Detection Of Genomic Variants Of SARS-CoV-2 Circulating In Wastewater By High-Throughput Sequencing

The use of SARS-CoV-2 metagenomics in wastewater can allow the detection of variants circulating at community level. After comparing with clinical databases, we identified three novel variants in the spike gene, and six new variants in the spike detected for the first time in Spain. We finally support the hypothesis that this approach allows the identification of unknown SARS-CoV-2 variants or detected at only low frequencies in clinical genomes.Abstract Figure

Download Full-text

High-Throughput Sequencing Reveals Further Diversity of Little Cherry Virus 1 with Implications for Diagnostics

Viruses ◽

10.3390/v10070385 ◽

2018 ◽

Vol 10 (7) ◽

pp. 385 ◽

Cited By ~ 4

Author(s):

Asimina Katsiani ◽

Varvara Maliogka ◽

Nikolaos Katis ◽

Laurence Svanella-Dumas ◽

Antonio Olmos ◽

...

Keyword(s):

Genetic Diversity ◽

High Throughput ◽

High Throughput Sequencing ◽

Sour Cherry ◽

Prunus Species ◽

Cherry Tree ◽

High Genetic Diversity ◽

Viral Genomes ◽

Virus Diversity ◽

First Time

Little cherry virus 1 (LChV1, Velarivirus, Closteroviridae) is a widespread pathogen of sweet or sour cherry and other Prunus species, which exhibits high genetic diversity and lacks a putative efficient transmission vector. Thus far, four distinct phylogenetic clusters of LChV1 have been described, including isolates from different Prunus species. The recent application of high throughput sequencing (HTS) technologies in fruit tree virology has facilitated the acquisition of new viral genomes and the study of virus diversity. In the present work, several new LChV1 isolates from different countries were fully sequenced using different HTS approaches. Our results reveal the presence of further genetic diversity within the LChV1 species. Interestingly, mixed infections of the same sweet cherry tree with different LChV1 variants were identified for the first time. Taken together, the high intra-host and intra-species diversities of LChV1 might affect its pathogenicity and have clear implications for its accurate diagnostics.

Download Full-text

7. High-Throughput Sequencing of AAV Proviral Libraries from the Human Population Reveals Novel Variants with Unprecedented Intra-and Inter-Tissue Diversity

Molecular Therapy ◽

10.1016/s1525-0016(16)32816-7 ◽

2016 ◽

Vol 24 ◽

pp. S4 ◽

Cited By ~ 3

Author(s):

Guangchao Xu ◽

Li Luo ◽

Phillip W.L. Tai ◽

Wanru Qin ◽

Yuanyuan Xiao ◽

...

Keyword(s):

High Throughput ◽

Human Population ◽

High Throughput Sequencing ◽

Novel Variants

Download Full-text

Rice Stripe Tenuivirus Has a Greater Tendency To Use the Prime-and-Realign Mechanism in Transcription of Genomic than in Transcription of Antigenomic Template RNAs

Journal of Virology ◽

10.1128/jvi.01414-17 ◽

2017 ◽

Vol 92 (1) ◽

Cited By ~ 3

Author(s):

Xiaojuan Liu ◽

Jing Jin ◽

Ping Qiu ◽

Fangluan Gao ◽

Wenzhong Lin ◽

...

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Rna Viruses ◽

Viral Mrnas ◽

Viral Pathogens ◽

Promising Target ◽

Cellular Mrnas ◽

Sequencing Strategy ◽

First Time ◽

Negative Sense

ABSTRACTMost segmented negative-sense RNA viruses employ a process termed cap snatching, during which they snatch capped RNA leaders from host cellular mRNAs and use the snatched leaders as primers for transcription, leading to the synthesis of viral mRNAs with 5′ heterogeneous sequences (HSs). With traditional methods, only a few HSs can be determined, and identification of their donors is difficult. Here, the mRNA 5′ ends ofRice stripe tenuivirus(RSV) andRice grassy stunt tenuivirus(RGSV) and those of their host rice were determined by high-throughput sequencing. Millions of tenuiviral HSs were obtained, and a large number of them mapped to the 5′ ends of corresponding host cellular mRNAs. Repeats of the dinucleotide AC, which are complementary to the U1G2of the tenuiviral template 3′-U1G2U3G4UUUCG, were found to be prevalent at the 3′ termini of tenuiviral HSs. Most of these ACs did not match host cellular mRNAs, supporting the idea that tenuiviruses use the prime-and-realign mechanism during cap snatching. We previously reported a greater tendency of RSV than RGSV to use the prime-and-realign mechanism in transcription with leaders cap snatched from a coinfecting reovirus. Besides confirming this observation in natural tenuiviral infections, the data here additionally reveal that RSV has a greater tendency to use this mechanism in transcribing genomic than in transcribing antigenomic templates. The data also suggest that tenuiviruses cap snatch host cellular mRNAs from translation- and photosynthesis-related genes, and capped RNA leaders snatched by tenuiviruses base pair with U1/U3or G2/G4of viral templates. These results provide unprecedented insights into the cap-snatching process of tenuiviruses.IMPORTANCEMany segmented negative-sense RNA viruses (segmented NSVs) are medically or agriculturally important pathogens. The cap-snatching process is a promising target for the development of antiviral strategies against this group of viruses. However, many details of this process remain poorly characterized. Tenuiviruses constitute a genus of agriculturally important segmented NSVs, several members of which are major viral pathogens of rice. Here, we for the first time adopted a high-throughput sequencing strategy to determine the 5′ heterogeneous sequences (HSs) of tenuiviruses and mapped them to host cellular mRNAs. Besides providing deep insights into the cap snatching of tenuiviruses, the data obtained provide clear evidence to support several previously proposed models regarding cap snatching. Curiously and importantly, the data here reveal that not only different tenuiviruses but also the same tenuivirus synthesizing different mRNAs use the prime-and-realign mechanism with different tendencies during their cap snatching.

Download Full-text

jackalope: a swift, versatile phylogenomic and high-throughput sequencing simulator

10.1101/650747 ◽

2019 ◽

Author(s):

Lucas A. Nell

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Population Genomics ◽

R Package ◽

Gene Trees ◽

Sequencing Platform ◽

Genomic Variants ◽

Pacific Biosciences ◽

Wide Range ◽

Reference Genomes

AbstractHigh-throughput sequencing (HTS) is central to the study of population genomics and has an increasingly important role in constructing phylogenies. Choices in research design for sequencing projects can include a wide range of factors, such as sequencing platform, depth of coverage, and bioinformatic tools. Simulating HTS data better informs these decisions. However, current standalone HTS simulators cannot generate genomic variants under even somewhat complex evolutionary scenarios, which greatly reduces their usefulness for fields such as population genomics and phylogenomics. Here I present the R package jackalope that simply and efficiently simulates (i) variants from reference genomes and (ii) reads from both Illumina and Pacific Biosciences (PacBio) platforms. Genomic variants can be simulated using phylogenies, gene trees, coalescent-simulation output, population-genomic summary statistics, and Variant Call Format (VCF) files. jackalope can simulate single, paired-end, or mate-pair Illumina reads, as well as reads from Pacific Biosciences. These simulations include sequencing errors, mapping qualities, multiplexing, and optical/PCR duplicates. It can read reference genomes from FASTA files and can simulate new ones, and all outputs can be written to standard file formats. jackalope is available for Mac, Windows, and Linux systems.

Download Full-text

High-throughput sequencing of pooled samples to determine community-level microbiome diversity

Annals of Epidemiology ◽

10.1016/j.annepidem.2019.09.002 ◽

2019 ◽

Vol 39 ◽

pp. 63-68 ◽

Cited By ~ 5

Author(s):

Kathryn J. Ray ◽

Sun Y. Cotter ◽

Ahmed M. Arzika ◽

Jessica Kim ◽

Nameywa Boubacar ◽

...

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Community Level ◽

Microbiome Diversity ◽

Pooled Samples

Download Full-text

Characterization of Spanish Olive Virome by High Throughput Sequencing Opens New Insights and Uncertainties

Viruses ◽

10.3390/v13112233 ◽

2021 ◽

Vol 13 (11) ◽

pp. 2233

Author(s):

Ana Belén Ruiz-García ◽

Celia Canales ◽

Félix Morán ◽

Manuel Ruiz-Torres ◽

Magdalena Herrera-Mármol ◽

...

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Biological Significance ◽

Euphyllura Olivina ◽

Leaf Yellowing ◽

Full Length Sequence ◽

Pros And Cons ◽

Olive Trees ◽

First Time ◽

Viral Sequences

The use of high throughput sequencing (HTS) for the analysis of Spanish olive trees showing leaf yellowing discoloration, defoliation, and/or decline has provided new insights into the olive viruses present in Spain and has opened discussions about the pros and cons of these technologies for diagnostic purposes. In this study, we report for the first time in Spanish orchards the presence of olive leaf yellowing-associated virus (OLYaV), for which the second full coding sequence has been determined. This virus has also been detected in a putative vector, the psyllid Euphyllura olivina. In addition, the presence in Spain of Olea europaea geminivirus (OEGV), recently reported in Italy, has been confirmed, and the full-length sequence of two isolates was obtained by HTS and Sanger sequencing. These results, as well as the detection of other viral sequences related to olive latent virus 3 (OLV-3) and olive viral satellite RNA, raises questions on the biological significance of the findings, about the requirement of standardization on the interpretation of HTS results, and the necessity of additional tests to confirm the relevance of the HTS detection of viral sequences.

Download Full-text

High-Throughput Sequencing for Detection of Subpopulations of Bacteria Not Previously Associated with Artisanal Cheeses

Applied and Environmental Microbiology ◽

10.1128/aem.00918-12 ◽

2012 ◽

Vol 78 (16) ◽

pp. 5717-5723 ◽

Cited By ~ 150

Author(s):

Lisa Quigley ◽

Orla O'Sullivan ◽

Tom P. Beresford ◽

R. Paul Ross ◽

Gerald F. Fitzgerald ◽

...

Keyword(s):

High Throughput ◽

Significant Influence ◽

High Throughput Sequencing ◽

Microbial Populations ◽

Bacterial Populations ◽

Content Type ◽

Pasteurized Milk ◽

First Time

ABSTRACTHere, high-throughput sequencing was employed to reveal the highly diverse bacterial populations present in 62 Irish artisanal cheeses and, in some cases, associated cheese rinds. Using this approach, we revealed the presence of several genera not previously associated with cheese, includingFaecalibacterium,Prevotella, andHelcococcusand, for the first time, detected the presence ofArthrobacterandBrachybacteriumin goats' milk cheese. Our analysis confirmed many previously observed patterns, such as the dominance of typical cheese bacteria, the fact that the microbiota of raw and pasteurized milk cheeses differ, and that the level of cheese maturation has a significant influence onLactobacilluspopulations. It was also noted that cheeses containing adjunct ingredients had lower proportions ofLactococcusspecies. It is thus apparent that high-throughput sequencing-based investigations can provide valuable insights into the microbial populations of artisanal foods.

Download Full-text

Use of High-Throughput Sequencing and Two RNA Input Methods to Identify Viruses Infecting Tomato Crops

Microorganisms ◽

10.3390/microorganisms9051043 ◽

2021 ◽

Vol 9 (5) ◽

pp. 1043

Author(s):

Ayoub Maachi ◽

Covadonga Torre ◽

Raquel N. Sempere ◽

Yolanda Hernando ◽

Miguel A. Aranda ◽

...

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Tomato Fruit ◽

Virus Detection ◽

Read Depth ◽

Cdna Libraries ◽

Virus Species ◽

Total Rna ◽

Viral Genomes ◽

First Time

We used high-throughput sequencing to identify viruses on tomato samples showing virus-like symptoms. Samples were collected from crops in the Iberian Peninsula. Either total RNA or double-stranded RNA (dsRNA) were used as starting material to build the cDNA libraries. In total, seven virus species were identified, with pepino mosaic virus being the most abundant one. The dsRNA input provided better coverage and read depth but missed one virus species compared with the total RNA input. By performing in silico analyses, we determined a minimum sequencing depth per sample of 0.2 and 1.5 million reads for dsRNA and rRNA-depleted total RNA inputs, respectively, to detect even the less abundant viruses. Primers and TaqMan probes targeting conserved regions in the viral genomes were designed and/or used for virus detection; all viruses were detected by qRT-PCR/RT-PCR in individual samples, with all except one sample showing mixed infections. Three virus species (Olive latent virus 1, Lettuce ring necrosis virus and Tomato fruit blotch virus) are herein reported for the first time in tomato crops in Spain.

Download Full-text

Identification and Characterization of Citrus Concave Gum-Associated Virus Infecting Citrus and Apple Trees by Serological, Molecular and High-Throughput Sequencing Approaches

Plants ◽

10.3390/plants10112390 ◽

2021 ◽

Vol 10 (11) ◽

pp. 2390

Author(s):

Maria Minutolo ◽

Maria Cinque ◽

Michela Chiumenti ◽

Francesco Di Serio ◽

Daniela Alioto ◽

...

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Rna Virus ◽

Polyclonal Antiserum ◽

Enzyme Linked Immunosorbent Assay ◽

Apple Trees ◽

The Usa ◽

Tissue Blot Immunoassay ◽

Apple Cultivars ◽

First Time

Citrus concave gum-associated virus (CCGaV) is a negative-stranded RNA virus, first reported a few years ago in citrus trees from Italy. It has been reported in apple trees in the USA and in Brazil, suggesting a wider host range and geographic distribution. Here, an anti-CCGaV polyclonal antiserum to specifically detect the virus has been developed and used in a standard double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) that has been validated as a sensitive and reliable method to detect this virus both in citrus and apple trees. In contrast, when the same antiserum was used in direct tissue-blot immunoassay, CCGaV was efficiently detected in citrus but not in apple. Using this antiserum, the first apple trees infected by CCGaV were identified in Italy and the presence of CCGaV in several apple cultivars in southern Italy was confirmed by field surveys. High-throughput sequencing (HTS) allowed for the assembling of the complete genome of one CCGaV Italian apple isolate (CE-c3). Phylogenetic analysis of Italian CCGaV isolates from apple and citrus and those available in the database showed close relationships between the isolates from the same genus (Citrus or Malus), regardless their geographical origin. This finding was further confirmed by the identification of amino acid signatures specific of isolates infecting citrus or apple hosts. Analysis of HTS reads also revealed that the CE-c3 Italian apple tree, besides CCGaV, was simultaneously infected by several viruses and one viroid, including apple rubbery wood virus 2 which is reported for the first time in Italy. The complete or almost complete genomic sequences of the coinfecting agents were determined.

Download Full-text