Curvularin derivatives from the marine mangrove derived fungus Penicillium sumatrense MA-325.

Sumalarins D-G (1-4), four previously undescribed curvularin derivatives, along with two known related metabolites, curvularin (5) and dehydrocurvularin (6), were isolated and identified from the mangrove-derived fungus Penicillium sumatrense MA-325. Among them, sumalarin D (1) represents a unique example of curvularin derivative featuring a 5-methylfuran-2-yl-methyl group. Their structures were elucidated based on analysis of NMR and MS data as well as comparison of ECD spectra and quantum chemical calculations of NMR, and compound 1 was confirmed by X-ray crystallographic analysis. Compounds 1, 2, 5, and 6 are active against aquatic pathogenic bacteria Vibrio alginolyticus and V. harveyi with MIC values ranging from 4 to 64 µg/mL, while compound 6 is cytotoxic against tumor cell lines 5673, HCT 116, 786-O, and Hela with IC50 values of 3.5, 10.6, 10.9, and 14.9 µM, respectively.

Quality variation and biosynthesis of anti-inflammatory compounds for Capparis spinosa based on the metabolome and transcriptome analysis.

Introduction: Capparis spinosa L. fruits as edible and medicinal plant, has anti-inflammatory activities. The different morphological characteristics of C. spinosa fruits from Ili, Turpan, and Karamay may affect their anti-inflammatory components and functions. Methods: The anti-inflammatory activity of C. spinosa fruit was assessed using an LPS-induced inflammatory cell model. Furthermore, the differences in anti-inflammatory compounds were analyzed by metabolome and RNA-seq. Additionally, the anti-inflammatory mechanism was elucidated using network pharmacology. Results: In the study, we found that the 95% ethanol extracts (CSE) obtained from the three kinds of fruits showed remarkable anti-inflammatory effects both in vivo and in vitro. However, the CSE derived from Ili fruits significantly reduced CD86 levels on DCs. As a result of metabolomic analysis, the metabolic profiles of Ili fruits differed significantly from those of the other two habitats, which were consistent with transcriptome analysis. A total of 15 compounds exhibiting anti-inflammatory activity were subjected to screening, revealing a greater accumulation of flavonoids in the Turpan and Karamay districts. Notably, phenolic compounds were identified as the principal anti-inflammatory components in C. spinosa. Conclusion: There were significant differences in the morphology, metabolites, transcriptional levels, and anti-inflammatory activity of C. spinosa from the three districts.

Photocoupled Electroreduction of CO2 over Photosensitizer-Decorated Covalent Organic Frameworks.

Introducing an external visible-light field would be a promising strategy to improve the activity of the electrocatalytic CO2 reduction reaction (CO2RR), but it still remains a challenge due to the short excited-state lifetime of active sites. Herein, Ru(bpy)3Cl2 struts as powerful photosensitive donors were immobilized into the backbones of Co-porphyrin-based covalent organic frameworks (named Co-Bpy-COF-Rux, x is the molar ratio of Ru and Co species, x = 1/2 and 2/3) via coordination bonds, for the photo-coupled CO2RR to produce CO. The optimal Co-Bpy-COF-Ru1/2 displays a high CO Faradaic efficiency of 96.7% at -0.7 V vs reversible hydrogen electrode (RHE) and a CO partial current density of 16.27 mA cm-2 at -1.1 V vs RHE under the assistance of light, both of which were far surpassing the values observed in the dark. The significantly enhanced activity is mainly attributed to the incorporation of a Ru(bpy)3Cl2 donor with long excited-state lifetime and concomitantly giant built-in electric field in Co-Bpy-COF-Ru1/2, which efficiently accelerate the photo-induced electron transfer from Ru(bpy)3Cl2 to the cobalt-porphyrin under the external light. Thus, the cobalt-porphyrin active sites have a longer excited-state lifetime to lower the rate-determining steps' energy occurring during the actual photo-coupled electrocatalytic CO2RR process. This is the first work of porphyrin-based COFs for photo-coupled CO2RR, opening a new frontier for the construction of efficient photo-coupled electrocatalysts.

Atomically Precise Copper Nanoclusters for Highly Efficient Electroreduction of CO2 towards Hydrocarbons via Breaking the Coordination Symmetry of Cu Site.

We propose an effective highest occupied d-orbital modulation strategy engendered by breaking the coordination symmetry of sites in the atomically precise Cu nanocluster (NC) to switch the product of CO2 electroreduction from HCOOH/CO to higher-valued hydrocarbons. An atomically well-defined Cu6 NC with symmetry-broken Cu-S2 N1 active sites (named Cu6 (MBD)6 , MBD=2-mercaptobenzimidazole) was designed and synthesized by a judicious choice of ligand containing both S and N coordination atoms. Different from the previously reported high HCOOH selectivity of Cu NCs with Cu-S3 sites, the Cu6 (MBD)6 with Cu-S2 N1 coordination structure shows a high Faradaic efficiency toward hydrocarbons of 65.5 % at -1.4 V versus the reversible hydrogen electrode (including 42.5 % CH4 and 23 % C2 H4 ), with the hydrocarbons partial current density of -183.4 mA cm-2 . Theoretical calculations reveal that the symmetry-broken Cu-S2 N1 sites can rearrange the Cu 3d orbitals with d x 2 - y 2 ${d_{x^2 - y^2 } }$ as the highest occupied d-orbital, thus favoring the generation of key intermediate *COOH instead of *OCHO to favor *CO formation, followed by hydrogenation and/or C-C coupling to produce hydrocarbons. This is the first attempt to regulate the coordination mode of Cu atom in Cu NCs for hydrocarbons generation, and provides new inspiration for designing atomically precise NCs for efficient CO2 RR towards highly-valued products.

Oxepine-containing pyrazinopyrimidine alkaloids and quinolinone derivatives produced by Aspergillus versicolor AS-212, a deep-sea-derived endozoic fungus.

Four new oxepine-containing pyrazinopyrimidine alkaloids, versicoxepines A - D (1-4), two quinolinone alkaloid analogs including 3-hydroxy-6-methoxy-4-phenylquinolin-2(1H)-one (5) and 3-methoxy-6-hydroxy-4-phenylquinolin-2(1H)-one (6) which were new naturally occurring compounds, together with two known compounds (7 and 8) were isolated from Aspergillus versicolor AS-212, an endozoic fungus isolated from the deep-sea coral Hemicorallium cf. imperiale, which was collected from the Magellan Seamounts in the Western Pacific Ocean. Their structures were determined by extensive analysis of the spectroscopic and X-ray crystallographic data as well as by chiral HPLC analysis, ECD calculation, and DP4+ probability prediction. Structurally, versicoxepines B and C (2 and 3) represent the first example of a new oxepine-containing pyrazinopyrimidine alkaloid whose cyclic dipeptide moiety is composed of the same type of amino acid (Val or Ile). Compound 5 displayed antibacterial activity against aquatic pathogens, Vibrio harveyi and V. alginolyticus, with MICs of 8 µg/mL.

Diketopiperazine Alkaloids and Bisabolene Sesquiterpenoids from Aspergillus versicolor AS-212, an Endozoic Fungus Associated with Deep-Sea Coral of Magellan Seamounts.

Two new quinazolinone diketopiperazine alkaloids, including versicomide E (2) and cottoquinazoline H (4), together with ten known compounds (1, 3, and 5-12) were isolated and identified from Aspergillus versicolor AS-212, an endozoic fungus associated with the deep-sea coral Hemicorallium cf. imperiale, which was collected from the Magellan Seamounts. Their chemical structures were determined by an extensive interpretation of the spectroscopic and X-ray crystallographic data as well as specific rotation calculation, ECD calculation, and comparison of their ECD spectra. The absolute configurations of (-)-isoversicomide A (1) and cottoquinazoline A (3) were not assigned in the literature reports and were solved in the present work by single-crystal X-ray diffraction analysis. In the antibacterial assays, compound 3 exhibited antibacterial activity against aquatic pathogenic bacteria Aeromonas hydrophilia with an MIC value of 18.6 µM, while compounds 4 and 8 exhibited inhibitory effects against Vibrio harveyi and V. parahaemolyticus with MIC values ranging from 9.0 to 18.1 µM.

Boosting Electroreduction of CO2 over Cationic Covalent Organic Frameworks: Hydrogen Bonding Effects of Halogen Ions.

We present the first example of charged imidazolium functionalized porphyrin-based covalent organic framework (Co-iBFBim-COF-X) for electrocatalytic CO2 reduction reaction, where the free anions (e.g., F- , Cl- , Br- , and I- ) of imidazolium ions nearby the active Co sites can stabilize the key intermediate *COOH and inhibit hydrogen evolution reaction. Thus, Co-iBFBim-COF-X exhibits higher activity than the neutral Co-BFBim-COF, following the trend of F-

Cistanche tubulosa phenylethanoid glycosides suppressed adipogenesis in 3T3-L1 adipocytes and improved obesity and insulin resistance in high-fat diet induced obese mice.

BACKGROUND: Cistanche tubulosa is an editable and medicinal traditional Chinese herb and phenylethanoid glycosides are its major components, which have shown various beneficial effects such as anti-tumor, anti-oxidant and neuroprotective activities. However, the anti-obesity effect of C. tubulosa phenylethanoid glycosides (CTPG) and their regulatory effect on gut microbiota are still unclear. In the present study, we investigated its anti-obesity effect and regulatory effect on gut microbiota by 3T3-L1 cell model and obesity mouse model. METHODS: 3T3-L1 adipocytes were used to evaluate CTPG effects on adipogenesis and lipids accumulation. Insulin resistant 3T3-L1 cells were induced and used to measure CTPG effects on glucose consumption and insulin sensitivity. High-fat diet (HFD)-induced C57BL/6 obese mice were used to investigate CTPG effects on fat deposition, glucose and lipid metabolism, insulin resistance and intestinal microorganism. RESULTS: In vitro data showed that CTPG significantly decreased the triglyceride (TG) and non-esterified fatty acid (NEFA) contents of the differentiated 3T3-L1 adipocytes in a concentration-dependent manner without cytotoxicity, and high concentration (100 µg/ml) of CTPG treatment dramatically suppressed the level of monocyte chemoattractant protein-1 (MCP-1) in 3T3-L1 mature adipocytes. Meanwhile, CTPG increased glucose consumption and decreased NEFA level in insulin resistant 3T3-L1 cells. We further found that CTPG protected mice from the development of obesity by inhibiting the expansion of adipose tissue and adipocyte hypertrophy, and improved hepatic steatosis by activating AMPKα to reduce hepatic fat accumulation. CTPG ameliorated HFD-induced hyperinsulinemia, hyperglycemia, inflammation and insulin resistance by activating IRS1/Akt/GLUT4 insulin signaling pathway in white adipose tissue. Moreover, gut microbiota structure and metabolic functions in HFD-induced obese mice was changed by CTPG, especially short chain fatty acids-producing bacteria including Blautia, Roseburia, Butyrivibrio and Bacteriodes were significantly increased by CTPG treatment. CONCLUSIONS: CTPG effectively suppressed adipogenesis and lipid accumulation in 3T3-L1 adipocytes and ameliorated HFD-induced obesity and insulin resistance through activating AMPKα and IRS1/AKT/GLUT4 signaling pathway and regulating the composition and metabolic functions of gut microbiota.

Effects of Forest Gaps on the Structure and Diversity of Soil Bacterial Communities in Weeping Cypress Forest Plantations.

The decline in forest ecological function caused by pure forest plantations planted in the Yangtze River basin is becoming increasingly serious. To investigate this problem, we selected the local low-efficiency weeping cypress plantations for forest gap transformation. Three forest gap sizes, specifically large, medium, and small gaps, were established, and the effects of gap sizes on soil bacterial community structure and diversity in winter and summer were studied compared to no gaps (CK; control). Compared to CK, forest gaps had a significant effect on soil organic carbon (SOC) and soil total nitrogen (TN), and the highest values of SOC and soil TN under two seasons occurred in large forest gaps. The interactions of forest gap sizes and seasons had significant effects on pH, SOC, TN, and alpha diversity indices, including Simpson, Chao1, and ACE indices. Compared to winter, forest gaps significantly increased the soil bacterial community diversity indices in summer. Forest gap sizes significantly affected the composition of the bacterial community, but the composition of the dominant bacteria at the phyla and genera levels was similar. Linear discriminant effect size (LEfSe) analysis showed that there were 32 indicator bacterial species in two seasons. Co-occurrence network analysis revealed that the relationship of the soil bacterial community at the phyla level was complex, and there was a significant positive correlation among bacterial species. Soil bulk density (BD) and soil moisture (SM) significantly affected the soil bacterial alpha diversity indices. The composition of the dominant bacteria at the phyla level was significantly affected by soil microbial carbon (MBC), whereas the composition of dominant bacteria at the genera level was affected by soil hydrolysable nitrogen (AN) and the carbon/nitrogen (C/N) ratio. In this study, compared to the other forest gaps, large forest gaps were more conducive to the accumulation of soil nutrients, thus improving the structure of the soil bacterial community. Importantly, changes in the soil bacterial community structure due to gap formation may have profound effects on soil biogeochemical processes in weeping cypress forest plantations.

Evaluation of Spike Protein Epitopes by Assessing the Dynamics of Humoral Immune Responses in Moderate COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic is caused by a novel coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The spike protein (S) of SARS-CoV-2 is a major target for diagnosis and vaccine development because of its essential role in viral infection and host immunity. Currently, time-dependent responses of humoral immune system against various S protein epitopes are poorly understood. In this study, enzyme-linked immunosorbent assay (ELISA), peptide microarray, and antibody binding epitope mapping (AbMap) techniques were used to systematically analyze the dynamic changes of humoral immune responses against the S protein in a small cohort of moderate COVID-19 patients who were hospitalized for approximately two months after symptom onset. Recombinant truncated S proteins, target S peptides, and random peptides were used as antigens in the analyses. The assays demonstrated the dynamic IgM- and IgG recognition and reactivity against various S protein epitopes with patient-dependent patterns. Comprehensive analysis of epitope distribution along the spike gene sequence and spatial structure of the homotrimer S protein demonstrated that most IgM- and IgG-reactive peptides were clustered into similar genomic regions and were located at accessible domains. Seven S peptides were generally recognized by IgG antibodies derived from serum samples of all COVID-19 patients. The dynamic immune recognition signals from these seven S peptides were comparable to those of the entire S protein or truncated S1 protein. This suggested that the humoral immune system recognized few conserved S protein epitopes in most COVID-19 patients during the entire duration of humoral immune response after symptom onset. Furthermore, in this cohort, individual patients demonstrated stable immune recognition to certain S protein epitopes throughout their hospitalization period. Therefore, the dynamic characteristics of humoral immune responses to S protein have provided valuable information for accurate diagnosis and immunotherapy of COVID-19 patients.

Single-Cell Transcriptome Analysis of H5N1-HA-Stimulated Alpaca PBMCs.

Avian influenza A virus H5N1 is a highly pathogenic and persistently a major threat to global health. Vaccines and antibodies targeting hemagglutinin (HA) protein are the primary management strategies for the epidemic virus. Although camelids possess unique immunological features, the immune response induced by specific antigens has not yet been thoroughly investigated. Herein, we immunized an alpaca with the HA antigen of the H5N1 virus and performed single-cell transcriptome profiling for analysis of longitudinal peripheral blood mononuclear cell (PBMCs) behavior using single-cell sequencing technology (scRNA-seq). We revealed multiple cellular immunities during the immunization. The monocytes continued to expand after immunization, while the plasma cells reached their peak three days after the second antigen stimulation. Both monocytes and B cells were stimulated by the HA antigen and produced cell-type-specific cytokines to participated in the immune response. To our knowledge, this is the first study to examine the HA-specific immunological dynamics of alpaca PBMCs at the single-cell level, which is beneficial for understanding the anti-viral immune system and facilitating the development of more potent vaccines and antibodies in camelid animals.

Unveiling the Microscopic Mechanism of Current Variation in the Sensing Region of the MspA Nanopore for DNA Sequencing.

Different nucleotides generate specific ionic currents that discriminate between the nucleotides while they are passing through the nanopore constriction. MspA is a commonly used nanopore for DNA sequencing. However, the reasons of the current variation remain ambiguous. Our work unveils the microscopic mechanism of current variation for an ssDNA passing through the MspA nanopore by all-atom molecular dynamic simulations. Besides the physical rigidity and dimensions of the nucleotides, nucleotide orientation is observed to induce nonignorable current variation. Besides the generally considered MspA nanopore constriction, it is also found that the region below constriction could be used to detect and differentiate single nucleotides when the single-stranded DNA translocates in the form of base-constriction-base meshing and ratcheting across the nanopore constriction compared to other regions. The work provides a novel insight into facilitating the development of low-cost and high-throughput nanopore DNA sequencing.

DNB-based on-chip motif finding: A high-throughput method to profile different types of protein-DNA interactions.

Here, we report a sensitive DocMF system that uses next-generation sequencing chips to profile protein-DNA interactions. Using DocMF, we successfully identified a variety of endonuclease recognition sites and the protospacer adjacent motif (PAM) sequences of different CRISPR systems. DocMF can simultaneously screen both 5' and 3' PAMs with high coverage. For SpCas9, we found noncanonical 5'-NAG-3' (~5%) and 5'-NGA-3' (~1.6%), in addition to its common PAMs, 5'-NGG-3' (~89.9%). More relaxed PAM sequences of two uncharacterized Cas endonucleases, VeCas9 and BvCas12a, were extensively characterized using DocMF. Moreover, we observed that dCas9, a DNA binding protein lacking endonuclease activity, preferably bound to the previously reported 5'-NGG-3' sequence. In summary, our studies demonstrate that DocMF is the first tool with the capacity to exhaustively assay both the binding and the cutting properties of different DNA binding proteins.

Elucidation of the Tailoring Steps in Julichrome Biosynthesis by Marine Gastropod Mollusk-Associated Streptomyces sampsonii SCSIO 054.

The biosynthetic gene cluster governing the production of antibacterial julichromes was identified from marine gastropod mollusk-associated Streptomyces sampsonii SCSIO 054. Post-PKS assembly/tailoring enzymes JuiL, JuiM, JuiI, and JuiN represent key assembly enzymes. JuiL serves as a ketoreductase. JuiM is an acetyltransferase. JuiI carries out an intriguing biaryl coupling of two julichrome Q6 units (immediate JuiL, JuiM product) to afford julichrome Q6-6. JuiN carries out tailoring steps on julichrome Q6-6, transforming Q6-6 into Q3-3.

Author Correction: Chronic Kidney Disease Impairs Bone Defect Healing in Rats.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Effects of Litterfall on the Accumulation of Extracted Soil Humic Substances in Subalpine Forests.

Plant litter is one of the main sources of soil humus, but which can also promote primary humus degradation by increasing microbial activity due to the higher availability of energy released, resulting in a confusing relationship between litterfall and soil humus. Therefore, an in situ incubation experiment was carried out in three subalpine forests (coniferous, mixed and broadleaved forests) on the eastern Qinghai-Tibetan Plateau. We set up two treatments. One that allowed litterfall to enter the soil normally and the other prevented litterfall to enter the soil. Soils were sampled in October (the end of the growing season), January (the onset of the freezing season), March (the end of the freezing season), and May (the start of the growing season) from May 2017 to May 2018. By assessing the litterfall production, the content of total extracted humus, humic acid (HA) and fulvic acid (FA) in the topsoil (0-20 cm) in each incubation period, we determined the impact of litterfall on the content of humus extracted from the soil during the freezing and the growing season. Over 1-year incubation, soil total extracted humus and HA showed considerable decreases in the treatment of retained litterfall in the mixed forest but not in the coniferous or broadleaved forests. Moreover, litterfall significantly reduced the contents of soil total extracted humus and HA during the growing season in all three forests, while only reduced soil HA content in the broadleaved forest in the freezing season. The relationship between litterfall and soil extracted humic substances was greatly regulated by the seasonal dynamics of litter types and litter production in all forest types. The larger the amount of litterfall was, the more litterfall could promote the reduction of soil extracted humic substances. Compared with a single type of broadleaf or needle litter, mixed litterfall could promote a higher degradation of soil humic substances. However, broadleaf litter might lead to much greater decreases in soil humic substance than needle litter because it is more decomposable. These results indicate that the effect of litterfall on soil humic substances are mainly regulated by litter types and litter production. Moreover, the effects of litterfall on soil humic substances are more significant during the growing season than winter. This suggests that the longer growing season and a shorter winter caused by ongoing global warming may alter the relationships between litterfall and extracted humic substances, further disrupting the carbon balance of forest ecosystems in the subalpine forests.

The roles of genes associated with regulation, transportation, and macrocyclization in desotamide biosynthesis in Streptomyces scopuliridis SCSIO ZJ46.

The deep-sea-derived microbe Streptomyces scopuliridis SCSIO ZJ46 produces desotamides A-D. Notably, desotamides A and B display antibacterial activities against pathogenic Gram-positive Streptococcus pneumoniae NCTC 7466, Staphylococcus aureus ATCC 29213, and the methicillin-resistant clinical isolate Staphylococcus epidermidis (MRSE) shhs-E1. The 39-kb desotamide biosynthetic gene cluster (dsa) has previously been identified and heterologously expressed in S. coelicolor M1152 for the purposes of assigning dsa gene functions. In this work, we identified seven genes in the dsa cluster including three regulatory genes (dsaA, dsaM, and dsaN), two transporter genes (dsaK and dsaL), and two other genes, dsaB (annotated as a phosphate synthase) and dsaJ (a PBP-type thioesterase). The DsaA and DsaN were unambiguously shown to be positive regulators of desotamide biosynthesis, and consistent with these roles, inactivation of either gene completely abolished desotamide production. Moreover, overexpression of dsaA or dsaN (independent of each other) was shown to improve desotamide titers. Production of desotamides in M1152/07-6H::dsaA strain was 2.4-fold greater than that in the heterologous dsa expression strain M1152/07-6H whereas desotamide titers from the M1152/07-6H::dsaN strain were about twice that of M1152/07-6H. In addition, inactivation of dsaB and dsaJ (independent of each other) completely abolished desotamide production, indicating their indispensability for desotamide assembly. These studies provide new insights into the functions and combinatorial biosynthetic potentials of seven key genes within the dsa biosynthetic gene cluster. Findings reported here are likely to facilitate further efforts aimed at assessing and developing the desotamides and related analogs for future applications.

Julichrome Monomers from Marine Gastropod Mollusk-Associated Streptomyces and Stereochemical Revision of Julichromes Q3 ⋠5 and Q3 ⋠3.

Two julichrome monomers, julichromes Q11 (1) and Q12 (2), along with five known julichromes (Q10 , Q3 ⋅ 5 , Q3 ⋅ 3 , Q6 ⋅ 6 , Q6 , 3-7) and four known anthraquinones (chrysophanol, 4-acetylchrysophanol, islandicin, huanglongmycin A, 8-11), were isolated from the marine gastropod mollusk Batillaria zonalis-associated Streptomyces sampsonii SCSIO 054. This is the first report of julichromes isolated from a marine source. Extensive dissection of 1D and 2D NMR datasets combined with X-ray crystallography enabled rigorous elucidation of the previously reported configurations of julichrome Q3 ⋅ 5 (4) and related julichrome Q3 ⋅ 3 (5); both of the configuration at C(9) needs to be revised. In addition, julichrome Q12 (2) was found to display antibacterial activity against Micrococcus luteus and Bacillus subtilis with MICs of 2.0 and 8.0 µg mL-1 ; four compounds (1, 3, 6, 7) also showed inhibitory activities against an array of methicillin-resistant Staphylococcus aureus, S. aureus and S. simulans AKA1 with MIC values ranging from 8 to 64 µg mL-1 .

Extraction and Analysis of Available Boron Isotopes in Soil Using Multicollector Inductively Coupled Plasma Mass Spectrometry.

As a result of the important roles of boron (B) in the growth of plants, the uptake of B by plants is dependent upon the existing form and content of available B in soil, which can bring about the local cycle of B isotope equilibrium. A method using water-heating extraction combined with three-step ion-exchange chromatography was developed for the extraction and isotopic analysis of available B in soil. The extraction efficiency and fractionation of B isotopic composition in the procedure were investigated. The results showed that, in the upper layers of soils, the change of δ11B values was opposite that of the mass concentration and a similar variation between δ11B and content occurred in the lower layers. The isotope of available B in soil can create a featured isotopic signature to further understand the geochemical details related to the soil properties and molecular mechanism of B uptake in plants.

Efficient and unique cobarcoding of second-generation sequencing reads from long DNA molecules enabling cost-effective and accurate sequencing, haplotyping, and de novo assembly.

Here, we describe single-tube long fragment read (stLFR), a technology that enables sequencing of data from long DNA molecules using economical second-generation sequencing technology. It is based on adding the same barcode sequence to subfragments of the original long DNA molecule (DNA cobarcoding). To achieve this efficiently, stLFR uses the surface of microbeads to create millions of miniaturized barcoding reactions in a single tube. Using a combinatorial process, up to 3.6 billion unique barcode sequences were generated on beads, enabling practically nonredundant cobarcoding with 50 million barcodes per sample. Using stLFR, we demonstrate efficient unique cobarcoding of more than 8 million 20- to 300-kb genomic DNA fragments. Analysis of the human genome NA12878 with stLFR demonstrated high-quality variant calling and phase block lengths up to N50 34 Mb. We also demonstrate detection of complex structural variants and complete diploid de novo assembly of NA12878. These analyses were all performed using single stLFR libraries, and their construction did not significantly add to the time or cost of whole-genome sequencing (WGS) library preparation. stLFR represents an easily automatable solution that enables high-quality sequencing, phasing, SV detection, scaffolding, cost-effective diploid de novo genome assembly, and other long DNA sequencing applications.