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1.
Genome Biol ; 23(1): 55, 2022 02 16.
Article in English | MEDLINE | ID: covidwho-1785167

ABSTRACT

BACKGROUND: Multiplexing of samples in single-cell RNA-seq studies allows a significant reduction of the experimental costs, straightforward identification of doublets, increased cell throughput, and reduction of sample-specific batch effects. Recently published multiplexing techniques using oligo-conjugated antibodies or -lipids allow barcoding sample-specific cells, a process called "hashing." RESULTS: Here, we compare the hashing performance of TotalSeq-A and -C antibodies, custom synthesized lipids and MULTI-seq lipid hashes in four cell lines, both for single-cell RNA-seq and single-nucleus RNA-seq. We also compare TotalSeq-B antibodies with CellPlex reagents (10x Genomics) on human PBMCs and TotalSeq-B with different lipids on primary mouse tissues. Hashing efficiency was evaluated using the intrinsic genetic variation of the cell lines and mouse strains. Antibody hashing was further evaluated on clinical samples using PBMCs from healthy and SARS-CoV-2 infected patients, where we demonstrate a more affordable approach for large single-cell sequencing clinical studies, while simultaneously reducing batch effects. CONCLUSIONS: Benchmarking of different hashing strategies and computational pipelines indicates that correct demultiplexing can be achieved with both lipid- and antibody-hashed human cells and nuclei, with MULTISeqDemux as the preferred demultiplexing function and antibody-based hashing as the most efficient protocol on cells. On nuclei datasets, lipid hashing delivers the best results. Lipid hashing also outperforms antibodies on cells isolated from mouse brain. However, antibodies demonstrate better results on tissues like spleen or lung.


Subject(s)
COVID-19/blood , Sequence Analysis, RNA/methods , Single-Cell Analysis/methods , Animals , Antibodies/chemistry , Case-Control Studies , Cell Line, Tumor , Cell Nucleus/chemistry , Humans , Lipids/chemistry , Mice, Inbred BALB C , Mice, Inbred C57BL , Neutrophils/chemistry , Neutrophils/immunology , Neutrophils/virology
2.
Nat Commun ; 13(1): 1722, 2022 03 31.
Article in English | MEDLINE | ID: covidwho-1773975

ABSTRACT

The rapidly growing popularity of RNA structure probing methods is leading to increasingly large amounts of available RNA structure information. This demands the development of efficient tools for the identification of RNAs sharing regions of structural similarity by direct comparison of their reactivity profiles, hence enabling the discovery of conserved structural features. We here introduce SHAPEwarp, a largely sequence-agnostic SHAPE-guided algorithm for the identification of structurally-similar regions in RNA molecules. Analysis of Dengue, Zika and coronavirus genomes recapitulates known regulatory RNA structures and identifies novel highly-conserved structural elements. This work represents a preliminary step towards the model-free search and identification of shared and conserved RNA structural features within transcriptomes.


Subject(s)
Zika Virus Infection , Zika Virus , Algorithms , Humans , Nucleic Acid Conformation , RNA/chemistry , RNA/genetics , RNA, Guide , Sequence Analysis, RNA/methods , Zika Virus/genetics
3.
Genome Biol ; 23(1): 33, 2022 01 24.
Article in English | MEDLINE | ID: covidwho-1649470

ABSTRACT

We consider an increasingly popular study design where single-cell RNA-seq data are collected from multiple individuals and the question of interest is to find genes that are differentially expressed between two groups of individuals. Towards this end, we propose a statistical method named IDEAS (individual level differential expression analysis for scRNA-seq). For each gene, IDEAS summarizes its expression in each individual by a distribution and then assesses whether these individual-specific distributions are different between two groups of individuals. We apply IDEAS to assess gene expression differences of autism patients versus controls and COVID-19 patients with mild versus severe symptoms.


Subject(s)
Autistic Disorder/genetics , COVID-19/genetics , Sequence Analysis, RNA/methods , Single-Cell Analysis/methods , Software , Autistic Disorder/metabolism , Autistic Disorder/pathology , COVID-19/metabolism , COVID-19/pathology , COVID-19/virology , Case-Control Studies , Gene Expression Profiling , Gene Expression Regulation , Humans , Microglia/metabolism , Microglia/pathology , Nerve Tissue Proteins/classification , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , SARS-CoV-2/pathogenicity , Severity of Illness Index , Whole Exome Sequencing
4.
PLoS One ; 17(1): e0262170, 2022.
Article in English | MEDLINE | ID: covidwho-1637228

ABSTRACT

The SARS-CoV-2 responsible for the ongoing COVID pandemic reveals particular evolutionary dynamics and an extensive polymorphism, mainly in Spike gene. Monitoring the S gene mutations is crucial for successful controlling measures and detecting variants that can evade vaccine immunity. Even after the costs reduction resulting from the pandemic, the new generation sequencing methodologies remain unavailable to a large number of scientific groups. Therefore, to support the urgent surveillance of SARS-CoV-2 S gene, this work describes a new feasible protocol for complete nucleotide sequencing of the S gene using the Sanger technique. Such a methodology could be easily adopted by any laboratory with experience in sequencing, adding to effective surveillance of SARS-CoV-2 spreading and evolution.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , COVID-19/epidemiology , Genes, Viral , Pandemics/prevention & control , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Sequence Analysis, RNA/methods , Spike Glycoprotein, Coronavirus/genetics , Base Sequence , Brazil/epidemiology , COVID-19/virology , Diagnostic Tests, Routine/methods , Electrophoresis, Agar Gel/methods , Epidemiological Monitoring , Humans , Mutation , RNA, Viral/genetics , RNA, Viral/isolation & purification
5.
PLoS One ; 16(12): e0260850, 2021.
Article in English | MEDLINE | ID: covidwho-1613341

ABSTRACT

Novel strains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) harboring nucleotide changes (mutations) in the spike gene have emerged and are spreading rapidly. These mutations are associated with SARS-CoV-2 transmissibility, virulence, or resistance to some neutralizing antibodies. Thus, the accurate detection of spike mutants is crucial for controlling SARS-CoV-2 transmission and identifying neutralizing antibody-resistance caused by amino acid changes in the receptor-binding domain. Here, we developed five SARS-CoV-2 spike gene primer pairs (5-SSG primer assay; 69S, 144S, 417S, 484S, and 570S) and verified their ability to detect nine key spike mutations (ΔH69/V70, T95I, G142D, ΔY144, K417T/N, L452R, E484K/Q, N501Y, and H655Y) using a Sanger sequencing-based assay. The 5-SSG primer assay showed 100% specificity and a conservative limit of detection with a median tissue culture infective dose (TCID50) values of 1.4 × 102 TCID50/mL. The accuracy of the 5-SSG primer assay was confirmed by next generation sequencing. The results of these two approaches showed 100% consistency. Taken together, the ability of the 5-SSG primer assay to accurately detect key SARS-CoV-2 spike mutants is reliable. Thus, it is a useful tool for detecting SARS-CoV-2 spike gene mutants in a clinical setting, thereby helping to improve the management of patients with COVID-19.


Subject(s)
Mutation , SARS-CoV-2/genetics , Sequence Analysis, RNA/methods , Spike Glycoprotein, Coronavirus/genetics , DNA Primers/genetics , High-Throughput Nucleotide Sequencing , Humans , Limit of Detection , Protein Domains , Spike Glycoprotein, Coronavirus/chemistry
6.
STAR Protoc ; 3(1): 101067, 2022 03 18.
Article in English | MEDLINE | ID: covidwho-1595326

ABSTRACT

N 6 -methylation of adenosine (m6A) is the most abundant internal mRNA modification and is an important post-transcriptional regulator of gene expression. Here, we describe a protocol for methylated RNA immunoprecipitation sequencing (MeRIP-Seq) to detect and quantify m6A modifications in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. The protocol is optimized for low viral RNA levels and is readily adaptable for other applications. For complete details on the use and execution of this protocol, please refer to Li et al. (2021).


Subject(s)
Adenosine/analogs & derivatives , Immunoprecipitation/methods , Sequence Analysis, RNA/methods , Adenosine/analysis , Adenosine/genetics , Animals , COVID-19/genetics , Caco-2 Cells , Chlorocebus aethiops , Gene Expression/genetics , Gene Expression Regulation/genetics , Genetic Techniques , HEK293 Cells , Humans , Methylation , RNA/chemistry , RNA/genetics , RNA Processing, Post-Transcriptional , RNA, Viral/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Vero Cells
7.
Sci Rep ; 11(1): 23928, 2021 12 14.
Article in English | MEDLINE | ID: covidwho-1585797

ABSTRACT

Identification of transcriptional regulatory mechanisms and signaling networks involved in the response of host cells to infection by SARS-CoV-2 is a powerful approach that provides a systems biology view of gene expression programs involved in COVID-19 and may enable the identification of novel therapeutic targets and strategies to mitigate the impact of this disease. In this study, our goal was to identify a transcriptional regulatory network that is associated with gene expression changes between samples infected by SARS-CoV-2 and those that are infected by other respiratory viruses to narrow the results on those enriched or specific to SARS-CoV-2. We combined a series of recently developed computational tools to identify transcriptional regulatory mechanisms involved in the response of epithelial cells to infection by SARS-CoV-2, and particularly regulatory mechanisms that are specific to this virus when compared to other viruses. In addition, using network-guided analyses, we identified kinases associated with this network. The results identified pathways associated with regulation of inflammation (MAPK14) and immunity (BTK, MBX) that may contribute to exacerbate organ damage linked with complications of COVID-19. The regulatory network identified herein reflects a combination of known hits and novel candidate pathways supporting the novel computational pipeline presented herein to quickly narrow down promising avenues of investigation when facing an emerging and novel disease such as COVID-19.


Subject(s)
COVID-19/genetics , Gene Expression Profiling/methods , SARS-CoV-2/pathogenicity , Sequence Analysis, RNA/methods , A549 Cells , Cell Line , Epithelial Cells/chemistry , Epithelial Cells/cytology , Epithelial Cells/virology , Gene Expression Regulation , Humans , Models, Biological , Systems Biology
8.
Sci Rep ; 11(1): 24042, 2021 12 15.
Article in English | MEDLINE | ID: covidwho-1574556

ABSTRACT

The microbiota of the nasopharyngeal tract (NT) play a role in host immunity against respiratory infectious diseases. However, scant information is available on interactions of SARS-CoV-2 with the nasopharyngeal microbiome. This study characterizes the effects of SARS-CoV-2 infection on human nasopharyngeal microbiomes and their relevant metabolic functions. Twenty-two (n = 22) nasopharyngeal swab samples (including COVID-19 patients = 8, recovered humans = 7, and healthy people = 7) were collected, and underwent to RNAseq-based metagenomic investigation. Our RNAseq data mapped to 2281 bacterial species (including 1477, 919 and 676 in healthy, COVID-19 and recovered metagenomes, respectively) indicating a distinct microbiome dysbiosis. The COVID-19 and recovered samples included 67% and 77% opportunistic bacterial species, respectively compared to healthy controls. Notably, 79% commensal bacterial species found in healthy controls were not detected in COVID-19 and recovered people. Similar dysbiosis was also found in viral and archaeal fraction of the nasopharyngeal microbiomes. We also detected several altered metabolic pathways and functional genes in the progression and pathophysiology of COVID-19. The nasopharyngeal microbiome dysbiosis and their genomic features determined by our RNAseq analyses shed light on early interactions of SARS-CoV-2 with the nasopharyngeal resident microbiota that might be helpful for developing microbiome-based diagnostics and therapeutics for this novel pandemic disease.


Subject(s)
Bacteria/classification , COVID-19/microbiology , Nasopharynx/microbiology , SARS-CoV-2/genetics , Sequence Analysis, RNA/methods , Adult , Aged , Bacteria/genetics , Bacteria/isolation & purification , Bacteria/pathogenicity , Case-Control Studies , Female , High-Throughput Nucleotide Sequencing , Humans , Male , Metagenomics , Middle Aged , Phylogeny , Symbiosis , Young Adult
9.
Front Immunol ; 12: 733539, 2021.
Article in English | MEDLINE | ID: covidwho-1572288

ABSTRACT

The response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is largely impacted by the level of virus exposure and status of the host immunity. The nature of protection shown by direct asymptomatic contacts of coronavirus disease 2019 (COVID-19)-positive patients is quite intriguing. In this study, we have characterized the antibody titer, SARS-CoV-2 surrogate virus neutralization, cytokine levels, single-cell T-cell receptor (TCR), and B-cell receptor (BCR) profiling in asymptomatic direct contacts, infected cases, and controls. We observed significant increase in antibodies with neutralizing amplitude in asymptomatic contacts along with cytokines such as Eotaxin, granulocyte-colony stimulating factor (G-CSF), interleukin 7 (IL-7), migration inhibitory factor (MIF), and macrophage inflammatory protein-1α (MIP-1α). Upon single-cell RNA (scRNA) sequencing, we explored the dynamics of the adaptive immune response in few representative asymptomatic close contacts and COVID-19-infected patients. We reported direct asymptomatic contacts to have decreased CD4+ naive T cells with concomitant increase in CD4+ memory and CD8+ Temra cells along with expanded clonotypes compared to infected patients. Noticeable proportions of class switched memory B cells were also observed in them. Overall, these findings gave an insight into the nature of protection in asymptomatic contacts.


Subject(s)
Adaptive Immunity/immunology , COVID-19/immunology , Genomics/methods , SARS-CoV-2/immunology , Single-Cell Analysis/methods , Adaptive Immunity/genetics , Adult , Antibodies, Viral/immunology , COVID-19/genetics , COVID-19/virology , Cytokines/immunology , Cytokines/metabolism , Female , Gene Expression Profiling/methods , Humans , Male , /metabolism , Middle Aged , SARS-CoV-2/physiology , Sequence Analysis, RNA/methods , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , T-Lymphocytes/virology , Young Adult
10.
PLoS One ; 16(12): e0261229, 2021.
Article in English | MEDLINE | ID: covidwho-1571989

ABSTRACT

In-depth study of the entire SARS-CoV-2 genome has uncovered many mutations, which have replaced the lineage that characterized the first wave of infections all around the world. In December 2020, the outbreak of variant of concern (VOC) 202012/01 (lineage B.1.1.7) in the United Kingdom defined a turning point during the pandemic, immediately posing a worldwide threat on the Covid-19 vaccination campaign. Here, we reported the evolution of B.1.1.7 lineage-related infections, analyzing samples collected from January 1st 2021, until April 15th 2021, in Friuli Venezia Giulia, a northeastern region of Italy. A cohort of 1508 nasopharyngeal swabs was analyzed by High Resolution Melting (HRM) and 479 randomly selected samples underwent Next Generation Sequencing analysis (NGS), uncovering a steady and continuous accumulation of B.1.1.7 lineage-related specimens, joined by sporadic cases of other known lineages (i.e. harboring the Spike glycoprotein p.E484K mutation). All the SARS-CoV-2 genome has been analyzed in order to highlight all the rare mutations that may eventually result in a new variant of interest. This work suggests that a thorough monitoring of the SARS-CoV-2 genome by NGS is essential to contain any new variant that could jeopardize all the efforts that have been made so far to resolve the emergence of the pandemic.


Subject(s)
COVID-19/diagnosis , Nasopharynx/virology , SARS-CoV-2/classification , Sequence Analysis, RNA/methods , COVID-19/epidemiology , Disease Outbreaks , High-Throughput Nucleotide Sequencing , Humans , Italy/epidemiology , Phylogeny , Phylogeography , RNA, Viral/genetics , SARS-CoV-2/genetics , United Kingdom/epidemiology
11.
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
12.
Nat Methods ; 18(6): 627-630, 2021 06.
Article in English | MEDLINE | ID: covidwho-1550331

ABSTRACT

We introduce the TRUST4 open-source algorithm for reconstruction of immune receptor repertoires in αß/γδ T cells and B cells from RNA-sequencing (RNA-seq) data. Compared with competing methods, TRUST4 supports both FASTQ and BAM format and is faster and more sensitive in assembling longer-even full-length-receptor repertoires. TRUST4 can also call repertoire sequences from single-cell RNA-seq (scRNA-seq) data without V(D)J enrichment, and is compatible with both SMART-seq and 5' 10x Genomics platforms.


Subject(s)
Algorithms , Receptors, Immunologic/genetics , Sequence Analysis, RNA/methods , Single-Cell Analysis/methods , Receptors, Antigen, B-Cell/genetics , Receptors, Antigen, T-Cell/genetics , V(D)J Recombination
13.
J Clin Epidemiol ; 142: 38-44, 2022 02.
Article in English | MEDLINE | ID: covidwho-1487821

ABSTRACT

OBJECTIVE: To evaluate the effectiveness of the Pfizer BNT162b2 vaccine against the SARS-Cov-2 Beta variant. STUDY DESIGN AND SETTING: Israel's mass vaccination program, using two doses of the Pfizer BNT162b2 vaccine, successfully curtailed the Alpha variant outbreak during winter 2020-2021, However, the virus may mutate and partially evade the immune system. To monitor this, sequencing of selected positive swab samples of interest was initiated. Comparing vaccinated with unvaccinated PCR positive persons, we estimated the odds ratio for a vaccinated case to have the Beta vs. the Alpha variant, using logistic regression, controlling for important confounders. RESULTS: There were 19 cases of Beta variant (3.2%) among those vaccinated more than 14 days before the positive sample and 79 (3.4%) among the unvaccinated. The estimated odds ratio was 1.26 (95% CI: 0.65-2.46). Assuming the effectiveness against the Alpha variant to be 95%, the estimated effectiveness against the Beta variant was 94% (95% CI: 88%-98%). CONCLUSION: Despite concerns over the Beta variant, the BNT162b2 vaccine seemed to provide substantial immunity against both the Beta and the Alpha variants. From 14 days following the second vaccine dose, the effectiveness of BNT162b2 vaccine was at most marginally affected by the Beta variant.


Subject(s)
/administration & dosage , COVID-19/virology , RNA, Viral/genetics , SARS-CoV-2/classification , Sequence Analysis, RNA/methods , Adult , Aged , Aged, 80 and over , COVID-19/prevention & control , Female , High-Throughput Nucleotide Sequencing , Humans , Israel , Logistic Models , Male , Mass Vaccination , Microbial Viability/drug effects , Middle Aged , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , SARS-CoV-2/growth & development , Young Adult
14.
Viruses ; 13(11)2021 10 20.
Article in English | MEDLINE | ID: covidwho-1481015

ABSTRACT

The causative agent of COVID-19 pandemic, SARS-CoV-2, has a 29,903 bases positive-sense single-stranded RNA genome. RNAs exhibit about 150 modified bases that are essential for proper function. Among internal modified bases, the N6-methyladenosine, or m6A, is the most frequent, and is implicated in SARS-CoV-2 immune response evasion. Although the SARS-CoV-2 genome is RNA, almost all genomes sequenced thus far are, in fact, reverse transcribed complementary DNAs. This process reduces the true complexity of these viral genomes because the incorporation of dNTPs hides RNA base modifications. Here, we present an initial exploration of Nanopore direct RNA sequencing to assess the m6A residues in the SARS-CoV-2 sequences of ORF3a, E, M, ORF6, ORF7a, ORF7b, ORF8, N, ORF10 and the 3'-untranslated region. We identified fifteen m6A methylated positions, of which, six are in ORF N. Additionally, because m6A is associated with the DRACH motif, we compared its distribution in major SARS-CoV-2 variants. Although DRACH is highly conserved among variants, we show that variants Beta and Eta have a fourth position C > U change in DRACH at 28,884b that could affect methylation. This is the first report of direct RNA sequencing of a Brazilian SARS-CoV-2 sample coupled with the identification of modified bases.


Subject(s)
Adenosine/analogs & derivatives , COVID-19/virology , Immune Evasion/genetics , RNA, Viral/metabolism , SARS-CoV-2/genetics , 3' Untranslated Regions , Adenosine/metabolism , Animals , Chlorocebus aethiops , Genome, Viral , Humans , Methylation , Nanopore Sequencing/methods , Open Reading Frames , Sequence Analysis, RNA/methods , Vero Cells
15.
Sci Rep ; 11(1): 20833, 2021 10 21.
Article in English | MEDLINE | ID: covidwho-1479812

ABSTRACT

Several single-cell RNA sequencing (scRNA-seq) studies analyzing immune response to COVID-19 infection have been recently published. Most of these studies have small sample sizes, which limits the conclusions that can be made with high confidence. By re-analyzing these data in a standardized manner, we validated 8 of the 20 published results across multiple datasets. In particular, we found a consistent decrease in T-cells with increasing COVID-19 infection severity, upregulation of type I Interferon signal pathways, presence of expanded B-cell clones in COVID-19 patients but no consistent trend in T-cell clonal expansion. Overall, our results show that the conclusions drawn from scRNA-seq data analysis of small cohorts of COVID-19 patients need to be treated with some caution.


Subject(s)
Biomarkers/metabolism , COVID-19/immunology , COVID-19/metabolism , RNA, Small Cytoplasmic , Single-Cell Analysis , Bronchoalveolar Lavage Fluid , Computational Biology , Databases, Factual , Gene Expression Profiling/methods , Genome, Human , Genome, Viral , Humans , Immunity , Leukocytes, Mononuclear/cytology , RNA-Seq , Reproducibility of Results , SARS-CoV-2 , Sequence Analysis, RNA/methods , Signal Transduction , Up-Regulation
16.
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
17.
PLoS One ; 16(9): e0257878, 2021.
Article in English | MEDLINE | ID: covidwho-1443847

ABSTRACT

Extracellular microRNAs (miRNAs) have been proposed to function in cross-kingdom gene regulation. Among these, plant-derived miRNAs of dietary origin have been reported to survive the harsh conditions of the human digestive system, enter the circulatory system, and regulate gene expression and metabolic function. However, definitive evidence supporting the presence of plant-derived miRNAs of dietary origin in mammals has been difficult to obtain due to limited sample sizes. We have developed a bioinformatics pipeline (ePmiRNA_finder) that provides strident miRNA classification and applied it to analyze 421 small RNA sequencing data sets from 10 types of human body fluids and tissues and comparative samples from carnivores and herbivores. A total of 35 miRNAs were identified that map to plants typically found in the human diet and these miRNAs were found in at least one human blood sample and their abundance was significantly different when compared to samples from human microbiome or cow. The plant-derived miRNA profiles were body fluid/tissue-specific and highly abundant in the brain and the breast milk samples, indicating selective absorption and/or the ability to be transported across tissue/organ barriers. Our data provide conclusive evidence for the presence of plant-derived miRNAs as a consequence of dietary intake and their cross-kingdom regulatory function within human circulating system.


Subject(s)
Computational Biology/methods , MicroRNAs/genetics , Plants/genetics , Sequence Analysis, RNA/methods , Animal Feed/analysis , Animals , Brain Chemistry , Carnivora/genetics , Diet , Female , Herbivory/genetics , Humans , Milk, Human/chemistry , Organ Specificity , RNA, Plant/genetics , Sample Size
19.
J Med Virol ; 94(1): 327-334, 2022 01.
Article in English | MEDLINE | ID: covidwho-1410052

ABSTRACT

Genomic surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays an important role in COVID-19 pandemic control and elimination efforts, especially by elucidating its global transmission network and illustrating its viral evolution. The deployment of multiplex PCR assays that target SARS-CoV-2 followed by either massively parallel or nanopore sequencing is a widely-used strategy to obtain genome sequences from primary samples. However, multiplex PCR-based sequencing carries an inherent bias of sequencing depth among different amplicons, which may cause uneven coverage. Here we developed a two-pool, long-amplicon 36-plex PCR primer panel with ~1000-bp amplicon lengths for full-genome sequencing of SARS-CoV-2. We validated the panel by assessing nasopharyngeal swab samples with a <30 quantitative reverse transcription PCR cycle threshold value and found that ≥90% of viral genomes could be covered with high sequencing depths (≥20% mean depth). In comparison, the widely-used ARTIC panel yielded 79%-88% high-depth genome regions. We estimated that ~5 Mbp nanopore sequencing data may ensure a >95% viral genome coverage with a ≥10-fold depth and may generate reliable genomes at consensus sequence levels. Nanopore sequencing yielded false-positive variations with frequencies of supporting reads <0.8, and the sequencing errors mostly occurred on the 5' or 3' ends of reads. Thus, nanopore sequencing could not elucidate intra-host viral diversity.


Subject(s)
Genome, Viral/genetics , Multiplex Polymerase Chain Reaction/methods , Nanopore Sequencing/methods , SARS-CoV-2/genetics , Whole Genome Sequencing/methods , COVID-19 , High-Throughput Nucleotide Sequencing/methods , Humans , Nasopharynx/virology , RNA, Viral/genetics , Sequence Analysis, RNA/methods
20.
Lab Invest ; 102(1): 4-13, 2022 01.
Article in English | MEDLINE | ID: covidwho-1402048

ABSTRACT

As one of the major approaches in combating the COVID-19 pandemics, the availability of specific and reliable assays for the SARS-CoV-2 viral genome and its proteins is essential to identify the infection in suspected populations, make diagnoses in symptomatic or asymptomatic individuals, and determine clearance of the virus after the infection. For these purposes, use of the quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) for detection of the viral nucleic acid remains the most valuable in terms of its specificity, fast turn-around, high-throughput capacity, and reliability. It is critical to update the sequences of primers and probes to ensure the detection of newly emerged variants. Various assays for increased levels of IgG or IgM antibodies are available for detecting ongoing or past infection, vaccination responses, and persistence and for identifying high titers of neutralizing antibodies in recovered individuals. Viral genome sequencing is increasingly used for tracing infectious sources, monitoring mutations, and subtype classification and is less valuable in diagnosis because of its capacity and high cost. Nanopore target sequencing with portable options is available for a quick process for sequencing data. Emerging CRISPR-Cas-based assays, such as SHERLOCK and AIOD-CRISPR, for viral genome detection may offer options for prompt and point-of-care detection. Moreover, aptamer-based probes may be multifaceted for developing portable and high-throughput assays with fluorescent or chemiluminescent probes for viral proteins. In conclusion, assays are available for viral genome and protein detection, and the selection of specific assays depends on the purposes of prevention, diagnosis and pandemic control, or monitoring of vaccination efficacy.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , Pandemics , SARS-CoV-2 , Antibodies, Viral/analysis , Antigens, Viral/analysis , COVID-19/epidemiology , COVID-19/virology , COVID-19 Nucleic Acid Testing/methods , COVID-19 Nucleic Acid Testing/trends , COVID-19 Serological Testing/methods , COVID-19 Serological Testing/trends , COVID-19 Testing/trends , Genome, Viral , Humans , Molecular Diagnostic Techniques/methods , Molecular Diagnostic Techniques/trends , Mutation , Nucleic Acid Amplification Techniques/methods , Nucleic Acid Amplification Techniques/trends , Open Reading Frames , RNA, Viral/analysis , RNA, Viral/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction/trends , SARS-CoV-2/classification , SARS-CoV-2/genetics , SARS-CoV-2/immunology , SARS-CoV-2/isolation & purification , Sequence Analysis, RNA/methods , Sequence Analysis, RNA/trends
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