scholarly journals MINERVA: A facile strategy for SARS-CoV-2 whole genome deep sequencing of clinical samples

2020 ◽  
Author(s):  
Chen Chen ◽  
Jizhou Li ◽  
Lin Di ◽  
Qiuyu Jing ◽  
Pengcheng Du ◽  
...  
Keyword(s):  
High Performance ◽  
Vaccine Development ◽  
Viral Evolution ◽  
High Sensitivity ◽  
High Yield ◽  
Clinical Samples ◽  
High Coverage ◽  
Preparation Methods ◽  
Viral Genomes ◽  
Viral Spread

AbstractThe novel coronavirus disease 2019 (COVID-19) pandemic poses a serious public health risk. Analyzing the genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from clinical samples is crucial for the understanding of viral spread and viral evolution, as well as for vaccine development. Existing sample preparation methods for viral genome sequencing are demanding on user technique and time, and thus not ideal for time-sensitive clinical samples; these methods are also not optimized for high performance on viral genomes. We have developed MetagenomIc RNA EnRichment VirAl sequencing (MINERVA), a facile, practical, and robust approach for metagenomic and deep viral sequencing from clinical samples. This approach uses direct tagmentation of RNA/DNA hybrids using Tn5 transposase to greatly simplify the sequencing library construction process, while subsequent targeted enrichment can generate viral genomes with high sensitivity, coverage, and depth. We demonstrate the utility of MINERVA on pharyngeal, sputum and stool samples collected from COVID-19 patients, successfully obtaining both whole metatranscriptomes and complete high-depth high-coverage SARS-CoV-2 genomes from these clinical samples, with high yield and robustness. MINERVA is compatible with clinical nucleic extracts containing carrier RNA. With a shortened hands-on time from sample to virus-enriched sequencing-ready library, this rapid, versatile, and clinic-friendly approach will facilitate monitoring of viral genetic variations during outbreaks, both current and future.

scholarly journals Rapid longitudinal SARS-CoV-2 intra-host emergence of novel haplotypes regardless of immune deficiencies

2021 ◽  
Author(s):  
Laura Manuto ◽  
Marco Grazioli ◽  
Andrea Spitaleri ◽  
Paolo Fontana ◽  
Luca Bianco ◽  
...  
Keyword(s):  
Viral Evolution ◽  
Viral Genomes ◽  
Network Analyses ◽  
Viral Spread ◽  
Minimum Spanning Network ◽  
Immune Deficiencies ◽  
Chinese Tourists ◽  
Containment Strategy ◽  
First Time ◽  
Viral Sequences

On February 2020, the municipality of Vo’, a small town near Padua (Italy), was quarantined due to the first coronavirus disease 19 (COVID-19)-related death detected in Italy. The entire population was swab tested in two sequential surveys. Here we report the analysis of the viral genomes, which revealed that the unique ancestor haplotype introduced in Vo’ belongs to lineage B and, more specifically, to the subtype found at the end of January 2020 in two Chinese tourists visiting Rome and other Italian cities, carrying mutations G11083T and G26144T. The sequences, obtained for 87 samples, allowed us to investigate viral evolution while being transmitted within and across households and the effectiveness of the non-pharmaceutical interventions implemented in Vo’. We report, for the first time, evidence that novel viral haplotypes can naturally arise intra-host within an interval as short as two weeks, in approximately 30% of the infected individuals, regardless of symptoms severity or immune system deficiencies. Moreover, both phylogenetic and minimum spanning network analyses converge on the hypothesis that the viral sequences evolved from a unique common ancestor haplotype, carried by an index case. The lockdown extinguished both viral spread and the emergence of new variants, confirming the efficiency of this containment strategy. The information gathered from household was used to reconstructs possible transmission events.

scholarly journals Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples

2017 ◽  
Author(s):  
Josh Quick ◽  
Nathan D Grubaugh ◽  
Steven T Pullan ◽  
Ingra M Claro ◽  
Andrew D Smith ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Genome Sequencing ◽  
Emerging Infectious Diseases ◽  
Design Tool ◽  
Clinical Samples ◽  
Metagenomic Sequencing ◽  
Bioinformatics Pipeline ◽  
Preparation Methods ◽  
Consensus Sequences ◽  
Viral Genomes

Genome sequencing has become a powerful tool for studying emerging infectious diseases; however, genome sequencing directly from clinical samples without isolation remains challenging for viruses such as Zika, where metagenomic sequencing methods may generate insufficient numbers of viral reads. Here we present a protocol for generating coding-sequence complete genomes comprising an online primer design tool, a novel multiplex PCR enrichment protocol, optimised library preparation methods for the portable MinION sequencer (Oxford Nanopore Technologies) and the Illumina range of instruments, and a bioinformatics pipeline for generating consensus sequences. The MinION protocol does not require an internet connection for analysis, making it suitable for field applications with limited connectivity. Our method relies on multiplex PCR for targeted enrichment of viral genomes from samples containing as few as 50 genome copies per reaction. Viral consensus sequences can be achieved starting with clinical samples in 1-2 days following a simple laboratory workflow. This method has been successfully used by several groups studying Zika virus evolution and is facilitating an understanding of the spread of the virus in the Americas.

scholarly journals MrHAMER yields highly accurate single molecule viral sequences enabling analysis of intra-host evolution

2021 ◽  
Author(s):  
CM Gallardo ◽  
S Wang ◽  
DJ Montiel-Garcia ◽  
SJ Little ◽  
DM Smith ◽  
...  
Keyword(s):  
Long Range ◽  
Single Molecule ◽  
Vaccine Development ◽  
Complex Mixture ◽  
Therapeutic Benefit ◽  
Read Length ◽  
Clinical Samples ◽  
Viral Genomes ◽  
Immune Pressure ◽  
Sequencing Platforms

AbstractTechnical challenges remain in the sequencing of RNA viruses due to their high intra-host diversity. This bottleneck is particularly pronounced when interrogating long-range co-evolution given the read-length limitations of next-generation sequencing platforms. This has hampered the direct observation of long-range genetic interactions that code for protein-protein interfaces with relevance in both drug and vaccine development. Here we overcome these technical limitations by developing a nanopore-based long-range viral sequencing pipeline that yields accurate single molecule sequences of circulating virions from clinical samples. We demonstrate its utility in observing the evolution of individual HIV Gag-Pol genomes in response to antiviral pressure. Our pipeline, called Multi-read Hairpin Mediated Error-correction Reaction (MrHAMER), yields >1000s viral genomes per sample at 99.9% accuracy, maintains the original proportion of sequenced virions present in a complex mixture, and allows the detection of rare viral genomes with their associated mutations present at <1% frequency. This method facilitates scalable investigation of genetic correlates of resistance to both antiviral therapy and immune pressure, and enable the identification of novel host-viral and viral-viral interfaces that can be modulated for therapeutic benefit.

scholarly journals Guidelines for accurate genotyping of SARS-CoV-2 using amplicon-based sequencing of clinical samples

2020 ◽  
Author(s):  
Slawomir Kubik ◽  
Ana Claudia Marques ◽  
Xiaobin Xing ◽  
Janine Silvery ◽  
Claire Bertelli ◽  
...  
Keyword(s):  
Viral Load ◽  
Clinical Samples ◽  
Sampling Location ◽  
Reliable Determination ◽  
Viral Genomes ◽  
Viral Spread ◽  
Broad Applicability ◽  
Input Material ◽  
Correct Sequence ◽  
The Impact

AbstractBackgroundSARS-CoV-2 genotyping has been instrumental to monitor virus evolution and transmission during the pandemic. The reliability of the information extracted from the genotyping efforts depends on a number of aspects, including the quality of the input material, applied technology and potential laboratory-specific biases. These variables must be monitored to ensure genotype reliability. The current lack of guidelines for SARS-CoV-2 genotyping leads to inclusion of error-containing genome sequences in studies of viral spread and evolution.ResultsWe used clinical samples and synthetic viral genomes to evaluate the impact of experimental factors, including viral load and sequencing depth, on correct sequence determination using an amplicon-based approach. We found that at least 1000 viral genomes are necessary to confidently detect variants in the genome at frequencies of 10% or higher. The broad applicability of our recommendations was validated in >200 clinical samples from six independent laboratories. The genotypes of clinical isolates with viral load above the recommended threshold cluster by sampling location and period. Our analysis also supports the rise in frequency of 20A.EU1 and 20A.EU2, two recently reported European strains whose dissemination was favoured by travelling during the summer 2020.ConclusionsWe present much-needed recommendations for reliable determination of SARS-CoV-2 genome sequence and demonstrate their broad applicability in a large cohort of clinical samples.

scholarly journals Rapid and extraction-free detection of SARS-CoV-2 from saliva with colorimetric LAMP

2020 ◽  
Author(s):  
Matthew A. Lalli ◽  
Xuhua Chen ◽  
S. Joshua Langmade ◽  
Catrina C. Fronick ◽  
Christopher S. Sawyer ◽  
...  
Keyword(s):  
High Performance ◽  
Colorimetric Assay ◽  
Rna Extraction ◽  
Human Saliva ◽  
Clinical Samples ◽  
Treatment Protocols ◽  
Diagnostic Assays ◽  
Viral Genomes ◽  
Nasal Swabs ◽  
Pre Treatment

AbstractRapid, reliable, and widespread testing is required to curtail the ongoing COVID-19 pandemic. Current gold standard diagnostic assays are hampered by supply shortages in critical reagents including nasal swabs, RNA extraction kits, personal protective equipment (PPE), instrumentation, and labor. Here we present an approach to overcome these challenges with the development of a rapid colorimetric assay using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) optimized on human saliva samples without an RNA purification step. We describe our optimizations of the LAMP reaction and saliva pre-treatment protocols that enabled rapid and sensitive detection of < 102 viral genomes per reaction in contrived saliva controls. We also observed high performance of this assay on a limited number of clinical saliva samples. While thorough validation on additional clinical samples will be needed before such an assay can be widely used, these preliminary results demonstrate a promising approach to overcome the current bottlenecks limiting widespread testing.

Determination of Selected Phthalates in Some Commercial Cosmetic Products by HPLC-UV

2020 ◽  
Vol 23 (10) ◽  
pp. 1010-1022
Author(s):  
Emrah Dural
Keyword(s):  
High Performance ◽  
Phthalate Esters ◽  
High Sensitivity ◽  
Hplc Method ◽  
Cosmetic Products ◽  
Limit Of Quantification ◽  
Nail Polish ◽  
Accuracy And Precision ◽  
Butyl Phthalate

Aim and scope: Due to the serious toxicological risks and their widespread use, quantitative determination of phthalates in cosmetic products have importance for public health. The aim of this study was to develop a validated simple, rapid and reliable high-performance liquid chromatography (HPLC) method for the determination of phthalates which are; dimethyl phthalate (DMP), diethyl phthalate (DEP), benzyl butyl phthalate (BBP), di-n-butyl phthalate (DBP), di(2- ethylhexyl) phthalate (DEHP), in cosmetic products and to investigate these phthalate (PHT) levels in 48 cosmetic products marketing in Sivas, Turkey. Materials and Methods: Separation was achieved by a reverse-phase ACE-5 C18 column (4.6 x 250 mm, 5.0 μm). As the mobile phase, 5 mM KH2PO4 and acetonitrile were used gradiently at 1.5 ml min-1. All PHT esters were detected at 230 nm and the run time was taking 21 minutes. Results: This method showed the high sensitivity value the limit of quantification (LOQ) values for which are below 0.64 μg mL-1 of all phthalates. Method linearity was ≥0.999 (r2). Accuracy and precision values of all phthalates were calculated between (-6.5) and 6.6 (RE%) and ≤6.2 (RSD%), respectively. Average recovery was between 94.8% and 99.6%. Forty-eight samples used for both babies and adults were successfully analyzed by the developed method. Results have shown that, DMP (340.7 μg mL-1 ±323.7), DEP (1852.1 μg mL-1 ± 2192.0), and DBP (691.3 μg mL-1 ± 1378.5) were used highly in nail polish, fragrance and cream products, respectively. Conclusion: Phthalate esters, which are mostly detected in the content of fragrance, cream and nail polish products and our research in general, are DEP (1852.1 μg mL-1 ± 2192.0), DBP (691.3 μg mL-1 ± 1378.5) and DMP (340.7 μg mL-1 ±323.7), respectively. Phthalates were found in the content of all 48 cosmetic products examined, and the most detected phthalates in general average were DEP (581.7 μg mL-1 + 1405.2) with a rate of 79.2%. The unexpectedly high phthalate content in the examined cosmetic products revealed a great risk of these products on human health. The developed method is a simple, sensitive, reliable and economical alternative for the determination of phthalates in the content of cosmetic products, it can be used to identify phthalate esters in different products after some modifications.

scholarly journals Latest Advances of Virology Research Using CRISPR/Cas9-Based Gene-Editing Technology and Its Application to Vaccine Development

Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 779
Author(s):  
Man Teng ◽  
Yongxiu Yao ◽  
Venugopal Nair ◽  
Jun Luo
Keyword(s):  
Biological Sciences ◽  
Gene Therapy ◽  
Genetic Engineering ◽  
Gene Function ◽  
Viral Genome ◽  
Gene Editing ◽  
Vaccine Development ◽  
Future Prospects ◽  
Dna Viruses ◽  
Viral Genomes

In recent years, the CRISPR/Cas9-based gene-editing techniques have been well developed and applied widely in several aspects of research in the biological sciences, in many species, including humans, animals, plants, and even in viruses. Modification of the viral genome is crucial for revealing gene function, virus pathogenesis, gene therapy, genetic engineering, and vaccine development. Herein, we have provided a brief review of the different technologies for the modification of the viral genomes. Particularly, we have focused on the recently developed CRISPR/Cas9-based gene-editing system, detailing its origin, functional principles, and touching on its latest achievements in virology research and applications in vaccine development, especially in large DNA viruses of humans and animals. Future prospects of CRISPR/Cas9-based gene-editing technology in virology research, including the potential shortcomings, are also discussed.

scholarly journals Design of a Sensitive Balloon Sensor for Safe Human–Robot Interaction

Sensors ◽  
2021 ◽  
Vol 21 (6) ◽  
pp. 2163
Author(s):  
Dongjin Kim ◽  
Seungyong Han ◽  
Taewi Kim ◽  
Changhwan Kim ◽  
Doohoe Lee ◽  
...  
Keyword(s):  
High Performance ◽  
Strain Sensor ◽  
High Sensitivity ◽  
Human Robot Interaction ◽  
Large Area ◽  
Tactile Sensors ◽  
Robot Interaction ◽  
Mechanical Crack ◽  
Flexible Strain Sensor ◽  
Contact Sensing

As the safety of a human body is the main priority while interacting with robots, the field of tactile sensors has expanded for acquiring tactile information and ensuring safe human–robot interaction (HRI). Existing lightweight and thin tactile sensors exhibit high performance in detecting their surroundings. However, unexpected collisions caused by malfunctions or sudden external collisions can still cause injuries to rigid robots with thin tactile sensors. In this study, we present a sensitive balloon sensor for contact sensing and alleviating physical collisions over a large area of rigid robots. The balloon sensor is a pressure sensor composed of an inflatable body of low-density polyethylene (LDPE), and a highly sensitive and flexible strain sensor laminated onto it. The mechanical crack-based strain sensor with high sensitivity enables the detection of extremely small changes in the strain of the balloon. Adjusting the geometric parameters of the balloon allows for a large and easily customizable sensing area. The weight of the balloon sensor was approximately 2 g. The sensor is employed with a servo motor and detects a finger or a sheet of rolled paper gently touching it, without being damaged.

scholarly journals SARS-CoV-2 Sequence Characteristics of COVID-19 Persistence and Reinfection

2021 ◽  
Author(s):  
Manish C Choudhary ◽  
Charles R Crain ◽  
Xueting Qiu ◽  
William Hanage ◽  
Jonathan Z Li
Keyword(s):  
Persistent Infection ◽  
Viral Evolution ◽  
Phylogenetic Reconstruction ◽  
Geographic Region ◽  
Antibody Treatment ◽  
Viral Genomes ◽  
Monoclonal Antibody Treatment ◽  
Viral Sequences ◽  
Sequence Characteristics ◽  
Baseline Health

Abstract Background Both SARS-CoV-2 reinfection and persistent infection have been reported, but sequence characteristics in these scenarios have not been described. We assessed published cases of SARS-CoV-2 reinfection and persistence, characterizing the hallmarks of reinfecting sequences and the rate of viral evolution in persistent infection. Methods A systematic review of PubMed was conducted to identify cases of SARS-CoV-2 reinfection and persistence with available sequences. Nucleotide and amino acid changes in the reinfecting sequence were compared to both the initial and contemporaneous community variants. Time-measured phylogenetic reconstruction was performed to compare intra-host viral evolution in persistent SARS-CoV-2 to community-driven evolution. Results Twenty reinfection and nine persistent infection cases were identified. Reports of reinfection cases spanned a broad distribution of ages, baseline health status, reinfection severity, and occurred as early as 1.5 months or &gt;8 months after the initial infection. The reinfecting viral sequences had a median of 17.5 nucleotide changes with enrichment in the ORF8 and N genes. The number of changes did not differ by the severity of reinfection and reinfecting variants were similar to the contemporaneous sequences circulating in the community. Patients with persistent COVID-19 demonstrated more rapid accumulation of sequence changes than seen with community-driven evolution with continued evolution during convalescent plasma or monoclonal antibody treatment. Conclusions Reinfecting SARS-CoV-2 viral genomes largely mirror contemporaneous circulating sequences in that geographic region, while persistent COVID-19 has been largely described in immunosuppressed individuals and is associated with accelerated viral evolution.

scholarly journals Iodine reduction for reproducible and high-performance perovskite solar cells and modules

2021 ◽  
Vol 7 (10) ◽  
pp. eabe8130
Author(s):  
Shangshang Chen ◽  
Xun Xiao ◽  
Hangyu Gu ◽  
Jinsong Huang
Keyword(s):  
Solar Cells ◽  
High Performance ◽  
High Efficiency ◽  
Electronic Materials ◽  
Substantial Reduction ◽  
Perovskite Solar Cells ◽  
High Yield ◽  
Operation Conditions ◽  
Record Value ◽  
Precursor Powders

Perovskite-based electronic materials and devices such as perovskite solar cells (PSCs) have notoriously bad reproducibility, which greatly impedes both fundamental understanding of their intrinsic properties and real-world applications. Here, we report that organic iodide perovskite precursors can be oxidized to I2 even for carefully sealed precursor powders or solutions, which markedly deteriorates the performance and reproducibility of PSCs. Adding benzylhydrazine hydrochloride (BHC) as a reductant into degraded precursor solutions can effectively reduce the detrimental I2 back to I−, accompanied by a substantial reduction of I3−-induced charge traps in the films. BHC residuals in perovskite films further stabilize the PSCs under operation conditions. BHC improves the stabilized efficiency of the blade-coated p-i-n structure PSCs to a record value of 23.2% (22.62 ± 0.40% certified by National Renewable Energy Laboratory), and the high-efficiency devices have a very high yield. A stabilized aperture efficiency of 18.2% is also achieved on a 35.8-cm2 mini-module.

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