Sequencing SARS-CoV-2 Genomes from Saliva.
Ontology highlight
ABSTRACT:
SUBMITTER: Alpert T
PROVIDER: S-EPMC8259911 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Genomic sequencing is crucial to understanding the epidemiology and evolution of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Often, genomic studies rely on remnant diagnostic material, typically nasopharyngeal (NP) swabs, as input into whole-genome SARS-CoV-2 next-generation sequencing pipelines. Saliva has proven to be a safe and stable specimen for the detection of SARS-CoV-2 RNA via traditional diagnostic assays; however, saliva is not commonly used for SARS-CoV-2 sequencing. Using the ARTIC Network amplicon-generation approach with sequencing on the Oxford Nanopore MinION, we demonstrate that sequencing SARS-CoV-2 from saliva produces genomes comparable to those from NP swabs, and that RNA extraction is necessary to generate complete genomes from saliva. In this study, we show that saliva is a useful specimen type for genomic studies of SARS-CoV-2.
| S-EPMC9074962 | biostudies-literature
Project description:We present CoronaHiT, a platform and throughput flexible method for sequencing SARS-CoV-2 genomes (≤ 96 on MinION or > 96 on Illumina NextSeq) depending on changing requirements experienced during the pandemic. CoronaHiT uses transposase-based library preparation of ARTIC PCR products. Method performance was demonstrated by sequencing 2 plates containing 95 and 59 SARS-CoV-2 genomes on nanopore and Illumina platforms and comparing to the ARTIC LoCost nanopore method. Of the 154 samples sequenced using all 3 methods, ≥ 90% genome coverage was obtained for 64.3% using ARTIC LoCost, 71.4% using CoronaHiT-ONT and 76.6% using CoronaHiT-Illumina, with almost identical clustering on a maximum likelihood tree. This protocol will aid the rapid expansion of SARS-CoV-2 genome sequencing globally.
| S-EPMC7871948 | biostudies-literature
Project description:Various next generation sequencing (NGS) based strategies have been successfully used in the recent past for tracing origins and understanding the evolution of infectious agents, investigating the spread and transmission chains of outbreaks, as well as facilitating the development of effective and rapid molecular diagnostic tests and contributing to the hunt for treatments and vaccines. The ongoing COVID-19 pandemic poses one of the greatest global threats in modern history and has already caused severe social and economic costs. The development of efficient and rapid sequencing methods to reconstruct the genomic sequence of SARS-CoV-2, the etiological agent of COVID-19, has been fundamental for the design of diagnostic molecular tests and to devise effective measures and strategies to mitigate the diffusion of the pandemic. Diverse approaches and sequencing methods can, as testified by the number of available sequences, be applied to SARS-CoV-2 genomes. However, each technology and sequencing approach has its own advantages and limitations. In the current review, we will provide a brief, but hopefully comprehensive, account of currently available platforms and methodological approaches for the sequencing of SARS-CoV-2 genomes. We also present an outline of current repositories and databases that provide access to SARS-CoV-2 genomic data and associated metadata. Finally, we offer general advice and guidelines for the appropriate sharing and deposition of SARS-CoV-2 data and metadata, and suggest that more efficient and standardized integration of current and future SARS-CoV-2-related data would greatly facilitate the struggle against this new pathogen. We hope that our 'vademecum' for the production and handling of SARS-CoV-2-related sequencing data, will contribute to this objective.
| S-EPMC7799330 | biostudies-literature
Project description:We introduce a target capture next-generation sequencing methodology, the ONETest Coronaviruses Plus, to sequence the SARS-CoV-2 genome and select loci of other respiratory viruses. We applied the ONETest on 70 respiratory samples (collected in Florida, USA between May and July, 2020), in which SARS-CoV-2 had been detected by a PCR assay. For 48 of the samples, we also applied the ARTIC protocol. Of the 70 ONETest libraries, 45 (64%) had a (near-)complete sequence (>29,000 bases and >90% covered by >9 reads). Of the 48 ARTIC libraries, 25 (52%) had a (near-)complete sequence. In 19 out of 25 (76%) samples in which both the ONETest and ARTIC yielded (near-)complete sequences, the lineages assigned were identical. As a target capture approach, the ONETest is less prone to loss of sequence coverage than amplicon approaches, and thus can provide complete genomic information more often to track and monitor SARS-CoV-2 variants.
| S-EPMC8299291 | biostudies-literature
Project description:In late 2019, a novel coronavirus began spreading in Wuhan, China, causing a potentially lethal respiratory viral infection. By early 2020, the novel coronavirus, called SARS-CoV-2, had spread globally, causing the COVID-19 pandemic. The infection and mutation rates of SARS-CoV-2 make it amenable to tracking introduction, spread and evolution by viral genome sequencing. Efforts to develop effective public health policies, therapeutics, or vaccines to treat or prevent COVID-19 are also expected to benefit from tracking mutations of the SARS-CoV-2 virus. Here we describe a set of comprehensive working protocols, from viral RNA extraction to analysis using established visualization tools, for high throughput sequencing of SARS-CoV-2 viral genomes using a MinION instrument. This set of protocols should serve as a reliable "how-to" reference for generating quality SARS-CoV-2 genome sequences with ARTIC primer sets and long-read nanopore sequencing technology. In addition, many of the preparation, quality control, and analysis steps will be generally applicable to other sequencing platforms.
| S-EPMC8493558 | biostudies-literature
Project description: Not available
| S-BSST379 | biostudies-other
Project description:COVID-19 patients produce circulating and mucosal antibodies. In adults, specific saliva antibodies have been detected. Nonetheless, seroprevalence is routinely investigated, while little attention has been paid to mucosal antibodies. We therefore assessed SARS-CoV-2-specific antibody prevalence in serum and saliva in children in the Netherlands. We assessed SARS-CoV-2 antibody prevalence in serum and saliva of 517 children attending medical services in the Netherlands (irrespective of COVID-19 exposure) from April to October 2020. The prevalence of SARS-CoV-2 spike (S), receptor binding domain (RBD), and nucleocapsid (N)-specific IgG and IgA were evaluated with an exploratory Luminex assay in serum and saliva and with the Wantai SARS-CoV-2 RBD total antibody enzyme-linked immunosorbent assay in serum. Using the Wantai assay, the RBD-specific antibody prevalence in serum was 3.3% (95% confidence interval [CI]. 1.9 to 5.3%). With the Luminex assay, we detected heterogeneity between antibodies for S, RBD, and N antigens, as IgG and IgA prevalence ranged between 3.6 and 4.6% in serum and between 0 and 4.4% in saliva. The Luminex assay also revealed differences between serum and saliva, with SARS-CoV-2-specific IgG present in saliva but not in serum for 1.5 to 2.7% of all children. Using multiple antigen assays, the IgG prevalence for at least two out of three antigens (S, RBD, or N) in serum or saliva can be calculated as 3.8% (95% CI, 2.3 to 5.6%). Our study displays the heterogeneity of the SARS-CoV-2 antibody response in children and emphasizes the additional value of saliva antibody detection and the combined use of different antigens. IMPORTANCE Comprehending humoral immunity to SARS-CoV-2, including in children, is crucial for future public health and vaccine strategies. Others have suggested that mucosal antibody measurement could be an important and more convenient tool to evaluate humoral immunity compared to circulating antibodies. Nonetheless, seroprevalence is routinely investigated, while little attention has been paid to mucosal antibodies. We show the heterogeneity of SARS-CoV-2 antibodies, in terms of both antigen specificity and differences between circulating and mucosal antibodies, emphasizing the additional value of saliva antibody detection next to detection of antibodies in serum.
| S-EPMC8557814 | biostudies-literature
Project description:We evaluated saliva (SAL) specimens for SARS-CoV-2 reverse transcriptase PCR (RT-PCR) testing by comparison of 459 prospectively paired nasopharyngeal (NP) or midturbinate (MT) swabs from 449 individuals with the aim of using saliva for asymptomatic screening. Samples were collected in a drive-through car line for symptomatic individuals (n = 380) and in the emergency department (ED) (n = 69). The percentages of positive and negative agreement of saliva compared to nasopharyngeal swab were 81.1% (95% confidence interval [CI], 65.8% to 90.5%) and 99.8% (95% CI, 98.7% to 100%), respectively. The percent positive agreement increased to 90.0% (95% CI, 74.4% to 96.5%) when considering only samples with moderate to high viral load (cycle threshold [CT ] for the NP, ≤34). Pools of five saliva specimens were also evaluated on three platforms, bioMérieux NucliSENS easyMAG with ABI 7500Fast (CDC assay), Hologic Panther Fusion, and Roche Cobas 6800. The average loss of signal upon pooling was 2 to 3 CT values across the platforms. The sensitivities of detecting a positive specimen in a pool compared with testing individually were 94%, 90%, and 94% for the CDC 2019-nCoV real-time RT-PCR, Panther Fusion SARS-CoV-2 assay, and Cobas SARS-CoV-2 test, respectively, with decreased sample detection trending with lower viral load. We conclude that although pooled saliva testing, as collected in this study, is not quite as sensitive as NP/MT testing, saliva testing is adequate to detect individuals with higher viral loads in an asymptomatic screening program, does not require swabs or viral transport medium for collection, and may help to improve voluntary screening compliance for those individuals averse to various forms of nasal collections.
| S-EPMC8106731 | biostudies-literature
Project description:We evaluated saliva (SAL) specimens for SARS-CoV-2 RT-PCR testing by comparison of 459 prospectively paired nasopharyngeal (NP) or mid-turbinate (MT) swabs from 449 individuals with the aim of using saliva for asymptomatic screening. Samples were collected in a drive-through car line for symptomatic individuals (N=380) and in the emergency department (ED) (N=69). The percent positive and negative agreement of saliva compared to nasopharyngeal swab were 81.1% (95% CI: 65.8% - 90.5%) and 99.8% (95% CI: 98.7% - 100%), respectively. The sensitivity increased to 90.0% (95% CI: 74.4% - 96.5%) when considering only samples with moderate to high viral load (Cycle threshold (Ct) for the NP <=34). Pools of five saliva specimens were also evaluated on three platforms: bioMérieux NucliSENS easyMAG with ABI 7500Fast (CDC assay), Hologic Panther Fusion, and Roche COBAS 6800. The median loss of signal upon pooling was 2-4 Ct values across the platforms. The sensitivity of detecting a positive specimen in a pool compared with testing individually was 100%, 93%, and 95% for CDC 2019-nCoV Real-Time RT-PCR, Panther Fusion® SARS-CoV-2 assay, and cobas® SARS-CoV-2 test respectively, with decreased sample detection trending with lower viral load. We conclude that although pooled saliva testing, as collected in this study, is not quite as sensitive as NP/MT testing, saliva testing is adequate to detect individuals with higher viral loads in an asymptomatic screening program, does not require swabs or viral transport media for collection, and may help to improve voluntary screening compliance for those individuals averse to various forms of nasal collections.
| S-EPMC7553188 | biostudies-literature
Project description:Obtaining complete, high-quality reference genomes is essential to the study of any organism. Recent advances in nanopore sequencing, as well as genome assembly and analysis methods, have made it possible to obtain complete bacterial genomes from metagenomic (i.e., multispecies) samples, including those from the human microbiome. In this study, methods are presented to obtain complete bacterial genomes from human saliva using complementary Oxford Nanopore (ONT) and Illumina sequencing. Applied to 3 human saliva samples, these methods resulted in 11 complete bacterial genomes: 3 Saccharibacteria clade G6 (also known as Ca. Nanogingivalaceae HMT-870), 1 Saccharibacteria clade G1 HMT-348, 2 Rothia mucilaginosa, 2 Actinomyces graevenitzii, 1 Mogibacterium diversum, 1 Lachnospiraceae HMT-096, and 1 Lancefieldella parvula; and one circular chromosome of Ruminococcaceae HMT-075 (which likely has at least 2 chromosomes). The 4 Saccharibacteria genomes, as well as the Actinomyces graeventizii genomes, represented the first complete genomes from their respective bacterial taxa. Aside from the complete genomes, the assemblies contained 147 contigs of over 500,000 bp each and thousands of smaller contigs, together representing a myriad of additional draft genomes including many which are likely nearly complete. The complete genomes enabled highly accurate pangenome analysis, which identified unique and missing features of each genome compared to its closest relatives with complete genomes available in public repositories. These features provide clues as to the lifestyle and ecological role of these bacteria within the human oral microbiota, which will be particularly useful in designing future studies of the taxa that have never been isolated or cultivated. IMPORTANCE Obtaining complete and accurate genomes is crucial to the study of any organism. Previously, obtaining complete genomes of bacteria, including those of the human microbiome, frequently required isolation of the organism, as well as low-throughput, manual sequencing methods to resolve repeat regions. Advancements in long-read sequencing technologies, including Oxford Nanopore (ONT), have made it possible to obtain complete, closed bacterial genomes from metagenomic samples. This study reports methods to obtain complete genomes from the human oral microbiome using complementary ONT and Illumina sequencing of saliva samples. Eleven complete genomes were obtained from 3 human saliva samples, with genomes of Saccharibacteria HMT-870, Saccharibacteria HMT-348, and Actinomyces graeventzii being the first complete genomes from their respective taxa. Obtaining complete bacterial genomes in a high-throughput manner will help illuminate the metabolic and ecological roles of important members of the human microbiota, particularly those that have remained recalcitrant to isolation and cultivation.
| S-EPMC9600527 | biostudies-literature