiple steps, due to the advantages of low reagent consumption and high automatization. Surface enhanced Raman scattering (SERS) has been proven as an attractive method for highly sensitive and multiplex detection, because of its remarkable signal amplification and excellent spatial resolution. Here we propose a SERS-based immunoassay with DMF for rapid, automated, and sensitive detection of disease biomarkers. SERS tags labeled with Raman reporter 4-mercaptobenzoic acid (4-MBA) were synthesized with a core@shell nanostructure and showed strong signals, good uniformity, and high stability. A sandwich immunoassay was designed, in which magnetic beads coated with antibodies were used as solid support to capture antigens from samples to form a beads–antibody–antigen immunocomplex. By labeling the immunocomplex with a detection antibody-functionalized SERS tag, antigen can be sensitively detected through the strong SERS signal. The automation capability of DMF can greatly simplify the assay procedure while reducing the risk of exposure to hazardous samples. Quantitative detection of avian influenza virus H5N1 in buffer and human serum was implemented to demonstrate the utility of the DMF-SERS method. The DMF-SERS method shows excellent sensitivity (LOD of 74 pg/mL) and selectivity for H5N1 detection with less assay time (<1 h) and lower reagent consumption (∼30 μL) compared to the standard ELISA method. Therefore, this DMF-SERS method holds great potentials for automated and sensitive detection of a variety of infectious diseases

Identification of a VWF gene mutation.

<p>Sequence analysis of the VWF gene in the proband detected a 6-bp nucleotides deletion in exon 28. The mutation caused D1529V1530 deletion (ΔD1529V1530) in VWF A2 domain. The ADAMTS13 cleavage site is indicated by an arrow.</p

Pedigree of the family with bleeding disorder.

<p>Squares and circles indicate males and females, respectively, and arrow indicates the proband. Black color denotes affected individuals. A slash through the symbol indicates decreased individuals.</p

Analysis of plasma and platelet VWF multimers.

<p>Plasma (A) and platelet (B) VWF multimers were assessed by 1.6% SDS-agarose gel electrophoresis and Western blotting. Normal plasma samples were diluted 1∶20 and patient plasma samples were diluted 1∶5. Samples from family members and a normal control (N) were indicated. Platelet samples were not available from IV-4.</p