Integrated characterization of SARS-CoV-2 genome, microbiome, antibiotic resistance and host response from single throat swabs

AbstractThe ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, poses a severe threat to humanity. Rapid and comprehensive analysis of both pathogen and host sequencing data is critical to track infection and inform therapies. In this study, we performed unbiased metatranscriptomic analysis of clinical samples from COVID-19 patients using a newly-developed RNA-seq library construction method (TRACE-seq), which utilizes tagmentation activity of Tn5 on RNA/DNA hybrids. This approach avoids the laborious and time-consuming steps in traditional RNA-seq procedure, and hence is fast, sensitive and convenient. We demonstrated that TRACE-seq allowed integrated characterization of full genome information of SARS-CoV-2, putative pathogens causing coinfection, antibiotic resistance and host response from single throat swabs. We believe that the integrated information will deepen our understanding of pathogenesis and improve diagnostic accuracy for infectious diseases.

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Integrated characterization of SARS-CoV-2 genome, microbiome, antibiotic resistance and host response from single throat swabs

Cell Discovery ◽

10.1038/s41421-021-00248-3 ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Bo Lu ◽

Yi Yan ◽

Liting Dong ◽

Lingling Han ◽

Yawei Liu ◽

...

Keyword(s):

Antibiotic Resistance ◽

Severe Acute Respiratory Syndrome ◽

Host Response ◽

Comprehensive Analysis ◽

Clinical Samples ◽

Full Genome ◽

Rna Seq ◽

Sequencing Data ◽

Genome Information

AbstractThe ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, poses a severe threat to humanity. Rapid and comprehensive analysis of both pathogen and host sequencing data is critical to track infection and inform therapies. In this study, we performed unbiased metatranscriptomic analysis of clinical samples from COVID-19 patients using a recently developed RNA-seq library construction method (TRACE-seq), which utilizes tagmentation activity of Tn5 on RNA/DNA hybrids. This approach avoids the laborious and time-consuming steps in traditional RNA-seq procedure, and hence is fast, sensitive, and convenient. We demonstrated that TRACE-seq allowed integrated characterization of full genome information of SARS-CoV-2, putative pathogens causing coinfection, antibiotic resistance, and host response from single throat swabs. We believe that the integrated information will deepen our understanding of pathogenesis and improve diagnostic accuracy for infectious diseases.

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TagSeqTools: a flexible and comprehensive analysis pipeline for NAD tagSeq data

10.1101/2020.03.09.982934 ◽

2020 ◽

Cited By ~ 1

Author(s):

Huan Zhong ◽

Zongwei Cai ◽

Zhu Yang ◽

Yiji Xia

Keyword(s):

Rna Sequencing ◽

Comprehensive Analysis ◽

Enzymatic Reactions ◽

Computational Tool ◽

Sequencing Data ◽

Analysis Pipeline ◽

Oxford Nanopore ◽

Long Read ◽

Identification And Characterization

AbstractNAD tagSeq has recently been developed for the identification and characterization of NAD+-capped RNAs (NAD-RNAs). This method adopts a strategy of chemo-enzymatic reactions to label the NAD-RNAs with a synthetic RNA tag before subjecting to the Oxford Nanopore direct RNA sequencing. A computational tool designed for analyzing the sequencing data of tagged RNA will facilitate the broader application of this method. Hence, we introduce TagSeqTools as a flexible, general pipeline for the identification and quantification of tagged RNAs (i.e., NAD+-capped RNAs) using long-read transcriptome sequencing data generated by NAD tagSeq method. TagSeqTools comprises two major modules, TagSeek for differentiating tagged and untagged reads, and TagSeqQuant for the quantitative and further characterization analysis of genes and isoforms. Besides, the pipeline also integrates some advanced functions to identify antisense or splicing, and supports the data reformation for visualization. Therefore, TagSeqTools provides a convenient and comprehensive workflow for researchers to analyze the data produced by the NAD tagSeq method or other tagging-based experiments using Oxford nanopore direct RNA sequencing. The pipeline is available at https://github.com/dorothyzh/TagSeqTools, under Apache License 2.0.

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Comprehensive analysis of RNA-seq and whole genome sequencing data reveals no evidence for SARS-CoV-2 integrating into host genome

10.1101/2021.06.06.447293 ◽

2021 ◽

Author(s):

Yu-Sheng Chen ◽

Shuaiyao Lu ◽

Bing Zhang ◽

Tingfu Du ◽

Wen-Jie Li ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Rna Virus ◽

Comprehensive Analysis ◽

Host Genome ◽

Whole Genome Sequencing Data ◽

Whole Genome ◽

Rna Seq ◽

Sequencing Data ◽

Infected Cells

SARS-CoV-2, as the causation of severe epidemic of COVID-19, is one kind of positive single-stranded RNA virus with high transmissibility. However, whether or not SARS-CoV-2 can integrate into host genome needs thorough investigation. Here, we performed both RNA sequencing (RNA-seq) and whole genome sequencing on SARS-CoV-2 infected human and monkey cells, and investigated the presence of host-virus chimeric events. Through RNA-seq, we did detect the chimeric host-virus reads in the infected cells. But further analysis using mixed libraries of infected cells and uninfected zebrafish embryos demonstrated that these reads are falsely generated during library construction. In support, whole genome sequencing also didn't identify the existence of chimeric reads in their corresponding regions. Therefore, the evidence for SARS-CoV-2's integration into host genome is lacking.

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Nucleotide sequence-based typing of meningococci directly from clinical samples

Journal of Medical Microbiology ◽

10.1099/jmm.0.05078-0 ◽

2003 ◽

Vol 52 (6) ◽

pp. 505-508 ◽

Cited By ~ 15

Author(s):

Mathew A. Diggle ◽

Carolyn M. Bell ◽

Stuart C. Clarke

Keyword(s):

Molecular Techniques ◽

Clinical Samples ◽

Nucleotide Sequencing ◽

Sequencing Data ◽

Conjugate Vaccines ◽

Laboratory Confirmation ◽

Highly Sensitive ◽

Culture Isolate ◽

Micro Organisms

The unpredictable characteristics of meningococcal disease (MD) make outbreaks complicated to monitor and consequently lead to high levels of public anxiety. Traditional molecular techniques have been utilized in order to understand better the epidemiology of MD, but some have disadvantages such as being highly specialized and labour-intensive, with low reproducibility. Some of these problems have been overcome by using multilocus sequence typing (MLST). This technique exploits the unambiguous nature and electronic portability of nucleotide sequencing data for the characterization of micro-organisms. The need for enhanced surveillance of MD after the introduction of serogroup C conjugate vaccines means that it is important to gain typing information from the infecting organism in the absence of a culture isolate. Here, the application of MLST for the laboratory confirmation and characterization of Neisseria meningitidis directly from clinical samples is described. This involved using a newly designed set of primers that were complementary to nucleotide sequences external to the existing MLST primers already in use for culture-based MLST of meningococci. This combination has produced a highly sensitive procedure to allow the efficient genotypic characterization of meningococci directly from clinical samples.

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Evaluation of Affymetrix Severe Acute Respiratory Syndrome Resequencing GeneChips in Characterization of the Genomes of Two Strains of Coronavirus Infecting Humans

Applied and Environmental Microbiology ◽

10.1128/aem.72.1.207-211.2006 ◽

2006 ◽

Vol 72 (1) ◽

pp. 207-211 ◽

Cited By ~ 10

Author(s):

Irshad M. Sulaiman ◽

Xin Liu ◽

Michael Frace ◽

Nikhat Sulaiman ◽

Melissa Olsen-Rasmussen ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Nucleotide Sequences ◽

Transmission Dynamics ◽

Clinical Samples ◽

Atypical Pneumonia ◽

Genome Sequences ◽

Multiple Alignments ◽

Novel Coronavirus ◽

Conventional Sequencing

ABSTRACT Severe acute respiratory syndrome (SARS) was discovered during a recent global outbreak of atypical pneumonia. A number of immunologic and molecular studies of the clinical samples led to the conclusion that a novel coronavirus (SARS-CoV) was associated with the outbreak. Later, a SARS resequencing GeneChip was developed by Affymetrix to characterize the complete genome of SARS-CoV on a single GeneChip. The present study was carried out to evaluate the performance of SARS resequencing GeneChips. Two human SARS-CoV strains (CDC#200301157 and Urbani) were resequenced by the SARS GeneChips. Five overlapping PCR amplicons were generated for each strain and hybridized with these GeneChips. The successfully hybridized GeneChips generated nucleotide sequences of nearly complete genomes for the two SARS-CoV strains with an average call rate of 94.6%. Multiple alignments of nucleotide sequences obtained from SARS GeneChips and conventional sequencing revealed full concordance. Furthermore, the GeneChip-based analysis revealed no additional polymorphic sites. The results of this study suggest that GeneChip-based genome characterization is fast and reproducible. Thus, SARS resequencing GeneChips may be employed as an alternate tool to obtain genome sequences of SARS-CoV strains pathogenic for humans in order to further understand the transmission dynamics of these viruses.

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Comprehensive Characterization of COVID-19 Patients with Repeatedly Positive SARS-CoV-2 Tests Using a Large U.S. Electronic Health Record Database

Microbiology Spectrum ◽

10.1128/spectrum.00327-21 ◽

2021 ◽

Author(s):

Xiao Dong ◽

Yujia Zhou ◽

Xiao-ou Shu ◽

Elmer V. Bernstam ◽

Rebecca Stern ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Electronic Health Record ◽

Health Record ◽

Health Consequences ◽

Sequencing Data ◽

Testing Data ◽

Electronic Health ◽

Comprehensive Characterization

The comprehensive characterization of clinical and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing data for patients with repeatedly positive SARS-CoV-2 tests can help prioritize suspected cases of reinfection for investigation in the absence of sequencing data and for continued surveillance of the potential long-term health consequences of SARS-CoV-2 infection.

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Antibiotic Resistance and mecA Gene Characterization of Coagulase-negative Staphylococci Isolated from Clinical Samples in Nepal

Infection and Drug Resistance ◽

10.2147/idr.s274163 ◽

2020 ◽

Vol Volume 13 ◽

pp. 3163-3169

Author(s):

Lok Bahadur Shrestha ◽

Narayan Raj Bhattarai ◽

Keshav Rai ◽

Basudha Khanal

Keyword(s):

Antibiotic Resistance ◽

Clinical Samples ◽

Gene Characterization ◽

Em Gene

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Dual RNA-Seq Enables Full-Genome Assembly of Measles Virus and Characterization of Host–Pathogen Interactions

Microorganisms ◽

10.3390/microorganisms9071538 ◽

2021 ◽

Vol 9 (7) ◽

pp. 1538

Author(s):

Timokratis Karamitros ◽

Vasiliki Pogka ◽

Gethsimani Papadopoulou ◽

Ourania Tsitsilonis ◽

Maria Evangelidou ◽

...

Keyword(s):

Immune Responses ◽

Viral Infection ◽

Peripheral Blood ◽

Measles Virus ◽

Full Genome ◽

Rna Seq ◽

Host Pathogen Interactions ◽

Dnase Treatment ◽

Host Pathogen

Measles virus (MeV) has a negative-sense 15 kb long RNA genome, which is generally conserved. Recent advances in high-throughput sequencing (HTS) and Dual RNA-seq allow the analysis of viral RNA genomes and the discovery of viral infection biomarkers, via the simultaneous characterization of the host transcriptome. However, these host–pathogen interactions remain largely unexplored in MeV infections. We performed untargeted Dual RNA-seq in 6 pharyngeal and 6 peripheral blood mononuclear cell (PBMCs) specimens from patients with MeV infection, as confirmed via routine real-time PCR testing. Following optimised DNase treatment of total nucleic acids, we used the pharyngeal samples to build poly-A-enriched NGS libraries. We reconstructed the viral genomes using the pharyngeal datasets and we further conducted differential expression, gene-ontology and pathways enrichment analysis to compare both the pharyngeal and the peripheral blood transcriptomes of the MeV-infected patients vs. control groups of healthy individuals. We obtained 6 MeV genotype-B3 full-genome sequences. We minutely analyzed the transcriptome of the MeV-infected pharyngeal epithelium, detecting all known viral infection biomarkers, but also revealing a functional cluster of local antiviral and inflammatory immune responses, which differ substantially from those observed in the PBMCs transcriptome. The application of Dual RNA-seq technologies in MeV-infected patients can potentially provide valuable information on the virus genome structure and the cellular innate immune responses and drive the discovery of new targets for antiviral therapy.

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The Isolation and Characterization of Proteus mirabilis from Different Clinical Samples

Journal of Biotechnology Research Center ◽

10.24126/jobrc.2013.7.2.261 ◽

2013 ◽

Vol 7 (2) ◽

pp. 24-30

Author(s):

Wasan W. Al-Bassam ◽

Abdul-Kareem Al-Kazaz

Keyword(s):

Antibiotic Resistance ◽

Nalidixic Acid ◽

High Resistance ◽

Proteus Mirabilis ◽

Urine Samples ◽

Clinical Samples ◽

Isolation And Characterization

A total of one hundred five samples were collected from hospitals of Baghdad city during the period from 10/12/2008 to 15/3/2009. These clinical samples included: urine (50) wound swabs (25), sputum (20), and ear swabs (10). These samples were collected from the Baghdad hospital/ Teaching Laboratories, and Al-Yarmook Hospital/ Teaching Laboratories. Twenty six isolates of Proteus mirabilis were characterized according to the morphology and microscopic characteristics, along with the biochemical and confirmatory APi 20 E tests. These isolates were obtained from: urine (19), wound swabs (6), ear swabs (2), and sputum (1). The twenty selected isolates were tested for resistance against ten antibiotics and only urine samples were tested for nalidixic acid and nitrofurantoin resistance. It was shown that there were differences in the antibiotic resistance of isolates. High resistance to nitrofurantoin and ampicillin were found among isolates as (100%) and (75%) respectively while the resistance of Proteus isolates to trimethoprin /sulphamethazol, were (65%). This study also showed that resistance of isolates to gentamicin, ciprofloxacin, ceftazidime, pipracillin, cefotaxime, nalidixic acid azteronam, imipenem and amikacin were (50, 40, 40, 40, 35,27, 20,15, 5)% respectively.

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