Efficient crop improvement depends on the application of accurate genetic information contained in diverse germplasm resources. Here we report a reference-grade genome of wild soybean accession W05, ...with a final assembled genome size of 1013.2 Mb and a contig N50 of 3.3 Mb. The analytical power of the W05 genome is demonstrated by several examples. First, we identify an inversion at the locus determining seed coat color during domestication. Second, a translocation event between chromosomes 11 and 13 of some genotypes is shown to interfere with the assignment of QTLs. Third, we find a region containing copy number variations of the Kunitz trypsin inhibitor (KTI) genes. Such findings illustrate the power of this assembly in the analysis of large structural variations in soybean germplasm collections. The wild soybean genome assembly has wide applications in comparative genomic and evolutionary studies, as well as in crop breeding and improvement programs.
Human papillomavirus (HPV) integration is a key genetic event in cervical carcinogenesis. By conducting whole-genome sequencing and high-throughput viral integration detection, we identified 3,667 ...HPV integration breakpoints in 26 cervical intraepithelial neoplasias, 104 cervical carcinomas and five cell lines. Beyond recalculating frequencies for the previously reported frequent integration sites POU5F1B (9.7%), FHIT (8.7%), KLF12 (7.8%), KLF5 (6.8%), LRP1B (5.8%) and LEPREL1 (4.9%), we discovered new hot spots HMGA2 (7.8%), DLG2 (4.9%) and SEMA3D (4.9%). Protein expression from FHIT and LRP1B was downregulated when HPV integrated in their introns. Protein expression from MYC and HMGA2 was elevated when HPV integrated into flanking regions. Moreover, microhomologous sequence between the human and HPV genomes was significantly enriched near integration breakpoints, indicating that fusion between viral and human DNA may have occurred by microhomology-mediated DNA repair pathways. Our data provide insights into HPV integration-driven cervical carcinogenesis.
Short-read sequencing has enabled the de novo assembly of several individual human genomes, but with inherent limitations in characterizing repeat elements. Here we sequence a Chinese individual HX1 ...by single-molecule real-time (SMRT) long-read sequencing, construct a physical map by NanoChannel arrays and generate a de novo assembly of 2.93 Gb (contig N50: 8.3 Mb, scaffold N50: 22.0 Mb, including 39.3 Mb N-bases), together with 206 Mb of alternative haplotypes. The assembly fully or partially fills 274 (28.4%) N-gaps in the reference genome GRCh38. Comparison to GRCh38 reveals 12.8 Mb of HX1-specific sequences, including 4.1 Mb that are not present in previously reported Asian genomes. Furthermore, long-read sequencing of the transcriptome reveals novel spliced genes that are not annotated in GENCODE and are missed by short-read RNA-Seq. Our results imply that improved characterization of genome functional variation may require the use of a range of genomic technologies on diverse human populations.
Fatty alcohols are important components of surfactants and cosmetic products. The production of fatty alcohols from sustainable resources using microbial fermentation could reduce dependence on ...fossil fuels and greenhouse gas emission. However, the industrialization of this process has been hampered by the current low yield and productivity of this synthetic pathway. As a result of metabolic engineering strategies, an Escherichia coli mutant containing Synechococcus elongatus fatty acyl-ACP reductase showed improved yield and productivity. Proteomics analysis and in vitro enzymatic assays showed that endogenous E. coli AdhP is a major contributor to the reduction of fatty aldehydes to fatty alcohols. Both in vitro and in vivo results clearly demonstrated that the activity and expression level of fatty acyl-CoA/ACP reductase is the rate-limiting step in the current protocol. In 2.5-L fed-batch fermentation with glycerol as the only carbon source, the most productive E. coli mutant produced 0.75g/L fatty alcohols (0.02g fatty alcohol/g glycerol) with a productivity of up to 0.06g/L/h. This investigation establishes a promising synthetic pathway for industrial microbial production of fatty alcohols.
•Synechococcus elongatus fatty acyl-ACP reductase was introduced in E. coli.•The fatty aldehyde and fatty alcohol pathway were reconstituted in vitro.•AdhP in E. coli can reduce fatty aldehydes to fatty alcohols.•The production of fatty alcohols can be significantly increased in E. coli.•The fatty alcohols inhibit several key enzymes' expression involved in the fatty alcohol biosynthesis.
Maduramicin is the most efficient and possesses the largest market share of all anti-coccidiosis polyether antibiotics (ionophore); however, its biosynthetic gene cluster (BGC) has yet to been ...identified, and the associated strains have not been genetically engineered. Herein, we performed whole-genome sequencing of a maduramicin-producing industrial strain of
Actinomadura
sp. J1-007 and identified its BGC. Additionally, we analyzed the identified BGCs
in silico
to predict the biosynthetic pathway of maduramicin. We then developed a conjugation method for the non-spore-forming
Actinomadura
sp. J1-007, consisting of a site-specific integration method for gene overexpression. The maduramicin titer increased by 30% to 7.16 g/L in shake-flask fermentation following overexpression of type II thioesterase MadTE that is the highest titer at present. Our findings provide insights into the biosynthetic mechanism of polyethers and provide a platform for the metabolic engineering of maduramicin-producing microorganisms for overproduction and development of maduramicin analogs in the future.
Pathogens identification is critical for the proper diagnosis and precise treatment of infective endocarditis (IE). Although blood and valve cultures are the gold standard for IE pathogens detection, ...many cases are culture-negative, especially in patients who had received long-term antibiotic treatment, and precise diagnosis has therefore become a major challenge in the clinic. Metagenomic sequencing can provide both information on the pathogenic strain and the antibiotic susceptibility profile of patient samples without culturing, offering a powerful method to deal with culture-negative cases.
To assess the feasibility of a metagenomic approach to detect the causative pathogens in resected valves from IE patients, we employed both next-generation sequencing and Oxford Nanopore Technologies MinION nanopore sequencing for pathogens and antimicrobial resistance detection in seven culture-negative IE patients. Using our in-house developed bioinformatics pipeline, we analyzed the sequencing results generated from both platforms for the direct identification of pathogens from the resected valves of seven clinically culture-negative IE patients according to the modified Duke criteria.
Our results showed both metagenomics methods can be applied for the causative pathogen detection in all IE samples. Moreover, we were able to simultaneously characterize respective antimicrobial resistance features.
Metagenomic methods for IE detection can provide clinicians with valuable information to diagnose and treat IE patients after valve replacement surgery. However, more efforts should be made to optimize protocols for sample processing, sequencing and bioinformatics analysis.
Unambiguous evidence indicates that microbes are closely linked to various human diseases, including cancer. Most prior work investigating the microbiome of breast tissue describes an association ...between compositional differences of microbial species in benign and malignant tissues, but few studies have examined the relative abundance of microbial communities within human breast tissue at the species level. In this work, a total of 44 breast tissue samples including benign and malignant tissues with adjacent normal breast tissue pairs were collected, and Oxford Nanopore long-read sequencing was employed to assess breast tissue microbial signatures. Nearly 900 bacterial species were detected from the four dominant phyla: Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes. The bacteria with the highest abundance in all breast tissues was
, and its relative abundance increased with decreasing malignancy. We further examined the breast-tissue microbiome composition with different hormone-receptor statuses, and the relative abundance of the genus
increased most significantly in breast tissues. Our study provides a rationale for exploring microbiomes associated with breast carcinogenesis and cancer development. Further large-cohort investigation of the breast microbiome is necessary to characterize a microbial risk signature and develop potential microbial-based prevention therapies.
The ongoing global novel coronavirus pneumonia COVID‐19 outbreak has engendered numerous cases of infection and death. COVID‐19 diagnosis relies upon nucleic acid detection; however, currently ...recommended methods exhibit high false‐negative rates and are unable to identify other respiratory virus infections, thereby resulting in patient misdiagnosis and impeding epidemic containment. Combining the advantages of targeted amplification and long‐read, real‐time nanopore sequencing, herein, nanopore targeted sequencing (NTS) is developed to detect SARS‐CoV‐2 and other respiratory viruses simultaneously within 6–10 h, with a limit of detection of ten standard plasmid copies per reaction. Compared with its specificity for five common respiratory viruses, the specificity of NTS for SARS‐CoV‐2 reaches 100%. Parallel testing with approved real‐time reverse transcription‐polymerase chain reaction kits for SARS‐CoV‐2 and NTS using 61 nucleic acid samples from suspected COVID‐19 cases show that NTS identifies more infected patients (22/61) as positive, while also effectively monitoring for mutated nucleic acid sequences, categorizing types of SARS‐CoV‐2, and detecting other respiratory viruses in the test sample. NTS is thus suitable for COVID‐19 diagnosis; moreover, this platform can be further extended for diagnosing other viruses and pathogens.
A detection technology, nanopore targeted sequencing (NTS), for the accurate and comprehensive detection of SARS‐CoV‐2 and other respiratory viruses within 6–10 h is developed, which is suitable for the identification of suspected cases and used as a supplementary technique for the SARS‐CoV‐2 test. NTS can also monitor mutations in the virus and the type of virus.
Background
Microorganism identification is critical for the early diagnosis and management of infectious endophthalmitis, but traditional culture can yield false‐negative results. Nanopore targeted ...sequencing (NTS) is a third‐generation sequencing technique with multiple advantages. This study aimed to test aqueous humour or vitreous fluid samples from presumed cases of infectious endophthalmitis using NTS to evaluate the feasibility of NTS in diagnosing endophthalmitis, especially for culture‐negative cases.
Methods
This prospective study enrolled patients who presented to the Department of Ophthalmology of Union Hospital (Wuhan, China) between June 2018 and December 2020. The samples were sent immediately for routine microbiology culture processing and NTS assay.
Results
NTS identified microorganisms in 17 of 18 cases (94.4%) (eight culture‐positive cases, nine culture‐negative cases, and one case unavailable for culture). There was a high‐quality match between culture and NTS for culture‐positive cases. In the eight culture‐negative cases and the case unavailable for culture, NTS detected either bacteria, fungi, or a mixture of bacteria and fungi in the intraocular fluids. The average waiting times for the results of bacterial and fungal cultures were 48 and 72 h, respectively. The average time for the NTS results was 12 h.
Conclusions
NTS appears to be a promising diagnostic platform for diagnosing infectious endophthalmitis, even for culture‐negative cases.
Traditional microbiological methodology has limited sensitivity, detection range, and turnaround times in diagnosis of bloodstream infection in Febrile Neutropenia (FN) patients. A more rapid and ...sensitive detection technology is urgently needed. Here we used the newly developed Nanapore targeted sequencing (NTS) to diagnose the pathogens in blood samples. The diagnostic performance (sensitivity, specificity and turnaround time) of NTS detection of 202 blood samples from FN patients with hematologic disease was evaluated in comparison to blood culture and nested Polymerase Chain Reaction (PCR) followed by sanger sequence. The impact of NTS results on antibiotic treatment modification, the effectivity and mortality of the patients under the guidance of NTS results were assessed. The data showed that NTS had clinical sensitivity of 92.11%, clinical specificity of 78.41% compared with the blood culture and PCR combination. Importantly, the turnaround time for NTS was <24 h for all specimens, and the pre‐report time within 6 h in emergency cases was possible in clinical practice. Among 118 NTS positive patients, 98.3% patients' antibiotic regimens were guided according to NTS results. There was no significant difference in effectivity and mortality rate between Antibiotic regimen switched according to NTS group and Antibiotic regimen covering pathogens detected by NTS group. Therefore, NTS could yield a higher sensitivity, specificity and shorter turnaround time for broad‐spectrum pathogens identification in blood samples detection compared with traditional tests. It's also a good guidance in clinical targeted antibiotic treatment for FN patients with hematologic disease, thereby emerging as a promising technology for detecting infectious disease.
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