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1.
BMC Genomics ; 23(1): 319, 2022 Apr 22.
Article in English | MEDLINE | ID: covidwho-1799119

ABSTRACT

BACKGROUND: Over 4 million SARS-CoV-2 genomes have been sequenced globally in the past 2 years. This has been crucial in elucidating transmission chains within communities, the development of new diagnostic methods, vaccines, and antivirals. Although several sequencing technologies have been employed, Illumina and Oxford Nanopore remain the two most commonly used platforms. The sequence quality between these two platforms warrants a comparison of the genomes produced by the two technologies. Here, we compared the SARS-CoV-2 consensus genomes obtained from the Oxford Nanopore Technology GridION and the Illumina MiSeq for 28 sequencing runs. RESULTS: Our results show that the MiSeq had a significantly higher number of consensus genomes classified by Nextclade as good and mediocre compared to the GridION. The MiSeq also had a significantly higher genome coverage and mutation counts than the GridION. CONCLUSION: Due to the low genome coverage, high number of indels, and sensitivity to SARS-CoV-2 viral load noted with the GridION when compared to MiSeq, we can conclude that the MiSeq is more favourable for SARS-CoV-2 genomic surveillance, as successful genomic surveillance is dependent on high quality, near-whole consensus genomes.


Subject(s)
COVID-19 , SARS-CoV-2 , Genome, Viral , High-Throughput Nucleotide Sequencing/methods , Humans , SARS-CoV-2/genetics , Whole Genome Sequencing/methods
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.
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
4.
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
5.
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
6.
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
7.
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
8.
Microbiol Spectr ; 10(2): e0256421, 2022 Apr 27.
Article in English | MEDLINE | ID: covidwho-1723567

ABSTRACT

Next-generation sequencing (NGS) is a powerful tool for detecting and investigating viral pathogens; however, analysis and management of the enormous amounts of data generated from these technologies remains a challenge. Here, we present VPipe (the Viral NGS Analysis Pipeline and Data Management System), an automated bioinformatics pipeline optimized for whole-genome assembly of viral sequences and identification of diverse species. VPipe automates the data quality control, assembly, and contig identification steps typically performed when analyzing NGS data. Users access the pipeline through a secure web-based portal, which provides an easy-to-use interface with advanced search capabilities for reviewing results. In addition, VPipe provides a centralized system for storing and analyzing NGS data, eliminating common bottlenecks in bioinformatics analyses for public health laboratories with limited on-site computational infrastructure. The performance of VPipe was validated through the analysis of publicly available NGS data sets for viral pathogens, generating high-quality assemblies for 12 data sets. VPipe also generated assemblies with greater contiguity than similar pipelines for 41 human respiratory syncytial virus isolates and 23 SARS-CoV-2 specimens. IMPORTANCE Computational infrastructure and bioinformatics analysis are bottlenecks in the application of NGS to viral pathogens. As of September 2021, VPipe has been used by the U.S. Centers for Disease Control and Prevention (CDC) and 12 state public health laboratories to characterize >17,500 and 1,500 clinical specimens and isolates, respectively. VPipe automates genome assembly for a wide range of viruses, including high-consequence pathogens such as SARS-CoV-2. Such automated functionality expedites public health responses to viral outbreaks and pathogen surveillance.


Subject(s)
COVID-19 , Viruses , Computational Biology/methods , High-Throughput Nucleotide Sequencing/methods , Humans , SARS-CoV-2/genetics , Viruses/genetics
9.
Sci Rep ; 12(1): 2419, 2022 02 14.
Article in English | MEDLINE | ID: covidwho-1684100

ABSTRACT

This study aimed to develop the feasible and effective universal screening strategy of the notable SARS-CoV-2 variants by Sanger Sequencing Strategy and then practically applied it for mass screening in Hiroshima, Japan. A total of 734 samples from COVID-19 confirmed cases in Hiroshima were screened for the notable SARS-CoV-2 variants (B.1.1.7, B.1.351, P.1, B.1.617.2, B.1.617.1, C.37, B.1.1.529, etc.). The targeted spike region is amplified by nested RT-PCR using in-house designed primer set hCoV-Spike-A and standard amplification protocol. Additionally, randomly selected 96 samples were also amplified using primer sets hCoV-Spike-B and hCoV-Spike-C. The negative amplified samples were repeated for second attempt of amplification by volume-up protocol. Thereafter, the amplified products were assigned for Sanger sequencing using corresponding primers. The positive amplification rate of primer set hCoV-Spike-A, hCoV-Spike-B and hCoV-Spike-C were 87.3%, 83.3% and 93.8% respectively for standard protocol and increased to 99.6%, 95.8% and 96.9% after second attempt by volume-up protocol. The readiness of genome sequences was 96.9%, 100% and 100% respectively. Among 48 mutant isolates, 26 were B.1.1.7 (Alpha), 7 were E484K single mutation and the rest were other types of mutation. Moreover, 5 cluster cases with single mutation at N501S were firstly reported in Hiroshima. This study indicates the reliability and effectiveness of Sanger sequencing to screen large number of samples for the notable SARS-CoV-2 variants. Compared to the Next Generation Sequencing (NGS), our method introduces the feasible, universally applicable, and practically useful tool for identification of the emerging variants with less expensive and time consuming especially in those countries where the NGS is not practically available. Our method allows not only to identify the pre-existing variants but also to examine other rare type of mutation or newly emerged variants and is crucial for prevention and control of pandemic.


Subject(s)
COVID-19/diagnosis , Mass Screening/methods , SARS-CoV-2/genetics , Sequence Analysis, DNA/methods , Spike Glycoprotein, Coronavirus/genetics , Amino Acid Sequence , COVID-19/epidemiology , COVID-19/virology , Feasibility Studies , High-Throughput Nucleotide Sequencing/methods , Humans , Japan/epidemiology , Pandemics/prevention & control , Reproducibility of Results , SARS-CoV-2/physiology , Sensitivity and Specificity , Sequence Homology, Amino Acid
10.
PLoS One ; 17(2): e0263794, 2022.
Article in English | MEDLINE | ID: covidwho-1674022

ABSTRACT

Genomic surveillance empowers agile responses to SARS-CoV-2 by enabling scientists and public health analysts to issue recommendations aimed at slowing transmission, prioritizing contact tracing, and building a robust genomic sequencing surveillance strategy. Since the start of the pandemic, real time RT-PCR diagnostic testing from upper respiratory specimens, such as nasopharyngeal (NP) swabs, has been the standard. Moreover, respiratory samples in viral transport media are the ideal specimen for SARS-CoV-2 whole-genome sequencing (WGS). In early 2021, many clinicians transitioned to antigen-based SARS-CoV-2 detection tests, which use anterior nasal swabs for SARS-CoV-2 antigen detection. Despite this shift in testing methods, the need for whole-genome sequence surveillance remains. Thus, we developed a workflow for whole-genome sequencing with antigen test-derived swabs as an input rather than nasopharyngeal swabs. In this study, we use excess clinical specimens processed using the BinaxNOW™ COVID-19 Ag Card. We demonstrate that whole-genome sequencing from antigen tests is feasible and yields similar results from RT-PCR-based assays utilizing a swab in viral transport media.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Culture Media/analysis , High-Throughput Nucleotide Sequencing/methods , SARS-CoV-2/genetics , Specimen Handling/methods , Whole Genome Sequencing/methods , COVID-19/genetics , COVID-19/virology , Culture Media/metabolism , Humans , SARS-CoV-2/isolation & purification
11.
Nucleic Acids Res ; 49(17): e102, 2021 09 27.
Article in English | MEDLINE | ID: covidwho-1594917

ABSTRACT

Rapidly evolving RNA viruses continuously produce minority haplotypes that can become dominant if they are drug-resistant or can better evade the immune system. Therefore, early detection and identification of minority viral haplotypes may help to promptly adjust the patient's treatment plan preventing potential disease complications. Minority haplotypes can be identified using next-generation sequencing, but sequencing noise hinders accurate identification. The elimination of sequencing noise is a non-trivial task that still remains open. Here we propose CliqueSNV based on extracting pairs of statistically linked mutations from noisy reads. This effectively reduces sequencing noise and enables identifying minority haplotypes with the frequency below the sequencing error rate. We comparatively assess the performance of CliqueSNV using an in vitro mixture of nine haplotypes that were derived from the mutation profile of an existing HIV patient. We show that CliqueSNV can accurately assemble viral haplotypes with frequencies as low as 0.1% and maintains consistent performance across short and long bases sequencing platforms.


Subject(s)
Algorithms , Computational Biology/methods , Haplotypes , High-Throughput Nucleotide Sequencing/methods , RNA Virus Infections/diagnosis , RNA Viruses/genetics , COVID-19/diagnosis , COVID-19/virology , Gene Frequency , HIV Infections/diagnosis , HIV Infections/virology , HIV-1/genetics , Humans , Mutation , Polymorphism, Single Nucleotide , RNA Virus Infections/virology , Reproducibility of Results , SARS-CoV-2/genetics , Sensitivity and Specificity
12.
Cells ; 11(1)2021 12 27.
Article in English | MEDLINE | ID: covidwho-1580992

ABSTRACT

Coronavirus disease 2019 (COVID-19) is a global infectious disease caused by the SARS-CoV-2 coronavirus. T cells play an essential role in the body's fighting against the virus invasion, and the T cell receptor (TCR) is crucial in T cell-mediated virus recognition and clearance. However, little has been known about the features of T cell response in convalescent COVID-19 patients. In this study, using 5'RACE technology and PacBio sequencing, we analyzed the TCR repertoire of COVID-19 patients after recovery for 2 weeks and 6 months compared with the healthy donors. The TCR clustering and CDR3 annotation were exploited to discover groups of patient-specific TCR clonotypes with potential SARS-CoV-2 antigen specificities. We first identified CD4+ and CD8+ T cell clones with certain clonal expansion after infection, and then observed the preferential recombination usage of V(D) J gene segments in CD4+ and CD8+ T cells of COVID-19 patients with different convalescent stages. More important, the TRBV6-5-TRBD2-TRBJ2-7 combination with high frequency was shared between CD4+ T and CD8+ T cells of different COVID-19 patients. Finally, we found the dominant characteristic motifs of the CDR3 sequence between recovered COVID-19 and healthy control. Our study provides novel insights on TCR in COVID-19 with different convalescent phases, contributing to our understanding of the immune response induced by SARS-CoV-2.


Subject(s)
COVID-19/immunology , High-Throughput Nucleotide Sequencing/methods , Immunity/immunology , Receptors, Antigen, T-Cell/immunology , SARS-CoV-2/immunology , T-Lymphocytes/immunology , Aged , Amino Acid Sequence , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/virology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/virology , COVID-19/metabolism , COVID-19/virology , Cells, Cultured , Complementarity Determining Regions/genetics , Complementarity Determining Regions/immunology , Convalescence , Female , Humans , Male , Middle Aged , Patient Acuity , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/metabolism , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Antigen, T-Cell, alpha-beta/immunology , Receptors, Antigen, T-Cell, alpha-beta/metabolism , SARS-CoV-2/physiology , T-Lymphocytes/metabolism , T-Lymphocytes/virology
13.
Cell Rep ; 38(3): 110242, 2022 01 18.
Article in English | MEDLINE | ID: covidwho-1588137

ABSTRACT

Characterization of COVID-19 antibodies has largely focused on memory B cells; however, it is the antibody-secreting plasma cells that are directly responsible for the production of serum antibodies, which play a critical role in resolving SARS-CoV-2 infection. Little is known about the specificity of plasma cells, largely because plasma cells lack surface antibody expression, thereby complicating their screening. Here, we describe a technology pipeline that integrates single-cell antibody repertoire sequencing and mammalian display to interrogate the specificity of plasma cells from 16 convalescent patients. Single-cell sequencing allows us to profile antibody repertoire features and identify expanded clonal lineages. Mammalian display screening is used to reveal that 43 antibodies (of 132 candidates) derived from expanded plasma cell lineages are specific to SARS-CoV-2 antigens, including antibodies with high affinity to the SARS-CoV-2 receptor-binding domain (RBD) that exhibit potent neutralization and broad binding to the RBD of SARS-CoV-2 variants (of concern/interest).


Subject(s)
Antibodies, Neutralizing/isolation & purification , Plasma Cells/metabolism , SARS-CoV-2/immunology , Single-Cell Analysis/methods , Animals , Antibodies, Viral/isolation & purification , COVID-19/immunology , COVID-19/prevention & control , Cells, Cultured , Cohort Studies , Gene Library , HEK293 Cells , High-Throughput Nucleotide Sequencing/methods , Humans , Mammals , Neutralization Tests , Peptide Library , Plasma Cells/chemistry
14.
Viruses ; 13(12)2021 12 19.
Article in English | MEDLINE | ID: covidwho-1580420

ABSTRACT

Rapid molecular surveillance of SARS-CoV-2 S-protein variants leading to immune escape and/or increased infectivity is of utmost importance. Among global bottlenecks for variant monitoring in diagnostic settings are sequencing and bioinformatics capacities. In this study, we aimed to establish a rapid and user-friendly protocol for high-throughput S-gene sequencing and subsequent automated identification of variants. We designed two new primer pairs to amplify only the immunodominant part of the S-gene for nanopore sequencing. Furthermore, we developed an automated "S-Protein-Typer" tool that analyzes and reports S-protein mutations on the amino acid level including a variant of concern indicator. Validation of our primer panel using SARS-CoV-2-positive respiratory specimens covering a broad Ct range showed successful amplification for 29/30 samples. Restriction to the region of interest freed sequencing capacity by a factor of 12-13, compared with whole-genome sequencing. Using either the MinION or Flongle flow cell, our sequencing strategy reduced the time required to identify SARS-CoV-2 variants accordingly. The S-Protein-Typer tool identified all mutations correctly when challenged with our sequenced samples and 50 deposited sequences covering all VOCs (December 2021). Our proposed S-protein variant screening offers a simple, more rapid, and low-cost entry into NGS-based SARS-CoV-2 analysis, compared with current whole-genome approaches.


Subject(s)
High-Throughput Nucleotide Sequencing/methods , Nanopore Sequencing/methods , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , COVID-19/epidemiology , COVID-19/virology , Epidemiological Monitoring , Genotype , Humans , Immune Evasion/genetics , Mutation , SARS-CoV-2/immunology
15.
Adv Sci (Weinh) ; 8(23): e2102593, 2021 12.
Article in English | MEDLINE | ID: covidwho-1559092

ABSTRACT

Fast and accurate identification of microbial pathogens is critical for the proper treatment of infections. Traditional culture-based diagnosis in clinics is increasingly supplemented by metagenomic next-generation-sequencing (mNGS). Here, RNA/cDNA-targeted sequencing (meta-transcriptomics using NGS (mtNGS)) is established to reduce the host nucleotide percentage in clinic samples and by combining with Oxford Nanopore Technology (ONT) platforms (meta-transcriptomics using third-generation sequencing, mtTGS) to improve the sequencing time. It shows that mtNGS improves the ratio of microbial reads, facilitates bacterial identification using multiple-strategies, and discovers fungi, viruses, and antibiotic resistance genes, and displaying agreement with clinical findings. Furthermore, longer reads in mtTGS lead to additional improvement in pathogen identification and also accelerate the clinical diagnosis. Additionally, primary tests utilizing direct-RNA sequencing and targeted sequencing of ONT show that ONT displays important potential but must be further developed. This study presents the potential of RNA-targeted pathogen identification in clinical samples, especially when combined with the newest developments in ONT.


Subject(s)
Bronchoalveolar Lavage Fluid/microbiology , High-Throughput Nucleotide Sequencing/methods , Infections/genetics , Metagenomics/methods , RNA/genetics , Sequence Analysis, RNA/methods , Aged , Bronchoalveolar Lavage/methods , Female , Humans , Male , Metagenome/genetics , Middle Aged
16.
PLoS Comput Biol ; 17(11): e1009594, 2021 11.
Article in English | MEDLINE | ID: covidwho-1518350

ABSTRACT

The growing number of next-generation sequencing (NGS) data presents a unique opportunity to study the combined impact of mitochondrial and nuclear-encoded genetic variation in complex disease. Mitochondrial DNA variants and in particular, heteroplasmic variants, are critical for determining human disease severity. While there are approaches for obtaining mitochondrial DNA variants from NGS data, these software do not account for the unique characteristics of mitochondrial genetics and can be inaccurate even for homoplasmic variants. We introduce MitoScape, a novel, big-data, software for extracting mitochondrial DNA sequences from NGS. MitoScape adopts a novel departure from other algorithms by using machine learning to model the unique characteristics of mitochondrial genetics. We also employ a novel approach of using rho-zero (mitochondrial DNA-depleted) data to model nuclear-encoded mitochondrial sequences. We showed that MitoScape produces accurate heteroplasmy estimates using gold-standard mitochondrial DNA data. We provide a comprehensive comparison of the most common tools for obtaining mtDNA variants from NGS and showed that MitoScape had superior performance to compared tools in every statistically category we compared, including false positives and false negatives. By applying MitoScape to common disease examples, we illustrate how MitoScape facilitates important heteroplasmy-disease association discoveries by expanding upon a reported association between hypertrophic cardiomyopathy and mitochondrial haplogroup T in men (adjusted p-value = 0.003). The improved accuracy of mitochondrial DNA variants produced by MitoScape will be instrumental in diagnosing disease in the context of personalized medicine and clinical diagnostics.


Subject(s)
Big Data , DNA, Mitochondrial/genetics , High-Throughput Nucleotide Sequencing/methods , Machine Learning , Genes, Mitochondrial , Humans
17.
Sci Rep ; 11(1): 21352, 2021 11 01.
Article in English | MEDLINE | ID: covidwho-1493207

ABSTRACT

The outcome of SARS-CoV-2 infection is determined by multiple factors, including the viral, host genetics, age, and comorbidities. This study investigated the association between prognostic factors and disease outcomes of patients infected by SARS-CoV-2 with multiple S protein mutations. Fifty-one COVID-19 patients were recruited in this study. Whole-genome sequencing of 170 full-genomes of SARS-CoV-2 was conducted with the Illumina MiSeq sequencer. Most patients (47%) had mild symptoms of COVID-19 followed by moderate (19.6%), no symptoms (13.7%), severe (4%), and critical (2%). Mortality was found in 13.7% of the COVID-19 patients. There was a significant difference between the age of hospitalized patients (53.4 ± 18 years) and the age of non-hospitalized patients (34.6 ± 19) (p = 0.001). The patients' hospitalization was strongly associated with hypertension, diabetes, and anticoagulant and were strongly significant with the OR of 17 (95% CI 2-144; p = 0.001), 4.47 (95% CI 1.07-18.58; p = 0.039), and 27.97 (95% CI 1.54-507.13; p = 0.02), respectively; while the patients' mortality was significantly correlated with patients' age, anticoagulant, steroid, and diabetes, with OR of 8.44 (95% CI 1.5-47.49; p = 0.016), 46.8 (95% CI 4.63-472.77; p = 0.001), 15.75 (95% CI 2-123.86; p = 0.009), and 8.5 (95% CI 1.43-50.66; p = 0.019), respectively. This study found the clade: L (2%), GH (84.3%), GR (11.7%), and O (2%). Besides the D614G mutation, we found L5F (18.8%), V213A (18.8%), and S689R (8.3%). No significant association between multiple S protein mutations and the patients' hospitalization or mortality. Multivariate analysis revealed that hypertension and anticoagulant were the significant factors influencing the hospitalization and mortality of patients with COVID-19 with an OR of 17.06 (95% CI 2.02-144.36; p = 0.009) and 46.8 (95% CI 4.63-472.77; p = 0.001), respectively. Moreover, the multiple S protein mutations almost reached a strong association with patients' hospitalization (p = 0.07). We concluded that hypertension and anticoagulant therapy have a significant impact on COVID-19 outcomes. This study also suggests that multiple S protein mutations may impact the COVID-19 outcomes. This further emphasized the significance of monitoring SARS-CoV-2 variants through genomic surveillance, particularly those that may impact the COVID-19 outcomes.


Subject(s)
COVID-19/mortality , Mutation , SARS-CoV-2/genetics , Severity of Illness Index , Spike Glycoprotein, Coronavirus/genetics , Adolescent , Adult , Aged , COVID-19/epidemiology , COVID-19/pathology , COVID-19/virology , Comorbidity , Female , High-Throughput Nucleotide Sequencing/methods , Hospitalization , Humans , Indonesia/epidemiology , Male , Middle Aged , Phylogeny , Prognosis , Retrospective Studies , Risk Factors , Whole Genome Sequencing/methods , Young Adult
18.
Genomics ; 113(6): 4109-4115, 2021 11.
Article in English | MEDLINE | ID: covidwho-1492759

ABSTRACT

Genetic variants of SARS-CoV-2 have been emerging and circulating in many places across the world. Rapid detection of these variants is essential since their dissemination can impact transmission rates, diagnostic procedures, disease severity, response to vaccines or patient management. Sanger sequencing has been used as the preferred approach for variant detection among circulating human immunodeficiency and measles virus genotypes. Using primers to amplify a fragment of the SARS-CoV-2 genome encoding part of the Spike protein, we showed that Sanger sequencing allowed us to rapidly detect the introduction and spread of three distinct SARS-CoV-2 variants in two major Brazilian cities. In both cities, after the predominance of variants closely related to the virus first identified in China, the emergence of the P.2 variant was quickly followed by the detection of the P1 variant, which became dominant in less than one month after it was first detected.


Subject(s)
COVID-19/virology , Genetic Variation , High-Throughput Nucleotide Sequencing/methods , SARS-CoV-2/genetics , Brazil/epidemiology , COVID-19/epidemiology , China , Cities , Humans , Mutation , Phylogeny , Spike Glycoprotein, Coronavirus/genetics
19.
Microbiol Spectr ; 9(2): e0050621, 2021 10 31.
Article in English | MEDLINE | ID: covidwho-1455679

ABSTRACT

Emerging SARS-CoV-2 (SC-2) variants with increased infectivity and vaccine resistance are of major concern. Rapid identification of such variants is important for the public health decision making and to provide valuable data for epidemiological and policy decision making. We developed a multiplex reverse transcriptase quantitative PCR (RT-qPCR) assay that can specifically identify and differentiate between the emerging B.1.1.7 and B.1.351 SC-2 variants. In a single assay, we combined four reactions-one that detects SC-2 RNA independently of the strain, one that detects the D3L mutation, which is specific to variant B.1.1.7, one that detects the 242 to 244 deletion, which is specific to variant B.1.351, and the fourth reaction, which identifies the human RNAseP gene, serving as an endogenous control for RNA extraction integrity. We show that the strain-specific reactions target mutations that are strongly associated with the target variants and not with other major known variants. The assay's specificity was tested against a panel of respiratory pathogens (n = 16), showing high specificity toward SC-2 RNA. The assay's sensitivity was assessed using both in vitro transcribed RNA and clinical samples and was determined to be between 20 and 40 viral RNA copies per reaction. The assay performance was corroborated with Sanger and whole-genome sequencing, showing complete agreement with the sequencing results. The new assay is currently implemented in the routine diagnostic work at the Central Virology Laboratory, and may be used in other laboratories to facilitate the diagnosis of these major worldwide-circulating SC-2 variants. IMPORTANCE This study describes the design and utilization of a multiplex reverse transcriptase quantitative PCR (RT-qPCR) to identify SARS-COV-2 (SC2) RNA in general and, specifically, to detect whether it is of lineage B.1.1.7 or B.1.351. Implementation of this method in diagnostic and research laboratories worldwide may help the efforts to contain the COVID-19 pandemic. The method can be easily scaled up and be used in high-throughput laboratories, as well as small ones. In addition to immediate help in diagnostic efforts, this method may also help in epidemiological studies focused on the spread of emerging SC-2 lineages.


Subject(s)
COVID-19/diagnosis , High-Throughput Nucleotide Sequencing/methods , High-Throughput Screening Assays/methods , SARS-CoV-2/classification , SARS-CoV-2/genetics , COVID-19/epidemiology , COVID-19/virology , Genome, Viral/genetics , Humans , Israel/epidemiology , RNA, Viral/genetics , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/immunology , SARS-CoV-2/isolation & purification , Sensitivity and Specificity , Whole Genome Sequencing
20.
Sci Rep ; 11(1): 19817, 2021 10 06.
Article in English | MEDLINE | ID: covidwho-1454815

ABSTRACT

Recent studies have focused their attention on conjunctivitis as one of the symptoms of coronavirus disease 2019 (COVID-19). Therefore, tear samples were taken from COVID-19 patients and the presence of SARS-CoV-2 was evidenced using Real Time reverse transcription polymerase chain reaction. The main aim of this study was to analyze mRNA expression in the tears of patients with COVID-19 compared with healthy subjects using Next Generation Sequencing (NGS). The functional evaluation of the transcriptome highlighted 25 genes that differ statistically between healthy individuals and patients affected by COVID-19. In particular, the NGS analysis identified the presence of several genes involved in B cell signaling and keratinization. In particular, the genes involved in B cell signaling were downregulated in the tears of COVID-19 patients, while those involved in keratinization were upregulated. The results indicated that SARS-CoV-2 may induce a process of ocular keratinization and a defective B cell response.


Subject(s)
COVID-19/genetics , Eye Diseases/virology , Tears/metabolism , Transcriptome , Aged , B-Lymphocytes/metabolism , COVID-19/pathology , COVID-19/virology , Eye Diseases/genetics , Female , High-Throughput Nucleotide Sequencing/methods , Humans , Keratins/metabolism , Male , SARS-CoV-2/isolation & purification , Sequence Analysis, RNA/methods , Skin/metabolism , Skin/pathology , Skin/virology , Tears/virology
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