scholarly journals High-Throughput Sequencing Reveals Bell Pepper Endornavirus Infection in Pepper (Capsicum annum) in Slovakia and Enables Its Further Molecular Characterization

Plants ◽  
2019 ◽  
Vol 9 (1) ◽  
pp. 41 ◽  
Author(s):  
Jana Tomašechová ◽  
Richard Hančinský ◽  
Lukáš Predajňa ◽  
Ján Kraic ◽  
Daniel Mihálik ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
De Novo ◽  
Genomic Sequence ◽  
Illumina Miseq ◽  
Bell Pepper ◽  
Watermelon Mosaic Virus ◽  
Pepper Plant ◽  
Close Phylogenetic Relationship

Ribosomal RNA-depleted total RNAs from a sweet pepper plant (Capsicum annuum, labelled as N65) grown in western Slovakia and showing severe virus-like symptoms (chlorosis, mottling and deformation of leaf lamina) were subjected to high-throughput sequencing (HTS) on an Illumina MiSeq platform. The de novo assembly of ca. 5.5 million reads, followed by mapping to the reference sequences, revealed the coinfection of pepper by several viruses; i.e., cucumber mosaic virus (CMV), watermelon mosaic virus (WMV), pepper cryptic virus 2 (PCV2) and bell pepper endornavirus (BPEV). A complete polyprotein-coding genomic sequence (14.6 kb) of BPEV isolate N65 was determined. A comparison of BPEV-N65 sequences with BPEV genomes available in GenBank showed 86.1% to 98.6% identity at the nucleotide level. The close phylogenetic relationship with isolates from India and China resulted in their distinct grouping compared to the other BPEV isolates. Further analysis has revealed the presence of BPEV in sweet or chili peppers obtained from various sources and locations in Slovakia (plants grown in gardens, greenhouse or retail shop). Additionally, the partial sequencing of two genomic portions from 15 BPEV isolates revealed that the Slovak isolates segregated into two molecular clusters, indicating a genetically distinct population (mean inter-group nucleotide divergence reaching 12.7% and 14.5%, respectively, based on the genomic region targeted). Due to the mix infections of BPEV-positive peppers by potato virus Y (PVY) and/or CMV, the potential role of individual viruses in the observed symptomatology could not be determined. This is the first evidence and characterization of BPEV from the central European region.

scholarly journals Molecular Characterization of Potato Virus Y (PVY) Using High-Throughput Sequencing: Constraints on Full Genome Reconstructions Imposed by Mixed Infection Involving Recombinant PVY Strains

Plants ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 753
Author(s):  
Miroslav Glasa ◽  
Richard Hančinský ◽  
Katarína Šoltys ◽  
Lukáš Predajňa ◽  
Jana Tomašechová ◽  
...  
Keyword(s):  
High Throughput ◽  
Potato Virus ◽  
Mixed Infection ◽  
High Throughput Sequencing ◽  
Potato Virus Y ◽  
De Novo ◽  
Sweet Pepper ◽  
Complete Sequence ◽  
Genome Region ◽  
Mapping Parameters

In recent years, high throughput sequencing (HTS) has brought new possibilities to the study of the diversity and complexity of plant viromes. Mixed infection of a single plant with several viruses is frequently observed in such studies. We analyzed the virome of 10 tomato and sweet pepper samples from Slovakia, all showing the presence of potato virus Y (PVY) infection. Most datasets allow the determination of the nearly complete sequence of a single-variant PVY genome, belonging to one of the PVY recombinant strains (N-Wi, NTNa, or NTNb). However, in three to-mato samples (T1, T40, and T62) the presence of N-type and O-type sequences spanning the same genome region was documented, indicative of mixed infections involving different PVY strains variants, hampering the automated assembly of PVY genomes present in the sample. The N- and O-type in silico data were further confirmed by specific RT-PCR assays targeting UTR-P1 and NIa genomic parts. Although full genomes could not be de novo assembled directly in this situation, their deep coverage by relatively long paired reads allowed their manual re-assembly using very stringent mapping parameters. These results highlight the complexity of PVY infection of some host plants and the challenges that can be met when trying to precisely identify the PVY isolates involved in mixed infection.

scholarly journals Zucchini yellow mosaic virus Genomic Sequences from Papua New Guinea: Lack of Genetic Connectivity with Northern Australian or East Timorese Genomes, and New Recombination Findings

Plant Disease ◽  
2019 ◽  
Vol 103 (6) ◽  
pp. 1326-1336 ◽  
Author(s):  
Solomon Maina ◽  
Martin J. Barbetti ◽  
Owain R. Edwards ◽  
David Minemba ◽  
Michael W. Areke ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Papua New Guinea ◽  
High Throughput Sequencing ◽  
Genomic Sequence ◽  
Zucchini Yellow Mosaic Virus ◽  
New Guinea ◽  
Yellow Mosaic Virus ◽  
Genetic Connectivity ◽  
Genomic Sequences ◽  
Nucleotide Identity

Zucchini yellow mosaic virus (ZYMV) isolates were obtained in Papua New Guinea (PNG) from cucumber (Cucumis sativus) or pumpkin (Cucurbita spp.) plants showing mosaic symptoms growing at Kongop in the Mount Hagen District, Western Highlands Province, or Zage in the Goroka District, Eastern Highlands Province. The samples were blotted onto FTA cards, which were sent to Australia, where they were subjected to high-throughput sequencing. When the coding regions of the nine new ZYMV genomic sequences found were compared with those of 64 other ZYMV sequences from elsewhere, they grouped together, forming new minor phylogroup VII within ZYMV’s major phylogroup A. Genetic connectivity was lacking between ZYMV genomic sequences from PNG and its neighboring countries, Australia and East Timor; the closest match between a PNG and any other genomic sequence was a 92.8% nucleotide identity with a sequence in major phylogroup A’s minor phylogroup VI from Japan. When the RDP5.2 recombination analysis program was used to compare 66 ZYMV sequences, evidence was obtained of 30 firm recombination events involving 41 sequences, and all isolates from PNG were recombinants. There were 21 sequences without recombination events in major phylogroup A, whereas there were only 4 such sequences within major phylogroup B. ZYMV’s P1, Cl, N1a-Pro, P3, CP, and NIb regions contained the highest evidence of recombination breakpoints. Following removal of recombinant sequences, seven minor phylogroups were absent (I, III, IV, V, VI, VII, and VIII), leaving only minor phylogroups II and IX. By contrast, when a phylogenetic tree was constructed using recombinant sequences with their recombinationally derived tracts removed before analysis, five previous minor phylogroups remained unchanged within major phylogroup A (II, III, IV, V, and VII) while four formed two new merged phylogroups (I/VI and VIII/IX). Absence of genetic connectivity between PNG, Australian, and East Timorese ZYMV sequences, and the 92.8% nucleotide identity between a PNG sequence and the closest sequence from elsewhere, suggest that a single introduction may have occurred followed by subsequent evolution to adapt to the PNG environment. The need for enhanced biosecurity measures to protect against potentially damaging virus movements crossing the seas separating neighboring countries in this region of the world is discussed.

scholarly journals Millet Could Be both a Weed and Serve as a Virus Reservoir in Crop Fields

Plants ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 954
Author(s):  
György Pasztor ◽  
Zsuzsanna Galbacs N. ◽  
Tamas Kossuth ◽  
Emese Demian ◽  
Erzsebet Nadasy ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Seed Dormancy ◽  
High Throughput ◽  
Small Rna ◽  
Diagnostic Method ◽  
High Throughput Sequencing ◽  
Infection Risk ◽  
Viral Diseases ◽  
Crop Fields ◽  
Special Control

Millet is a dangerous weed in crop fields. A lack of seed dormancy helps it to spread easily and be present in maize, wheat, and other crop fields. Our previous report revealed the possibility that millet can also play a role as a virus reservoir. In that study, we focused on visual symptoms and detected the presence of several viruses in millet using serological methods, which can only detect the presence of the investigated pathogen. In this current work, we used small RNA high-throughput sequencing as an unbiased virus diagnostic method to uncover presenting viruses in randomly sampled millet grown as a volunteer weed in two maize fields, showing stunting, chlorosis, and striped leaves. Our results confirmed the widespread presence of wheat streak mosaic virus at both locations. Moreover, barley yellow striate mosaic virus and barley virus G, neither of which had been previously described in Hungary, were also identified. As these viruses can cause severe diseases in wheat and other cereals, their presence in a weed implies a potential infection risk. Our study indicates that the presence of millet in fields requires special control to prevent the emergence of new viral diseases in crop fields.

Analysis of the effects of nanosilver on bacterial community in the intestinal fluid of silkworms using high-throughput sequencing

2019 ◽  
Vol 110 (3) ◽  
pp. 309-320
Author(s):  
Chen Lin ◽  
Zhou Wei ◽  
Zhou Yi ◽  
Tan Tingting ◽  
Du Huamao ◽  
...  
Keyword(s):  
Bacterial Community ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Intestinal Bacteria ◽  
Illumina Miseq ◽  
The Body ◽  
Dependent Manner ◽  
Negative Effects ◽  
Silkworm Larvae ◽  
Intestinal Bacterial Community

AbstractNanosilver is an environment-friendly, harmless alternative of traditional disinfectants which can be potentially applied in the sericulture industry. However, the effects of nanosilver on the intestinal bacterial community of the silkworms (Bombyx mori L.) are unclear. In this study, Illumina MiSeq high-throughput sequencing technology was used to assess the intestinal bacterial community in both male and female silkworms while treated with different concentrations of nanosilver. We found that nanosilver significantly influenced the composition of silkworm intestinal bacterial community on the different taxonomic levels. Most conspicuously, the abundance of Firmicutes was increased by the treatment of 20 mg L−1 nanosilver but decreased by that of 100 mg L−1 nanosilver at the phylum level. The same trend was observed in Bacilli at the class level and in Enterococcus at the genus level. In some extreme cases, application of nanosilver eliminated the bacterium, e.g., Brevibacillus, but increased the population of several other bacteria in the host intestine, such as Blautia, Terrisporobacter, Faecalibacterium, and some bacteria could only be found in nanosilver treatment groups, e.g., Dialister. In addition, although nanosilver generally showed negative effects on the cocooning rate in a dose-dependent manner, we found that 20 mg L−1 nanosilver treatment significantly increased the body weight of silkworms and did not show negative effects on the survival rate. These results indicated that the intestinal bacteria community of silkworm larvae was significantly changed after nanosilver treatment which might consequently influence host growth and development.

scholarly journals DNA metabarcoding of insects and allies: an evaluation of primers and pipelines

2015 ◽  
Vol 105 (6) ◽  
pp. 717-727 ◽  
Author(s):  
G.-J. Brandon-Mong ◽  
H.-M. Gan ◽  
K.-W. Sing ◽  
P.-S. Lee ◽  
P.-E. Lim ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Illumina Miseq ◽  
Ease Of Use ◽  
Malaise Trap ◽  
Detection Rates ◽  
Operational Taxonomic Units ◽  
Arthropod Species ◽  
Primer Sets ◽  
Arthropod Biodiversity

AbstractMetabarcoding, the coupling of DNA-based species identification and high-throughput sequencing, offers enormous promise for arthropod biodiversity studies but factors such as cost, speed and ease-of-use of bioinformatic pipelines, crucial for making the leapt from demonstration studies to a real-world application, have not yet been adequately addressed. Here, four published and one newly designed primer sets were tested across a diverse set of 80 arthropod species, representing 11 orders, to establish optimal protocols for Illumina-based metabarcoding of tropical Malaise trap samples. Two primer sets which showed the highest amplification success with individual specimen polymerase chain reaction (PCR, 98%) were used for bulk PCR and Illumina MiSeq sequencing. The sequencing outputs were subjected to both manual and simple metagenomics quality control and filtering pipelines. We obtained acceptable detection rates after bulk PCR and high-throughput sequencing (80–90% of input species) but analyses were complicated by putative heteroplasmic sequences and contamination. The manual pipeline produced similar or better outputs to the simple metagenomics pipeline (1.4 compared with 0.5 expected:unexpected Operational Taxonomic Units). Our study suggests that metabarcoding is slowly becoming as cheap, fast and easy as conventional DNA barcoding, and that Malaise trap metabarcoding may soon fulfill its potential, providing a thermometer for biodiversity.

Minirmd: accurate and fast duplicate removal tool for short reads via multiple minimizers

2020 ◽  
Author(s):  
Yuansheng Liu ◽  
Xiaocai Zhang ◽  
Quan Zou ◽  
Xiangxiang Zeng
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
De Novo ◽  
Supplementary Information ◽  
Supplementary Data ◽  
Complementary Strand ◽  
Short Reads ◽  
Sequencing Technologies ◽  
Computational Resources

Abstract Summary Removing duplicate and near-duplicate reads, generated by high-throughput sequencing technologies, is able to reduce computational resources in downstream applications. Here we develop minirmd, a de novo tool to remove duplicate reads via multiple rounds of clustering using different length of minimizer. Experiments demonstrate that minirmd removes more near-duplicate reads than existing clustering approaches and is faster than existing multi-core tools. To the best of our knowledge, minirmd is the first tool to remove near-duplicates on reverse-complementary strand. Availability and implementation https://github.com/yuansliu/minirmd. Supplementary information Supplementary data are available at Bioinformatics online.

The Novel FLT3-N676K Mutant Induces Acute Leukemia Independently of the Inv(16) Chimeric Gene CBFB-MYH11

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1383-1383
Author(s):  
Kezhi Huang ◽  
Min Yang ◽  
Zengkai Pan ◽  
Florian H. Heidel ◽  
Michaela Scherr ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
De Novo ◽  
Chimeric Gene ◽  
Leukemic Cells ◽  
Single Amino Acid ◽  
Hematopoietic Stem ◽  
Transforming Activity

Abstract Using high-throughput sequencing, an increased number of gene mutations has been identified in cancer. Among the up to hundreds of acquired mutations in cancer clones, only a few cooperating mutations are believed to be needed for initiation of the malignant disease. Recently, we reported a single amino acid substitution at position 676 (N676K) within the FLT3 kinase domain as the sole cause of resistance to PKC412 in one patient with FLT3-ITD associated acute myeloid leukemia (AML). The FLT3-N676K mutation was more recently identified independently in up to 6% of de novo AML patients with inv(16) by other groups. As FLT3-TKD mutations are strongly associated with inv(16) in AML and particularly FLT3-N676K was found almost exclusively in AML patients with inv(16), this prompted us to investigate the transforming activity of FLT3-N676K and to test whether FLT3-N676K would cooperate with inv(16) to promote AML. First, we analyzed in vivo leukemogenesis mediated by FLT3-N676K. Retroviral expression of FLT3-N676K in myeloid 32D cells induced AML in syngeneic C3H/HeJ mice (n=11/13, latency ~8 weeks), with a transforming activity similar to FLT3-ITD (n=8/8), FLT3-TKD D835Y (n=8/9), and FLT3-ITD-N676K (n=9/9) mutations. Three out of 14 C57BL/6J mice transplanted with FLT3-N676K-transduced primary lineage negative (Lin-) bone marrow cells died of acute leukemia (latency of 68, 77, and 273 days), while none of 16 animals in the control groups including FLT3-ITD and CBFß-SMMHC developed any hematological malignancy. Secondly, co-expression of FLT3-N676K and CBFß-SMMHC did not promote acute leukemia in 3 independent experiments using C3H/HeJ and C57BL/6J mice (n=16). So far only 1 out of 11 C57BL/6J mice co-expressing FLT3-N676K and CBFß-SMMHC developed acute leukemia (AML with latency of 166 days). In comparison with FLT3-ITD, FLT3-N676K tended to result in stronger phosphorylation of FLT3, MAPK and AKT, and diseased animals carrying FLT3-N676K demonstrated much lower frequency of leukemic stem cells in the majority of analyzed cases. Importantly, leukemic cells co-expressing FLT3-N676K and CBFß-SMMHC were still highly sensitive to the FLT3 inhibitor AC220. Taken together, we show that FLT3-N676K mutant is potent to transform murine hematopoietic stem/progenitor cells in vivo independently of the inv(16) chimeric gene CBFB-MYH11. This is the first report of acute leukemia induced by an activating FLT3 mutation in C57BL/6J mice. Moreover, our data suggest that targeting FLT3-N676K mutation may be an attractive treatment option for FLT3-N676K-positive patients without concurrent ITD. Our data emphasize more careful analysis of the cooperating network of mutations identified in AML by high-throughput sequencing. This work was supported by DJCLS (grant: 13/22) and the Deutsche Forschungsgemeinschaft (grant: Li 1608/2-1). KH and ZP were supported by the China Scholarship Council (2011638024 and 201406100008). Disclosures Heidel: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals Direct Metatranscriptomic Survey of the Sunflower Microbiome and Virome

Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1867
Author(s):  
Ziyi Wang ◽  
Achal Neupane ◽  
Jiuhuan Feng ◽  
Connor Pedersen ◽  
Shin-Yi Lee Marzano
Keyword(s):  
South Dakota ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Genetic Resource ◽  
De Novo ◽  
Fungal Infections ◽  
Yellow Mosaic Virus ◽  
Protein Database ◽  
Dna Virus ◽  
Field Production

Sunflowers (Helianthus annuus L.) are susceptible to multiple diseases in field production. In this study, we collected diseased sunflower leaves in fields located in South Dakota, USA, for virome investigation. The leaves showed visible symptoms on the foliage, indicating phomopsis and rust infections. To identify the viruses potentially associated with the disease diagnosed, symptomatic leaves were obtained from diseased plants. Total RNA was extracted corresponding to each disease diagnosed to generate libraries for paired-end high throughput sequencing. Short sequencing reads were assembled de novo and the contigs with similarities to viruses were identified by aligning against a custom protein database. We report the discovery of two novel mitoviruses, four novel partitiviruses, one novel victorivirus, and nine novel totiviruses based on similarities to RNA-dependent RNA polymerases and capsid proteins. Contigs similar to bean yellow mosaic virus and Sclerotinia sclerotiorum hypovirulence-associated DNA virus were also detected. To the best of our knowledge, this is the first report of direct metatranscriptomics discovery of viruses associated with fungal infections of sunflowers bypassing culturing. These newly discovered viruses represent a natural genetic resource from which we can further develop potential biopesticide to control sunflower diseases.

Sign in / Sign up

Export Citation Format

Share Document