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 Small RNA-Omics for Virome Reconstruction and Antiviral Defense Characterization in Mixed Infections of Cultivated Solanum Plants

2018 ◽  
Vol 31 (7) ◽  
pp. 707-723 ◽  
Author(s):  
Silvia Turco ◽  
Victor Golyaev ◽  
Jonathan Seguin ◽  
Céline Gilli ◽  
Laurent Farinelli ◽  
...  
Keyword(s):  
Potato Virus ◽  
Potato Virus Y ◽  
De Novo ◽  
Cross Protection ◽  
Mixed Infections ◽  
Antiviral Defense ◽  
Viral Quasispecies ◽  
Genome Sequences ◽  
Depth Analysis

In plants, RNA silencing–based antiviral defense generates viral small RNAs (sRNAs) faithfully representing the viral genomes. We employed sRNA sequencing and bioinformatics (sRNA-omics) to characterize antiviral defense and to reconstruct the full genomic sequences and their variants in the evolving viral quasispecies in cultivated solanaceous plants carrying mixed infections. In naturally infected Solanum tuberosum (potato), one case study revealed a virome comprising Potato virus Y (genus Potyvirus) and Potato virus X (genus Potexvirus), which was reconstructed by de novo–assembling separate genome-size sRNA contigs. Another case study revealed a virome comprising NTN and O strains of Potato virus Y, whose sRNAs assembled in chimeric contigs, which could be disentangled on the basis of reference genome sequences. Both viromes were stable in vegetative potato progeny. In a cross-protection trial of Solanum lycopersicum (tomato), the supposedly protective mild strain CH2 of Pepino mosaic virus (genus Potexvirus) was tested for protection against strain LP of the same virus. Reciprocal mechanical inoculations eventually resulted in co-infection of all individual plants with CH2 and LP strains, reconstructed as separate sRNA contigs. LP invasions into CH2-preinfected plants and vice versa were accompanied by alterations of consensus genome sequences in viral quasispecies, indicating a potential risk of cross-protection measures. Additionally, the study also revealed, by reconstruction from sRNAs, the presence of the mechanically nontransmissible Southern tomato virus (genus Amalgavirus) in some plants. Our in-depth analysis of sRNA sizes, 5′-nucleotide frequencies and hotspot maps revealed similarities in sRNA-generating mechanisms in potato and tomato, differential silencing responses to virome components and potential for sRNA-directed cross-targeting between viral strains which could not, however, prevent the formation of stable viromes.

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.

scholarly journals Molecular Fitness Landscapes from High-Coverage Sequence Profiling

2019 ◽  
Vol 48 (1) ◽  
pp. 1-18 ◽  
Author(s):  
Celia Blanco ◽  
Evan Janzen ◽  
Abe Pressman ◽  
Ranajay Saha ◽  
Irene A. Chen
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
High Throughput Sequencing ◽  
Fitness Landscape ◽  
Complete Sequence ◽  
Fitness Landscapes ◽  
Future Research ◽  
High Coverage

The function of fitness (or molecular activity) in the space of all possible sequences is known as the fitness landscape. Evolution is a random walk on the fitness landscape, with a bias toward climbing hills. Mapping the topography of real fitness landscapes is fundamental to understanding evolution, but previous efforts were hampered by the difficulty of obtaining large, quantitative data sets. The accessibility of high-throughput sequencing (HTS) has transformed this study, enabling large-scale enumeration of fitness for many mutants and even complete sequence spaces in some cases. We review the progress of high-throughput studies in mapping molecular fitness landscapes, both in vitro and in vivo, as well as opportunities for future research. Such studies are rapidly growing in number. HTS is expected to have a profound effect on the understanding of real molecular fitness landscapes.

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.

scholarly journals Evaluation of direct metagenomics and target enriched approaches for high-throughput sequencing of field rabies viruses

2019 ◽  
Vol 63 (4) ◽  
pp. 471-479
Author(s):  
Anna Orłowska ◽  
Ewelina Iwan ◽  
Marcin Smreczak ◽  
Jerzy Rola
Keyword(s):  
High Throughput ◽  
Deep Sequencing ◽  
High Throughput Sequencing ◽  
De Novo ◽  
High Reliability ◽  
Rna Extraction ◽  
Genetic Material ◽  
Target Enrichment ◽  
Red Foxes ◽  
Field Samples

AbstractIntroductionHigh-throughput sequencing (HTS) identifies random viral fragments in environmental samples metagenomically. High reliability gains it broad application in virus evolution, host-virus interaction, and pathogenicity studies. Deep sequencing of field samples with content of host genetic material and bacteria often produces insufficient data for metagenomics and must be preceded by target enrichment. The main goal of the study was the evaluation of HTS for complete genome sequencing of field-case rabies viruses (RABVs).Material and MethodsThe material was 23 RABVs isolated mainly from red foxes and one European bat lyssavirus-1 isolate propagated in neuroblastoma cells. Three methods of RNA isolation were tested for the direct metagenomics and RABV-enriched approaches. Deep sequencing was performed with a MiSeq sequencer (Illumina) and reagent v3 kit. Bioinformatics data were evaluated by Kraken and Centrifuge software and de novo assembly was done with metaSPAdes.ResultsTesting RNA extraction procedures revealed the deep sequencing scope superiority of the combined TRIzol/column method. This HTS methodology made it possible to obtain complete genomes of all the RABV isolates collected in the field. Significantly greater rates of RABV genome coverages (over 5,900) were obtained with RABV enrichment. Direct metagenomic studies sequenced the full length of 6 out of 16 RABV isolates with a medium coverage between 1 and 71.ConclusionDirect metagenomics gives the most realistic illustration of the field sample microbiome, but with low coverage. For deep characterisation of viruses, e.g. for spatial and temporal phylogeography during outbreaks, target enrichment is recommended as it covers sequences much more completely.

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