scholarly journals High-Throughput Sequencing Reveals Cyclamen persicum Mill. as a Natural Host for Fig Mosaic Virus

Viruses ◽  
2018 ◽  
Vol 10 (12) ◽  
pp. 684 ◽  
Author(s):  
Toufic Elbeaino ◽  
Armelle Marais ◽  
Chantal Faure ◽  
Elisa Trioano ◽  
Thierry Candresse ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Viral Infections ◽  
Small Scale ◽  
Rt Pcr ◽  
Cyclamen Persicum ◽  
Sequence Variations ◽  
Pcr Products ◽  
Complete Sequences ◽  
Fig Mosaic Virus

In a search for viral infections, double-stranded RNA (dsRNA) were recovered from a diseased cyclamen (Cyclamen persicum Mill.) accession (Cic) and analyzed by high-throughput sequencing (HTS) technology. Analysis of the HTS data showed the presence of Fig mosaic emaravirus (FMV) in this accession. The complete sequences of six FMV-Cic RNA genomic segments were determined from the HTS data and using Sanger sequencing. All FMV-Cic RNA segments are similar in size to those of FMV from fig (FMV-Gr10), with the exception of RNA-6 that is one nucleotide longer. The occurrence of FMV in cyclamen was investigated through a small-scale survey, from which four plants (out of 18 tested) were found RT-PCR positive. To study sequence variations of cyclamen isolates of FMV, RT-PCR products generated through the amplification of the partially RNA-dependent RNA polymerase (RdRp, RNA-1), glycoprotein (GP, RNA-2), and nucleocapsid (NCP, RNA-3) genes were explored. The nucleotide sequence identities for cyclamen isolates ranged between 86% and 99% in RNA-1, 93% and 99% in RNA-2, and 98% and 99% in RNA-3, while lower identity levels were observed with the sequences of fig isolates. Based on the phylogenetic tree obtained with a 304-nt fragment of RNA3, all FMV isolates from cyclamens were assigned to a single cluster close to fig isolates from the Mediterranean. FMV was graft-transmitted to healthy cyclamens eliciting symptoms similar to those observed in the Cic accession, thus suggesting a causal role of FMV in the symptoms that prompted the investigation. This is the first report of FMV in a non-fig host, Cyclamen persicum, a finding that may help in the control of the mosaic and mosaic-like diseases of fig and cyclamen, respectively.

Comparison of high throughput sequencing to standard protocols for virus detection in berry crops

Plant Disease ◽  
2021 ◽  
Author(s):  
Dan Edward Veloso Villamor ◽  
Karen E Keller ◽  
Robert Martin ◽  
Ioannis Emmanouil Tzanetakis
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Wide Spectrum ◽  
Virus Detection ◽  
Rt Pcr ◽  
Host Interactions ◽  
Growing Seasons ◽  
Berry Crops ◽  
Sampling Times ◽  
Better Than

A comprehensive study comparing virus detection between high throughput sequencing (HTS) and standard protocols in 30 berry selections (12 Fragaria, 10 Vaccinium and 8 Rubus) with known virus profiles was completed. The study examined temporal detection of viruses at four sampling times encompassing two growing seasons. Within the standard protocols, RT-PCR proved better than biological indexing. Detection of known viruses by HTS and RT-PCR nearly mirrored each other. HTS provided superior detection compared to RT-PCR on a wide spectrum of virus variants and discovery of novel viruses. More importantly, in most cases where the two protocols showed parallel virus detection, 11 viruses in 16 berry selections were not consistently detected by both methods at all sampling points. Based on these data we propose a four sampling times/two-year testing requirement for berry and potentially other crops to ensure that no virus remains undetected independent of titer, distribution or other virus/virus or virus/host interactions.

scholarly journals A cell-free DNA metagenomic sequencing assay that integrates the damage response to infection

2019 ◽  
Author(s):  
Alexandre Pellan Cheng ◽  
Philip Burnham ◽  
John Richard Lee ◽  
Matthew Pellan Cheng ◽  
Manikkam Suthanthiran ◽  
...  
Keyword(s):  
Urinary Tract ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Viral Infections ◽  
Clinical Samples ◽  
Metagenomic Sequencing ◽  
Kidney Transplant Recipients ◽  
Bladder Tissue ◽  
Cell Free Dna ◽  
Free Dna

ABSTRACTHigh-throughput metagenomic sequencing offers an unbiased approach to identify pathogens in clinical samples. Conventional metagenomic sequencing however does not integrate information about the host, which is often critical to distinguish infection from infectious disease, and to assess the severity of disease. Here, we explore the utility of high-throughput sequencing of cell-free DNA after bisulfite conversion to map the tissue and cell types of origin of host-derived cell-free DNA, and to profile the bacterial and viral metagenome. We applied this assay to 51 urinary cfDNA isolates collected from a cohort of kidney transplant recipients with and without bacterial and viral infection of the urinary tract. We find that the cell and tissue types of origin of urinary cell-free DNA can be derived from its genome-wide profile of methylation marks, and strongly depend on infection status. We find evidence of kidney and bladder tissue damage due to viral and bacterial infection, respectively, and of the recruitment of neutrophils to the urinary tract during infection. Through direct comparison to conventional metagenomic sequencing as well as clinical tests of infection, we find this assay accurately captures the bacterial and viral composition of the sample. The assay presented here is straightforward to implement, offers a systems view into bacterial and viral infections of the urinary tract, and can find future use as a tool for the differential diagnosis of infections.

scholarly journals First Report of Grapevine Kizil Sapak Virus in Grapevine in China

Plant Disease ◽  
2021 ◽  
Author(s):  
Fang Ren ◽  
Zunping Zhang ◽  
Xudong Fan ◽  
Guojun Hu ◽  
Yafeng Dong
Keyword(s):  
Complete Genome ◽  
High Throughput Sequencing ◽  
Pairwise Comparison ◽  
Bioinformatic Analysis ◽  
Rt Pcr ◽  
Illumina Hiseq ◽  
First Report ◽  
Hop Stunt Viroid ◽  
Pcr Products ◽  
Rapid Amplification

Grapevine Kizil Sapak virus (GKSV) is a novel member of the family Betaflexiviridae classified into the proposed genus Fivivirus within the subfamily Trivirinae. It was first discovered in USA from a grapevine originating from Turkmenistan (Al Rwahnih et al. 2019) and later in France from a grapevine accession from Iran (Marais et al. 2020). In October 2019, an asymptomatic grapevine cv. ‘Crimson Seedless’ (native to USA) was collected from Xinjiang province in China and analyzed by high-throughput sequencing (HTS). Ribosome-depleted RNA preparations were used for library synthesis followed by HTS on an Illumina HiSeq X-ten platform. A total of 29,141,024 cleaned reads were obtained, and 7,878 contigs were generated using CLC Genomics Workbench 9.5 (QIAGEN). One long contig (7,328 bp) showed 88.2% nucleotide (nt) identity with the sequence of GKSV-127 (MN172165) via Blastx, with an average coverage of 284-X. Bioinformatic analysis of the remaining contigs showed the presence of Grapevine leafroll-associated virus 4, Grapevine rupestris vein feathering virus, Grapevine fabavirus, grapevine yellow speckle viroid-1 (GYSVd-1), GYSVd-2 and Hop stunt viroid in the sample. The presence of GKSV was checked by RT-PCR using the primer GKSV-F/R (Al Rwahnih et al. 2019); the 1,240 bp PCR product was cloned using a pTOPO-T vector (Aidlab, China) and sequenced. In pairwise comparison, the obtained nt sequences shared 92.6 to 95.2% identity to the corresponding HTS sequence, confirming the presence of GKSV in the sample. The complete GKSV genome sequence was obtained as two pieces of overlapping DNA sequence using primers GKSV-20A/20B (5’-TAGTCTGGATTTCCCTACCT/5’-CTCCCTAAACTGATTTGATG) and GKSV-25A/25B (5’-GCCACTGGTGAATGAAAAGA/5’-CTAAATGAATGGGCAGGTAT) designed based on the HTS-generated sequence. The 5’ and 3’ termini were determined by rapid amplification of cDNA ends using SMARTer RACE 5’/3’ Kit (Takara, Dalian, China). The complete genome of GKSV isolate CS (MW582898) comprised 7,604 nt (without the polyA tail) and shared 77.8 to 89.2% identities with the other nine reported GKSV isolates, among which it shared the highest nt identity (89.2%) with GKSV-127. In phylogenetic analysis based on complete or nearly complete genome sequences of representative members of Betaflexiviridae, GKSV-CS clustered with the nine known GKSV isolates, forming a subclade with GKSV-127 (Supplementary Fig. 1). To determine the incidence and distribution of GKSV in China, 476 grapevine samples of 75 cultivars were collected from 20 provinces and tested by RT-PCR using primers GKSV-F/R (Al Rwahnih et al. 2019) and Vini-F1/R1 (Marais et al. 2020). The results showed that 0.42% (2 of 476) of the samples tested positive with both primers, including samples GKSV-CS and a ‘Black Monukka’ grape (native to India) also sampled from Xinjiang. Both PCR products of ‘Black Monukka’ were cloned and sequenced (MZ311588 to MZ311602) and they showed 85.1 to 88.9% nt identities to the GKSV-CS sequence. This is the first report of GKSV infecting grapevine in China. Although the pathogenicity of GKSV is yet to be determined, it has been found in several countries such as USA (Al Rwahnih et al. 2019), France (Marais et al. 2020) and China (this study). Both positive samples in this study were collected from Nanjiang region in Xinjiang province, indicating the sporadic occurrence of GKSV in this area.

scholarly journals High-throughput sequencing identifies distinct fecal and mucosal gut microbiota correlating with different mucosal proteins

2016 ◽  
Author(s):  
Li-na Dong ◽  
Jun-ping Wang ◽  
Ping Liu ◽  
Yun-feng Yang ◽  
Jing Feng ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Flexible Sigmoidoscopy ◽  
Local Effect ◽  
Chinese People ◽  
Rt Pcr ◽  
Clinical Indices ◽  
16Srrna Gene ◽  
Firmicutes Phylum

The intestinal microbiota is associated with human health. The luminal microbiota (LM) and mucosa-associated microbiota (MAM) are distinct ecosystems with different metabolic and immunological functions. Several studies have examined the correlations between the gut microbiota and clinical indices, but few have investigated the relationships between the microbiota and mucosal proteins. We characterized the intestinal LM and MAM in Chinese people and examined the association between these communities and the expression of mucosal proteins. Fresh fecal samples and distal colonic mucosal biopsies were collected from 32 subjects before (fecal) and during (mucosal) flexible sigmoidoscopy. We used high-throughput sequencing targeting the 16SrRNA gene V3–V4 region to analyze the samples and reverse transcription(RT)–PCR to detect the expression of colonic proteins BDNF, ZO1, TLR2, TLR4, AQP3, and AQP8. Differences in the stool and mucosal microbiota were identified and a correlation network analysis performed. The LM and MAM populations differed significantly. In LM, the microbiota composition correlated significantly positively with host age, and Firmicutes (phylum) correlated positively with body mass index (BMI), but inversely with ZO1.At the genus level, systemic indices, such as age, BMI, and BDNF, correlated predominantly with LM, whereas systemic and local indices, such as TLR2, correlated with both MAM and LM. ZO1 and TLR4 which usually exert a local effect, mainly correlated with MAM. Different bacteria were associated with the expression of different proteins. Our data suggest that The microbial compositions of LM and MAM differed. Different gut bacteria may play different roles by regulating the expression of different proteins.

scholarly journals Investigation of the Plasma Virome from Cases of Unexplained Febrile Illness in Tanzania from 2013 to 2014: a Comparative Analysis between Unbiased and VirCapSeq-VERT High-Throughput Sequencing Approaches

mSphere ◽  
2018 ◽  
Vol 3 (4) ◽  
Author(s):  
Simon H. Williams ◽  
Samuel Cordey ◽  
Nishit Bhuva ◽  
Florian Laubscher ◽  
Mary-Anne Hartley ◽  
...  
Keyword(s):  
Comparative Analysis ◽  
Positive Selection ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Viral Infections ◽  
Febrile Illness ◽  
Clinical Samples ◽  
Barr Virus ◽  
Outbreak Investigations ◽  
Epstein Barr

ABSTRACT High-throughput sequencing can provide insights into epidemiology and medicine through comprehensive surveys of viral genetic sequences in environmental and clinical samples. Here, we characterize the plasma virome of Tanzanian patients with unexplained febrile illness by using two high-throughput sequencing methods: unbiased sequencing and VirCapSeq-VERT (a positive selection system). Sequences from dengue virus 2, West Nile virus, human immunodeficiency virus type 1, human pegivirus, and Epstein-Barr virus were identified in plasma. Both sequencing strategies recovered nearly complete genomes in samples containing multiple viruses. Whereas VirCapSeq-VERT had better sensitivity, unbiased sequencing provided better coverage of genome termini. Together, these data demonstrate the utility of high-throughput sequencing strategies in outbreak investigations. IMPORTANCE Characterization of the viruses found in the blood of febrile patients provides information pertinent to public health and diagnostic medicine. PCR and culture have historically played an important role in clinical microbiology; however, these methods require a targeted approach and may lack the capacity to identify novel or mixed viral infections. High-throughput sequencing can overcome these constraints. As the cost of running multiple samples continues to decrease, the implementation of high-throughput sequencing for diagnostic purposes is becoming more feasible. Here we present a comparative analysis of findings from an investigation of unexplained febrile illness using two strategies: unbiased high-throughput sequencing and VirCapSeq-VERT, a positive selection high-throughput sequencing system.

High-throughput sequencing of kDNA amplicons for the analysis ofLeishmaniaminicircles and identification of Neotropical species

Parasitology ◽  
2017 ◽  
Vol 145 (5) ◽  
pp. 585-594 ◽  
Author(s):  
ARTHUR KOCHER ◽  
SOPHIE VALIÈRE ◽  
ANNE-LAURE BAÑULS ◽  
JÉRÔME MURIENNE
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Pcr Primers ◽  
Genomic Region ◽  
Kinetoplast Dna ◽  
Sequence Comparisons ◽  
Conserved Sequence ◽  
Species Complexes ◽  
Pcr Products ◽  
Minicircle Sequence

SUMMARYLeishmaniakinetoplast DNA contains thousands of small circular molecules referred to as kinetoplast DNA (kDNA) minicercles. kDNA minicircles are the preferred targets for sensitiveLeishmaniadetection, because they are present in high copy number and contain conserved sequence blocks in which polymerase chain reaction (PCR) primers can be designed. On the other hand, the heterogenic nature of minicircle networks has hampered the use of this peculiar genomic region for strain typing. The characterization ofLeishmaniaminicirculomes used to require isolation and cloning steps prior to sequencing. Here, we show that high-throughput sequencing of single minicircle PCR products allows bypassing these laborious laboratory tasks. The 120 bp long minicircle conserved region was amplified by PCR from 18Leishmaniastrains representative of the major species complexes found in the Neotropics. High-throughput sequencing of PCR products enabled recovering significant numbers of distinct minicircle sequences from each strain, reflecting minicircle class diversity. Minicircle sequence analysis revealed patterns that are congruent with current hypothesis ofLeishmaniarelationships. Then, we show that a barcoding-like approach based on minicircle sequence comparisons may allow reliable identifications ofLeishmaniaspp. This work opens up promising perspectives for the study of kDNA minicercles and a variety of applications inLeishmaniaresearch.

scholarly journals Small-scale high-throughput sequencing–based identification of new therapeutic tools in cystic fibrosis

2015 ◽  
Vol 17 (10) ◽  
pp. 796-806 ◽  
Author(s):  
Jennifer Bonini ◽  
Jessica Varilh ◽  
Caroline Raynal ◽  
Corinne Thèze ◽  
Emmanuelle Beyne ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Small Scale ◽  
Therapeutic Tools

scholarly journals A novel emaravirus comprising five RNA segments is associated with ringspot disease in oak

2021 ◽  
Vol 166 (3) ◽  
pp. 987-990
Author(s):  
Marius Rehanek ◽  
Susanne von Bargen ◽  
Martina Bandte ◽  
David G. Karlin ◽  
Carmen Büttner
Keyword(s):  
High Throughput Sequencing ◽  
Bioinformatic Analysis ◽  
High Plains ◽  
Rt Pcr ◽  
Viral Origin ◽  
Complete Sequences ◽  
Novel Virus ◽  
Rapid Amplification ◽  
Full Length Genome ◽  
Common Oak

AbstractWe report the complete nucleotide sequence of the genome of a novel virus in ringspot-diseased common oak (Quercus robur L.). The newly identified pathogen is associated with leaf symptoms such as mottle, chlorotic spots and ringspots on diseased trees. High-throughput sequencing (HTS, Illumina RNASeq) was used to explore the virome of a ringspot-diseased oak that had chlorotic ringspots of suspected viral origin on leaves for several years. Bioinformatic analysis of the HTS dataset followed by RT-PCR enabled us to determine complete sequences of four RNA genome segments of a novel virus. These sequences showed high similarity to members of the genus Emaravirus, which includes segmented negative-stranded RNA viruses of economic importance. To verify the ends of each RNA, we conducted rapid amplification of cDNA ends (RACE). We identified an additional genome segment (RNA 5) by RT-PCR using a genus-specific primer (PDAP213) to the conserved 3´ and 5´termini in order to amplify full-length genome segments. RNA 5 encodes a 21-kDa protein that is homologous to the silencing suppressor P8 of High Plains wheat mosaic virus. The five viral RNAs were consistently detected by RT-PCR in ringspot-diseased oaks in Germany, Sweden, and Norway. We conclude that the virus represents a new member of the genus Emaravirus affecting oaks in Germany and in Scandinavia, and we propose the name “common oak ringspot-associated emaravirus” (CORaV).

scholarly journals CoronaHiT: high-throughput sequencing of SARS-CoV-2 genomes

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Dave J. Baker ◽  
Alp Aydin ◽  
Thanh Le-Viet ◽  
Gemma L. Kay ◽  
Steven Rudder ◽  
...  
Keyword(s):  
Maximum Likelihood ◽  
Genome Sequencing ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Rapid Expansion ◽  
Library Preparation ◽  
Method Performance ◽  
Genome Coverage ◽  
Maximum Likelihood Tree ◽  
Pcr Products

AbstractWe 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.

Sign in / Sign up

Export Citation Format

Share Document