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1.
PLoS One ; 17(2): e0263563, 2022.
Article in English | MEDLINE | ID: covidwho-1793526

ABSTRACT

Deletions frequently occur in the six accessory genes of SARS-CoV-2, but most genomes with deletions are sporadic and have limited spreading capability. Here, we analyze deletions in the ORF7a of the N.7 lineage, a unique Uruguayan clade from the Brazilian B.1.1.33 lineage. Thirteen samples collected during the early SARS-CoV-2 wave in Uruguay had deletions in the ORF7a. Complete genomes were obtained by Illumina next-generation sequencing, and deletions were confirmed by Sanger sequencing and capillary electrophoresis. The N.7 lineage includes several individuals with a 12-nucleotide deletion that removes four amino acids of the ORF7a. Notably, four individuals underwent an additional 68-nucleotide novel deletion that locates 44 nucleotides downstream in the terminal region of the same ORF7a. The simultaneous occurrence of the 12 and 68-nucleotide deletions fuses the ORF7a and ORF7b, two contiguous accessory genes that encode transmembrane proteins with immune-modulation activity. The fused ORF retains the signal peptide and the complete Ig-like fold of the 7a protein and the transmembrane domain of the 7b protein, suggesting that the fused protein plays similar functions to original proteins in a single format. Our findings evidence the remarkable dynamics of SARS-CoV-2 and the possibility that single and consecutive deletions occur in accessory genes and promote changes in the genomic organization that help the virus explore genetic variations and select for new, higher fit changes.


Subject(s)
COVID-19/virology , Cell Lineage , Gene Deletion , Genome, Viral , Open Reading Frames/genetics , SARS-CoV-2/genetics , Viral Proteins/genetics , Adult , Aged , COVID-19/epidemiology , COVID-19/genetics , Child , Female , High-Throughput Nucleotide Sequencing , Humans , Male , Middle Aged , Phylogeny , SARS-CoV-2/classification , SARS-CoV-2/isolation & purification , Uruguay/epidemiology
2.
Front Public Health ; 9: 738412, 2021.
Article in English | MEDLINE | ID: covidwho-1775888

ABSTRACT

Background: Unbiased metagenomic next-generation sequencing (mNGS) detects pathogens in a target-independent manner. It is not well-understood whether mNGS has comparable sensitivity to target-dependent nucleic acid test for pathogen identification. Methods: This study included 31 patients with chickenpox and neurological symptoms for screening of possible varicella-zoster virus (VZV) central nervous system (CNS) infection. Microbiological diagnosing of VZV cerebrospinal fluid (CSF) infection was performed on stored CSF samples using mNGS, quantitative and qualitative VZV-specific PCR assays, and VZV IgM antibodies test. Results: The median age was 30.0 [interquartile range (IQR), 24.3-33.3] years. 51.6% of the patients were men. About 80.6% of the patients had normal CSF white blood cell counts (≤ 5 × 106/L). VZV IgM antibodies presented in 16.1% of the CSF samples, and nucleic acids were detectable in 16.1 and 9.7% using two different VZV-specific real-time PCR protocols. Intriguingly, maximal identification of VZV elements was achieved by CSF mNGS (p = 0.001 and p = 007; compared with qualitative PCR and VZV IgM antibody test, respectively), with sequence reads of VZV being reported in 51.6% (16/31) of the CSF samples. All VZV PCR positive samples were positive when analyzed by mNGS. Of note, human betaherpesvirus 6A with clinical significance was unexpectedly detected in one CSF sample. Conclusions: Our study suggests that CSF mNGS may have higher sensitivity for VZV detection than CSF VZV PCR and antibody tests, and has the advantage of identifying unexpected pathogens.


Subject(s)
Central Nervous System Infections , Chickenpox , Adult , Central Nervous System , Herpesvirus 3, Human/genetics , High-Throughput Nucleotide Sequencing/methods , Humans , Male
3.
PLoS One ; 17(4): e0265220, 2022.
Article in English | MEDLINE | ID: covidwho-1775443

ABSTRACT

As different SARS-CoV-2 variants emerge and with the continuous evolvement of sub lineages of the delta variant, it is crucial that all countries carry out sequencing of at least >1% of their infections, in order to detect emergence of variants with higher transmissibility and with ability to evade immunity. However, due to limited resources as many resource poor countries are unable to sequence adequate number of viruses, we compared to usefulness of a two-step commercially available multiplex real-time PCR assay to detect important single nucleotide polymorphisms (SNPs) associated with the variants and compared the sensitivity, accuracy and cost effectiveness of the Illumina sequencing platform and the Oxford Nanopore Technologies' (ONT) platform. 138/143 (96.5%) identified as the alpha and 36/39 (92.3%) samples identified as the delta variants due to the presence of lineage defining SNPs by the multiplex real time PCR, were assigned to the same lineage by either of the two sequencing platforms. 34/37 of the samples sequenced by ONT had <5% ambiguous bases, while 21/37 samples sequenced using Illumina generated <5%. However, the mean PHRED scores averaged at 32.35 by Illumina reads but 10.78 in ONT. This difference results in a base error probability of 1 in 10 by the ONT and 1 in 1000 for Illumina sequencing platform. Sub-consensus single nucleotide variations (SNV) are highly correlated between both platforms (R2 = 0.79) while indels appear to have a weaker correlation (R2 = 0.13). Although the ONT had a slightly higher error rate compared to the Illumina technology, it achieved higher coverage with a lower number or reads, generated less ambiguous bases and was significantly less expensive than Illumina sequencing technology.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , COVID-19/genetics , High-Throughput Nucleotide Sequencing , Humans , Real-Time Polymerase Chain Reaction , SARS-CoV-2/genetics , Whole Genome Sequencing/methods
4.
Viruses ; 14(3)2022 03 14.
Article in English | MEDLINE | ID: covidwho-1765953

ABSTRACT

Virus pandemics have happened, are happening and will happen again. In recent decades, the rate of zoonotic viral spillover into humans has accelerated, mirroring the expansion of our global footprint and travel network, including the expansion of viral vectors and the destruction of natural spaces, bringing humans closer to wild animals. Once viral cross-species transmission to humans occurs, transmission cannot be stopped by cement walls but by developing barriers based on knowledge that can prevent or reduce the effects of any pandemic. Controlling a local transmission affecting few individuals is more efficient that confronting a community outbreak in which infections cannot be traced. Genetic detection, identification, and characterization of infectious agents using next-generation sequencing (NGS) has been proven to be a powerful tool allowing for the development of fast PCR-based molecular assays, the rapid development of vaccines based on mRNA and DNA, the identification of outbreaks, transmission dynamics and spill-over events, the detection of new variants and treatment of vaccine resistance mutations, the development of direct-acting antiviral drugs, the discovery of relevant minority variants to improve knowledge of the viral life cycle, strengths and weaknesses, the potential for becoming dominant to take appropriate preventive measures, and the discovery of new routes of viral transmission.


Subject(s)
Hepatitis C, Chronic , Viruses , Animals , Antiviral Agents , High-Throughput Nucleotide Sequencing , Pandemics
5.
Microb Pathog ; 165: 105506, 2022 Apr.
Article in English | MEDLINE | ID: covidwho-1763898

ABSTRACT

Since its first appearance, the SARS-CoV-2 has spread rapidly in the human population, reaching the pandemic scale with >280 million confirmed infections and more than 5 million deaths to date (https://covid19.who.int/). These data justify the urgent need to enhance our understanding of SARS-CoV-2 effects in the respiratory system, including those linked to co-infections. The principal aim of our study is to investigate existing correlations in the nasopharynx between the bacterial community, potential pathogens, and SARS-CoV-2 infection. The main aim of this study was to provide evidence pointing to possible relationships between components of the bacterial community and SARS-CoV-2 in the nasopharynx. Meta-transcriptomic profiling of the nasopharyngeal microbial community was carried out in 89 SARS-Cov-2 positive subjects from the Campania Region in Italy. To this end, RNA extracted from nasopharyngeal swabs collected at different times during the initial phases of the pandemic was analyzed by Next-Generation Sequencing (NGS). Results show a consistently high presence of members of the Proteobacteria (41.85%), Firmicutes (28.54%), and Actinobacteria (16.10%) phyla, and an inverted correlation between the host microbiome, co-infectious bacteria, and super-potential pathogens such as Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, and Neisseria gonorrhoeae. In depth characterization of microbiota composition in the nasopharynx can provide clues to understand its potential contribution to the clinical phenotype of Covid-19, clarifying the interaction between SARS-Cov-2 and the bacterial flora of the host, and highlighting its dysbiosis and the presence of pathogens that could affect the patient's disease progression and outcome.


Subject(s)
COVID-19 , Coinfection , Microbiota , Bacteria/genetics , Coinfection/epidemiology , High-Throughput Nucleotide Sequencing , Humans , Italy/epidemiology , Microbiota/genetics , Nasopharynx/microbiology , Pandemics , SARS-CoV-2/genetics
6.
Int J Mol Sci ; 23(6)2022 Mar 17.
Article in English | MEDLINE | ID: covidwho-1760650

ABSTRACT

The recent covid crisis has provided important lessons for academia and industry regarding digital reorganization. Among the fascinating lessons from these times is the huge potential of data analytics and artificial intelligence. The crisis exponentially accelerated the adoption of analytics and artificial intelligence, and this momentum is predicted to continue into the 2020s and beyond. Drug development is a costly and time-consuming business, and only a minority of approved drugs generate returns exceeding the research and development costs. As a result, there is a huge drive to make drug discovery cheaper and faster. With modern algorithms and hardware, it is not too surprising that the new technologies of artificial intelligence and other computational simulation tools can help drug developers. In only two years of covid research, many novel molecules have been designed/identified using artificial intelligence methods with astonishing results in terms of time and effectiveness. This paper reviews the most significant research on artificial intelligence in de novo drug design for COVID-19 pharmaceutical research.


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Artificial Intelligence , COVID-19/drug therapy , COVID-19/virology , Drug Design , SARS-CoV-2/drug effects , Antiviral Agents/therapeutic use , Drug Discovery/methods , Drug Evaluation, Preclinical , High-Throughput Nucleotide Sequencing , Humans , Ligands , SARS-CoV-2/physiology , Small Molecule Libraries , Structure-Activity Relationship
7.
Arch Virol ; 167(4): 1175-1179, 2022 Apr.
Article in English | MEDLINE | ID: covidwho-1748474

ABSTRACT

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) amplicon contamination was discovered due to next-generation sequencing (NGS) reads mapping in the negative controls. Environmental screening was undertaken to determine the source of contamination, which was suspected to be evaporation during polymerase chain reaction (PCR) assays while using the coronavirus disease 2019 (COVID-19) ARTIC protocol. A decontamination strategy is hereby documented to assist laboratories that may experience similar amplicon contamination. Routine molecular laboratory environmental screening as a quality control is highly recommended.


Subject(s)
COVID-19 , Laboratories , COVID-19/prevention & control , Decontamination , High-Throughput Nucleotide Sequencing , Humans , SARS-CoV-2/genetics
8.
Sci Rep ; 12(1): 4631, 2022 03 17.
Article in English | MEDLINE | ID: covidwho-1747175

ABSTRACT

Using next generation sequencing technology, we identified a novel SARS-CoV-2 variant with a truncated ORF8 protein mutation near the end of the viral genome from nucleotides 27,878 to 27,958. This point mutation from C to T at nucleotide 27,956 changed the amino acid codon CAA (glutamine) to a stop codon, TAA, created a novel stop codon in ORF8 gene, resulting in a much smaller ORF8 protein (26 aa) than the wild type ORF8 protein (121 aa). This variant belongs to Pango lineage B.1.1291, which also contains the D614G mutation in the Spike (S) gene. The B.1.1291 lineage is predominantly circulated in the United States of America (97.18%), although it was also found in other counties (Russia, Canada, Latvia, Chile, India, Japan, Colombia, Germany, Greece, Mexico, and UK). A total of 340 closely related variants to this novel variant were identified in GISAID database with collection dates ranged from 3/6/2020 to 10/21/2020. In addition, a search within NCBI Genbank database found that 108,405 of 873,230 (12.4%) SAR-CoV-2 complete genomes contain this truncated ORF8 protein mutation, indicating this mutation may arise spontaneously in other lineages as well. The wide distribution of this mutation indicates that this truncated ORF8 protein mutation may provide the virus a growth advantage and adaptive evolution.


Subject(s)
COVID-19 , Chiroptera , Animals , COVID-19/epidemiology , COVID-19/genetics , High-Throughput Nucleotide Sequencing , Humans , SARS-CoV-2/genetics
9.
Genome Biol ; 22(1): 324, 2021 11 29.
Article in English | MEDLINE | ID: covidwho-1745431

ABSTRACT

High-throughput single-cell technologies hold the promise of discovering novel cellular relationships with disease. However, analytical workflows constructed for these technologies to associate cell proportions with disease often employ unsupervised clustering techniques that overlook the valuable hierarchical structures that have been used to define cell types. We present treekoR, a framework that empirically recapitulates these structures, facilitating multiple quantifications and comparisons of cell type proportions. Our results from twelve case studies reinforce the importance of quantifying proportions relative to parent populations in the analyses of cytometry data - as failing to do so can lead to missing important biological insights.


Subject(s)
Flow Cytometry/methods , Phenotype , CD8 Antigens , CD8-Positive T-Lymphocytes , COVID-19 , Cluster Analysis , Gene Expression Profiling , High-Throughput Nucleotide Sequencing , Humans , Single-Cell Analysis/methods
10.
Microb Genom ; 8(3)2022 03.
Article in English | MEDLINE | ID: covidwho-1746154

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is adaptively evolving to ensure its persistence within human hosts. It is therefore necessary to continuously monitor the emergence and prevalence of novel variants that arise. Importantly, some mutations have been associated with both molecular diagnostic failures and reduced or abrogated next-generation sequencing (NGS) read coverage in some genomic regions. Such impacts are particularly problematic when they occur in genomic regions such as those that encode the spike (S) protein, which are crucial for identifying and tracking the prevalence and dissemination dynamics of concerning viral variants. Targeted Sanger sequencing presents a fast and cost-effective means to accurately extend the coverage of whole-genome sequences. We designed a custom set of primers to amplify a 401 bp segment of the receptor-binding domain (RBD) (between positions 22698 and 23098 relative to the Wuhan-Hu-1 reference). We then designed a Sanger sequencing wet-laboratory protocol. We applied the primer set and wet-laboratory protocol to sequence 222 samples that were missing positions with key mutations K417N, E484K, and N501Y due to poor coverage after NGS sequencing. Finally, we developed SeqPatcher, a Python-based computational tool to analyse the trace files yielded by Sanger sequencing to generate consensus sequences, or take preanalysed consensus sequences in fasta format, and merge them with their corresponding whole-genome assemblies. We successfully sequenced 153 samples of 222 (69 %) using Sanger sequencing and confirmed the occurrence of key beta variant mutations (K417N, E484K, N501Y) in the S genes of 142 of 153 (93 %) samples. Additionally, one sample had the Y508F mutation and four samples the S477N. Samples with RT-PCR C t scores ranging from 13.85 to 37.47 (mean=25.70) could be Sanger sequenced efficiently. These results show that our method and pipeline can be used to improve the quality of whole-genome assemblies produced using NGS and can be used with any pairs of the most used NGS and Sanger sequencing platforms.


Subject(s)
Genome, Viral , SARS-CoV-2/genetics , Sequence Analysis, DNA/methods , High-Throughput Nucleotide Sequencing , Mutation
11.
Int J Mol Sci ; 23(6)2022 Mar 15.
Article in English | MEDLINE | ID: covidwho-1742492

ABSTRACT

SARS-CoV-2 variants surveillance is a worldwide task that has been approached with techniques such as Next Generation Sequencing (NGS); however, this technology is not widely available in developing countries because of the lack of equipment and limited funding in science. An option is to deploy a RT-qPCR screening test which aids in the analysis of a higher number of samples, in a shorter time and at a lower cost. In this study, variants present in samples positive for SARS-CoV-2 were identified with a RT-qPCR mutation screening kit and were later confirmed by NGS. A sample with an abnormal result was found with the screening test, suggesting the simultaneous presence of two viral populations with different mutations. The DRAGEN Lineage analysis identified the Delta variant, but there was no information about the other three mutations previously detected. When the sequenced data was deeply analyzed, there were reads with differential mutation patterns, that could be identified and classified in terms of relative abundance, whereas only the dominant population was reported by DRAGEN software. Since most of the software developed to analyze SARS-CoV-2 sequences was aimed at obtaining the consensus sequence quickly, the information about viral populations within a sample is scarce. Here, we present a faster and deeper SARS-CoV-2 surveillance method, from RT-qPCR screening to NGS analysis.


Subject(s)
COVID-19/diagnosis , DNA Mutational Analysis/methods , Genome, Viral/genetics , Mutation , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , COVID-19/epidemiology , COVID-19/virology , High-Throughput Nucleotide Sequencing/methods , Humans , Pandemics/prevention & control , Reproducibility of Results , SARS-CoV-2/physiology , Sensitivity and Specificity
12.
Nat Commun ; 13(1): 1321, 2022 03 14.
Article in English | MEDLINE | ID: covidwho-1740438

ABSTRACT

Infectious disease monitoring on Oxford Nanopore Technologies (ONT) platforms offers rapid turnaround times and low cost. Tracking low frequency intra-host variants provides important insights with respect to elucidating within-host viral population dynamics and transmission. However, given the higher error rate of ONT, accurate identification of intra-host variants with low allele frequencies remains an open challenge with no viable computational solutions available. In response to this need, we present Variabel, a novel approach and first method designed for rescuing low frequency intra-host variants from ONT data alone. We evaluate Variabel on both synthetic data (SARS-CoV-2) and patient derived datasets (Ebola virus, norovirus, SARS-CoV-2); our results show that Variabel can accurately identify low frequency variants below 0.5 allele frequency, outperforming existing state-of-the-art ONT variant callers for this task. Variabel is open-source and available for download at: www.gitlab.com/treangenlab/variabel .


Subject(s)
COVID-19 , Nanopore Sequencing , Nanopores , High-Throughput Nucleotide Sequencing/methods , Humans , SARS-CoV-2/genetics
13.
PLoS One ; 17(3): e0264855, 2022.
Article in English | MEDLINE | ID: covidwho-1736511

ABSTRACT

Since December 2019 the world has been facing the outbreak of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Identification of infected patients and discrimination from other respiratory infections have so far been accomplished by using highly specific real-time PCRs. Here we present a rapid multiplex approach (RespiCoV), combining highly multiplexed PCRs and MinION sequencing suitable for the simultaneous screening for 41 viral and five bacterial agents related to respiratory tract infections, including the human coronaviruses NL63, HKU1, OC43, 229E, Middle East respiratory syndrome coronavirus, SARS-CoV, and SARS-CoV-2. RespiCoV was applied to 150 patient samples with suspected SARS-CoV-2 infection and compared with specific real-time PCR. Additionally, several respiratory tract pathogens were identified in samples tested positive or negative for SARS-CoV-2. Finally, RespiCoV was experimentally compared to the commercial RespiFinder 2SMART multiplex screening assay (PathoFinder, The Netherlands).


Subject(s)
Bacteria/genetics , COVID-19/diagnosis , High-Throughput Nucleotide Sequencing/methods , RNA Viruses/genetics , Respiratory Tract Infections/diagnosis , SARS-CoV-2/genetics , Bacteria/isolation & purification , COVID-19/virology , Coronavirus/genetics , Coronavirus/isolation & purification , DNA, Bacterial/chemistry , DNA, Bacterial/metabolism , Herpesvirus 1, Human/genetics , Herpesvirus 1, Human/isolation & purification , Humans , Multiplex Polymerase Chain Reaction , Nanopores , Orthomyxoviridae/genetics , Orthomyxoviridae/isolation & purification , RNA Viruses/isolation & purification , RNA, Viral/chemistry , RNA, Viral/metabolism , Respiratory Tract Infections/microbiology , Respiratory Tract Infections/virology , SARS-CoV-2/isolation & purification
14.
J Virol ; 96(5): e0218621, 2022 03 09.
Article in English | MEDLINE | ID: covidwho-1736028

ABSTRACT

Recent emergence of SARS-CoV-1 variants demonstrates the potential of this virus for targeted evolution, despite its overall genomic stability. Here we show the dynamics and the mechanisms behind the rapid adaptation of SARS-CoV-2 to growth in Vero E6 cells. The selective advantage for growth in Vero E6 cells is due to increased cleavage efficiency by cathepsins at the mutated S1/S2 site. S1/S2 site also constitutes a heparan sulfate (HS) binding motif that influenced virus growth in Vero E6 cells, but HS antagonist did not inhibit virus adaptation in these cells. The entry of Vero E6-adapted virus into human cells is defective because the mutated spike variants are poorly processed by furin or TMPRSS2. Minor subpopulation that lack the furin cleavage motif in the spike protein rapidly become dominant upon passaging through Vero E6 cells, but wild type sequences are maintained at low percentage in the virus swarm and mediate a rapid reverse adaptation if the virus is passaged again on TMPRSS2+ human cells. Our data show that the spike protein of SARS-CoV-2 can rapidly adapt itself to available proteases and argue for deep sequence surveillance to identify the emergence of novel variants. IMPORTANCE Recently emerging SARS-CoV-2 variants B.1.1.7 (alpha variant), B.1.617.2 (delta variant), and B.1.1.529 (omicron variant) harbor spike mutations and have been linked to increased virus pathogenesis. The emergence of these novel variants highlights coronavirus adaptation and evolution potential, despite the stable consensus genotype of clinical isolates. We show that subdominant variants maintained in the virus population enable the virus to rapidly adapt to selection pressure. Although these adaptations lead to genotype change, the change is not absolute and genomes with original genotype are maintained in the virus swarm. Thus, our results imply that the relative stability of SARS-CoV-2 in numerous independent clinical isolates belies its potential for rapid adaptation to new conditions.


Subject(s)
COVID-19/metabolism , Furin/metabolism , SARS-CoV-2/physiology , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Adaptation, Physiological , Animals , COVID-19/genetics , COVID-19/virology , Chlorocebus aethiops , Cytopathogenic Effect, Viral , Furin/genetics , HEK293 Cells , High-Throughput Nucleotide Sequencing , Humans , Mutation , SARS-CoV-2/genetics , Serine Endopeptidases/genetics , Spike Glycoprotein, Coronavirus/genetics , Vero Cells , Virus Replication
15.
Trends Biotechnol ; 40(4): 463-481, 2022 04.
Article in English | MEDLINE | ID: covidwho-1735006

ABSTRACT

Humoral immunity is divided into the cellular B cell and protein-level antibody responses. High-throughput sequencing has advanced our understanding of both these fundamental aspects of B cell immunology as well as aspects pertaining to vaccine and therapeutics biotechnology. Although the protein-level serum and mucosal antibody repertoire make major contributions to humoral protection, the sequence composition and dynamics of antibody repertoires remain underexplored. This limits insight into important immunological and biotechnological parameters such as the number of antigen-specific antibodies, which are for example, relevant for pathogen neutralization, microbiota regulation, severity of autoimmunity, and therapeutic efficacy. High-resolution mass spectrometry (MS) has allowed initial insights into the antibody repertoire. We outline current challenges in MS-based sequence analysis of antibody repertoires and propose strategies for their resolution.


Subject(s)
Antibodies , High-Throughput Nucleotide Sequencing , Antibodies/chemistry , Antigens , B-Lymphocytes , High-Throughput Nucleotide Sequencing/methods , Mass Spectrometry
16.
Sci Rep ; 12(1): 4082, 2022 03 08.
Article in English | MEDLINE | ID: covidwho-1735288

ABSTRACT

The Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), also known as 2019 novel coronavirus (2019-nCoV), is a highly infectious RNA virus. A percentage of patients develop coronavirus disease 2019 (COVID-19) after infection, whose symptoms include fever, cough, shortness of breath and fatigue. Acute and life-threatening respiratory symptoms are experienced by 10-20% of symptomatic patients, particularly those with underlying medical conditions. One of the main challenges in the containment of COVID-19 is the identification and isolation of asymptomatic/pre-symptomatic individuals. A number of molecular assays are currently used to detect SARS-CoV-2. Many of them can accurately test hundreds or even thousands of patients every day. However, there are presently no testing platforms that enable more than 10,000 tests per day. Here, we describe the foundation for the REcombinase Mediated BaRcoding and AmplificatioN Diagnostic Tool (REMBRANDT), a high-throughput Next Generation Sequencing-based approach for the simultaneous screening of over 100,000 samples per day. The REMBRANDT protocol includes direct two-barcoded amplification of SARS-CoV-2 and control amplicons using an isothermal reaction, and the downstream library preparation for Illumina sequencing and bioinformatics analysis. This protocol represents a potentially powerful approach for community screening of COVID-19 that may be modified for application to any infectious or non-infectious genome.


Subject(s)
COVID-19/diagnosis , DNA-Binding Proteins/metabolism , Membrane Proteins/metabolism , Nucleic Acid Amplification Techniques/methods , SARS-CoV-2/genetics , Viral Proteins/metabolism , COVID-19/virology , High-Throughput Nucleotide Sequencing , Humans , Mass Screening , RNA, Viral/analysis , RNA, Viral/metabolism , SARS-CoV-2/isolation & purification
17.
J Transl Med ; 20(1): 105, 2022 03 03.
Article in English | MEDLINE | ID: covidwho-1724506

ABSTRACT

BACKGROUND: The COVID-19 pandemic has highlighted the importance of whole genome sequencing (WGS) of SARS-CoV-2 to inform public health policy. By enabling definition of lineages it facilitates tracking of the global spread of the virus. The evolution of new variants can be monitored and knowledge of specific mutations provides insights into the mechanisms through which the virus increases transmissibility or evades immunity. To date almost 1 million SARS-CoV-2 genomes have been sequenced by members of the COVID-19 Genomics UK (COG-UK) Consortium. To achieve similar feats in a more cost-effective and sustainable manner in future, improved high throughput virus sequencing protocols are required. We have therefore developed a miniaturized library preparation protocol with drastically reduced consumable use and costs. RESULTS: We present the 'Mini-XT' miniaturized tagmentation-based library preparation protocol available on protocols.io ( https://doi.org/10.17504/protocols.io.bvntn5en ). SARS-CoV-2 RNA was amplified using the ARTIC nCov-2019 multiplex RT-PCR protocol and purified using a conventional liquid handling system. Acoustic liquid transfer (Echo 525) was employed to reduce reaction volumes and the number of tips required for a Nextera XT library preparation. Sequencing was performed on an Illumina MiSeq. The final version of Mini-XT has been used to sequence 4384 SARS-CoV-2 samples from N. Ireland with a COG-UK QC pass rate of 97.4%. Sequencing quality was comparable and lineage calling consistent for replicate samples processed with full volume Nextera DNA Flex (333 samples) or using nanopore technology (20 samples). SNP calling between Mini-XT and these technologies was consistent and sequences from replicate samples paired together in maximum likelihood phylogenetic trees. CONCLUSIONS: The Mini-XT protocol maintains sequence quality while reducing library preparation reagent volumes eightfold and halving overall tip usage from sample to sequence to provide concomitant cost savings relative to standard protocols. This will enable more efficient high-throughput sequencing of SARS-CoV-2 isolates and future pathogen WGS.


Subject(s)
COVID-19 , SARS-CoV-2 , High-Throughput Nucleotide Sequencing/methods , Humans , Pandemics , Phylogeny , RNA, Viral/genetics , SARS-CoV-2/genetics
18.
Sci Rep ; 12(1): 1824, 2022 02 03.
Article in English | MEDLINE | ID: covidwho-1713207

ABSTRACT

The human gut contains a complex microbiota dominated by bacteriophages but also containing other viruses and bacteria and fungi. There are a growing number of techniques for the extraction, sequencing, and analysis of the virome but currently no standardized protocols. This study established an effective workflow for virome analysis to investigate the virome of stool samples from two understudied ethnic groups from Malaysia: the Jakun and Jehai Orang Asli. By using the virome extraction and analysis workflow with the Oxford Nanopore Technology, long-read sequencing successfully captured close to full-length viral genomes. The virome composition of the two indigenous Malaysian communities were remarkably different from those found in other parts of the world. Additionally, plant viruses found in the viromes of these individuals were attributed to traditional food-seeking methods. This study establishes a human gut virome workflow and extends insights into the healthy human gut virome, laying the groundwork for comparative studies.


Subject(s)
Gastrointestinal Microbiome/genetics , Genome, Viral , Indigenous Peoples , Viruses/genetics , Feces/virology , Female , High-Throughput Nucleotide Sequencing , Humans , Malaysia , Metagenomics/methods , Phylogeny , Virome/genetics , Viruses/classification
19.
Nucleic Acids Res ; 50(D1): D387-D390, 2022 01 07.
Article in English | MEDLINE | ID: covidwho-1705079

ABSTRACT

The Sequence Read Archive (SRA, https://www.ncbi.nlm.nih.gov/sra/) stores raw sequencing data and alignment information to enhance reproducibility and facilitate new discoveries through data analysis. Here we note changes in storage designed to increase access and highlight analyses that augment metadata with taxonomic insight to help users select data. In addition, we present three unanticipated applications of taxonomic analysis.


Subject(s)
Bacteria/genetics , Databases, Genetic , Metadata/statistics & numerical data , Software , Viruses/genetics , Bacteria/classification , Base Sequence , High-Throughput Nucleotide Sequencing , Internet , Phylogeny , Reproducibility of Results , SARS-CoV-2/genetics , Sequence Analysis, RNA , Viruses/classification
20.
Genes (Basel) ; 13(2)2022 02 14.
Article in English | MEDLINE | ID: covidwho-1686686

ABSTRACT

The use of high-throughput small RNA sequencing is well established as a technique to unveil the miRNAs in various tissues. The miRNA profiles are different between infected and non-infected tissues. We compare the SARS-CoV-2 positive and SARS-CoV-2 negative RNA samples extracted from human nasopharynx tissue samples to show different miRNA profiles. We explored differentially expressed miRNAs in response to SARS-CoV-2 in the RNA extracted from nasopharynx tissues of 10 SARS-CoV-2-positive and 10 SARS-CoV-2-negative patients. miRNAs were identified by small RNA sequencing, and the expression levels of selected miRNAs were validated by real-time RT-PCR. We identified 943 conserved miRNAs, likely generated through posttranscriptional modifications. The identified miRNAs were expressed in both RNA groups, NegS and PosS: miR-148a, miR-21, miR-34c, miR-34b, and miR-342. The most differentially expressed miRNA was miR-21, which is likely closely linked to the presence of SARS-CoV-2 in nasopharynx tissues. Our results contribute to further understanding the role of miRNAs in SARS-CoV-2 pathogenesis, which may be crucial for understanding disease symptom development in humans.


Subject(s)
MicroRNAs/metabolism , Nasopharynx/metabolism , SARS-CoV-2/physiology , COVID-19/pathology , COVID-19/virology , Down-Regulation , High-Throughput Nucleotide Sequencing , Humans , MicroRNAs/chemistry , Nasopharynx/virology , Principal Component Analysis , RNA, Viral/metabolism , Real-Time Polymerase Chain Reaction , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Sequence Analysis, RNA , Transcriptome , Up-Regulation
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