scholarly journals Reconstruction and Characterization of Full-Length Begomovirus and Alphasatellite Genomes Infecting Pepper through Metagenomics

Viruses ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 202 ◽  
Author(s):  
Verónica A. Bornancini ◽  
José M. Irazoqui ◽  
Ceferino R. Flores ◽  
Carlos G. Vaghi Medina ◽  
Ariel F. Amadio ◽  
...  
Keyword(s):  
Rolling Circle Amplification ◽  
Full Length ◽  
Yellow Spot ◽  
Streak Virus ◽  
Rolling Circle ◽  
Viral Particles ◽  
Bipartite Genome ◽  
First Time ◽  
Leaf Virus

In northwestern Argentina (NWA), pepper crops are threatened by the emergence of begomoviruses due to the spread of its vector, Bemisia tabaci (Gennadius). The genus Begomovirus includes pathogens that can have a monopartite or bipartite genome and are occasionally associated with sub-viral particles called satellites. This study characterized the diversity of begomovirus and alphasatellite species infecting pepper in NWA using a metagenomic approach. Using RCA-NGS (rolling circle amplification-next generation sequencing), 19 full-length begomovirus genomes (DNA-A and DNA-B) and one alphasatellite were assembled. This ecogenomic approach revealed six begomoviruses in single infections: soybean blistering mosaic virus (SbBMV), tomato yellow spot virus (ToYSV), tomato yellow vein streak virus (ToYVSV), tomato dwarf leaf virus (ToDfLV), sida golden mosaic Brazil virus (SiGMBRV), and a new proposed species, named pepper blistering leaf virus (PepBLV). SbBMV was the most frequently detected species, followed by ToYSV. Moreover, a new alphasatellite associated with ToYSV, named tomato yellow spot alphasatellite 2 (ToYSA-2), was reported for the first time in Argentina. For the Americas, this was the first report of an alphasatellite found in a crop (pepper) and in a weed (Leonurus japonicus). We also detected intra-species and inter-species recombination.

scholarly journals Development of a Novel Rolling-Circle Amplification Technique to Detect Banana streak virus that also Discriminates Between Integrated and Episomal Virus Sequences

Plant Disease ◽  
2011 ◽  
Vol 95 (1) ◽  
pp. 57-62 ◽  
Author(s):  
A. P. James ◽  
R. J. Geijskes ◽  
J. L. Dale ◽  
R. M. Harding
Keyword(s):  
Rolling Circle Amplification ◽  
Cauliflower Mosaic Virus ◽  
Diagnostic Methods ◽  
Streak Virus ◽  
Banana Streak Virus ◽  
Rolling Circle ◽  
Sugarcane Bacilliform Virus ◽  
Amplification Technique ◽  
Banana Streak Ol Virus

Banana plants are hosts to a large number of Banana streak virus (BSV) species. However, diagnostic methods for BSV are inadequate because of the considerable genetic and serological diversity among BSV isolates and the presence of integrated BSV sequences in some banana cultivars which leads to false positives. In this study, a sequence-nonspecific, rolling-circle amplification (RCA) technique was developed and shown to overcome these limitations for the detection and subsequent characterization of BSV isolates infecting banana. This technique was shown to discriminate between integrated and episomal BSV DNA, specifically detecting the latter in several banana cultivars known to contain episomal or integrated sequences of Banana streak Mysore virus (BSMyV), Banana streak OL virus (BSOLV), and Banana streak GF virus (BSGFV). Using RCA, the presence of BSMyV and BSOLV was confirmed in Australia, while BSOLV, BSGFV, Banana streak Uganda I virus (BSUgIV), Banana streak Uganda L virus (BSUgLV), and Banana streak Uganda M virus (BSUgMV) were detected in Uganda. This is the first confirmed report of episomally-derived BSUglV, BSUgLV, and BSUgMV in Uganda. As well as its ability to detect BSV, RCA was shown to detect two other pararetroviruses, Sugarcane bacilliform virus in sugarcane and Cauliflower mosaic virus in turnip.

scholarly journals Characterization of Two Novel Polyomaviruses of Birds by Using Multiply Primed Rolling-Circle Amplification of Their Genomes

2006 ◽  
Vol 80 (7) ◽  
pp. 3523-3531 ◽  
Author(s):  
Reimar Johne ◽  
Walter Wittig ◽  
Daniel Fernández-de-Luco ◽  
Ursula Höfle ◽  
Hermann Müller
Keyword(s):  
Rolling Circle Amplification ◽  
Large Tumor ◽  
Rolling Circle ◽  
Reading Frame ◽  
Field Samples ◽  
Close Relationship ◽  
Avian Polyomavirus ◽  
Avian Group ◽  
Pyrrhula Pyrrhula

ABSTRACT Polyomaviruses are small nonenveloped particles with a circular double-stranded genome, approximately 5 kbp in size. The mammalian polyomaviruses mainly cause persistent subclinical infections in their natural nonimmunocompromised hosts. In contrast, the polyomaviruses of birds—avian polyomavirus (APV) and goose hemorrhagic polyomavirus (GHPV)—are the primary agents of acute and chronic disease with high mortality rates in young birds. Screening of field samples of diseased birds by consensus PCR revealed the presence of two novel polyomaviruses in the liver of an Eurasian bullfinch (Pyrrhula pyrrhula griseiventris) and in the spleen of a Eurasian jackdaw (Corvus monedula), tentatively designated as finch polyomavirus (FPyV) and crow polyomavirus (CPyV), respectively. The genomes of the viruses were amplified by using multiply primed rolling-circle amplification and cloned. Analysis of the FPyV and CPyV genome sequences revealed a close relationship to APV and GHPV, indicating the existence of a distinct avian group among the polyomaviruses. The main characteristics of this group are (i) involvement in fatal disease, (ii) the existence of an additional open reading frame in the 5′ region of the late mRNAs, and (iii) a different manner of DNA binding of the large tumor antigen compared to that of the mammalian polyomaviruses.

Rolling circle amplification and nanopore-based deep sequencing of full-length HBV genomes

2018 ◽  
Vol 68 ◽  
pp. S762 ◽  
Author(s):  
A. Mcnaughton ◽  
D. Bonsall ◽  
M.D. Cesare ◽  
A. Brown ◽  
D. Parkes ◽  
...  
Keyword(s):  
Deep Sequencing ◽  
Rolling Circle Amplification ◽  
Full Length ◽  
Rolling Circle

scholarly journals Circular genomes related to anelloviruses identified in human and animal samples by using a combined rolling-circle amplification/sequence-independent single primer amplification approach

2007 ◽  
Vol 88 (10) ◽  
pp. 2696-2701 ◽  
Author(s):  
Philippe Biagini ◽  
Rathviro Uch ◽  
Mourad Belhouchet ◽  
Houssam Attoui ◽  
Jean-François Cantaloube ◽  
...  
Keyword(s):  
Human Plasma ◽  
Genetic Divergence ◽  
Rolling Circle Amplification ◽  
Plasma Samples ◽  
Rolling Circle ◽  
Human Plasma Samples ◽  
Single Primer ◽  
Divergent Sequences

A combined rolling-circle amplification (RCA) and sequence-independent single primer amplification (SISPA) approach was applied to four samples of human plasma and one sample of saliva from a cat. This approach permitted the characterization of nine anelloviruses. Most of them were identified as highly divergent strains that were classified into species of the genus Anellovirus. The smallest anellovirus described so far in humans was characterized (2PoSMA, 2002 nt; ‘small anellovirus’ species). Two highly divergent sequences belonging to the species Torque Teno Mini Virus (LIL-y1, 2887 nt; LIL-y2, 2871 nt), which clustered into a new phylogenetic branch, were also identified in human plasma samples. Finally, two genomes that are separated by a genetic divergence of 46 % were characterized in the cat's saliva, one of these creating a distinct phylogenetic branch (PRA1, 2019 nt). These results highlight the potential of RCA–SISPA for detecting circular (or circularized) genomes.

scholarly journals High-throughput, cost-effective verification of structural DNA assembly

2013 ◽  
Vol 42 (4) ◽  
pp. e22-e22 ◽  
Author(s):  
Yandi Dharmadi ◽  
Kedar Patel ◽  
Elaine Shapland ◽  
Daniel Hollis ◽  
Todd Slaby ◽  
...  
Keyword(s):  
High Throughput ◽  
Low Cost ◽  
Rolling Circle Amplification ◽  
Cost Effective ◽  
Building Blocks ◽  
Pathway Engineering ◽  
Dna Assembly ◽  
Rolling Circle ◽  
Enzyme Screening ◽  
First Time

Abstract DNA ‘assembly’ from ‘building blocks’ remains a cornerstone in synthetic biology, whether it be for gene synthesis (∼1 kb), pathway engineering (∼10 kb) or synthetic genomes (>100 kb). Despite numerous advances in the techniques used for DNA assembly, verification of the assembly is still a necessity, which becomes cost-prohibitive and a logistical challenge with increasing scale. Here we describe for the first time a comprehensive, high-throughput solution for structural DNA assembly verification by restriction digest using exhaustive in silico enzyme screening, rolling circle amplification of plasmid DNA, capillary electrophoresis and automated digest pattern recognition. This low-cost and robust methodology has been successfully used to screen over 31 000 clones of DNA constructs at <$1 per sample.

scholarly journals Comprehensive transcriptome characterization of Grus japonensis using PacBio SMRT and Illumina sequencing

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Wentao Ye ◽  
Wei Xu ◽  
Nan Xu ◽  
Rong Chen ◽  
Changhu Lu ◽  
...  
Keyword(s):  
Transcriptional Regulatory Network ◽  
Full Length ◽  
Sequencing Data ◽  
Consensus Sequences ◽  
Grus Japonensis ◽  
Transcriptional Regulatory ◽  
Illumina Data ◽  
Transmission Pathways ◽  
First Time

AbstractThe red-crowned crane (Grus japonensis) is an endangered species distributed across southeast Russia, northeast China, Korea, and Japan. Here, we sequenced for the first time the full-length unreferenced transcriptome of red-crowned crane mixed samples using a PacBio Sequel platform. A total of 359,136 circular consensus sequences (CCS) were obtained via clustering to remove redundancy. A total of 303,544 full-length non-chimeric sequences were identified by judging whether CCS contained 5′ and 3′ adapters, and the poly(A) tail. Eight samples were sequenced using Illumina, and PacBio sequencing data were corrected according to the collected Illumina data to obtain more accurate full-length transcripts. A total of 4,100 long non-coding RNAs, 13,115 simple sequences repeat loci and 29 transcription factor families were identified. The expression of lncRNAs and TFs in pancreas was lowest comparing with other tissues. Many enriched immune-related transmission pathways (MHC and IL receptors) were identified in the spleen. This study will contribute to a better understanding of the gene structure and post-transcriptional regulatory network, and provide references for future studies on red-crowned cranes.

scholarly journals First Detection and Molecular Characterization of Apple Stem Grooving Virus, Apple Chlorotic Leaf Spot Virus, and Apple Hammerhead Viroid in Loquat in Spain

Plants ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 2293
Author(s):  
Celia Canales ◽  
Félix Morán ◽  
Antonio Olmos ◽  
Ana Belén Ruiz-García
Keyword(s):  
Leaf Spot ◽  
High Throughput Sequencing ◽  
Eriobotrya Japonica ◽  
Spot Virus ◽  
Apple Stem Grooving Virus ◽  
Apple Stem ◽  
Chlorotic Leaf ◽  
First Time ◽  
Leaf Virus

Loquat (Eriobotrya japonica) is an important crop in Spain. To date, only one viral species, apple stem pitting virus (ASPV), has been detected in Spanish loquat orchards. In this study, the presence of additional viruses infecting this crop in Spain was investigated. RT-PCR and high-throughput sequencing (HTS) of symptomatic loquat plants led to first-time detection and characterization of apple stem grooving virus (ASGV), also known as citrus tatter leaf virus (CTLV), and apple chlorotic leaf spot virus (ACLSV) from Spain with description of nearly complete genomic sequences. The frequency of ACLSV infection was the highest, with over 30% of the samples testing positive and were also detected as coinfections with ASGV and ASPV, although most of the samples infected were symptomless. Studies on all the full-length sequences available in the databases were performed in order to establish the phylogenetic relationships of the Spanish isolates of these two viral species. Moreover, apple hammerhead viroid (AHVd) was also detected to infect loquat, the first host different from apple reported for this viroid to date.

scholarly journals Rolling Circle cDNA Synthesis Uncovers Circular RNA Splice Variants

2019 ◽  
Vol 20 (16) ◽  
pp. 3988 ◽  
Author(s):  
Aniruddha Das ◽  
Pranita K. Rout ◽  
Myriam Gorospe ◽  
Amaresh C. Panda
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Splice Variants ◽  
Rolling Circle Amplification ◽  
Rnase H ◽  
Full Length ◽  
Accurate Identification ◽  
Junction Sequence ◽  
Rolling Circle ◽  
Forward Primer

High-throughput RNA sequencing and novel bioinformatic pipelines have identified thousands of circular (circ)RNAs containing backsplice junction sequences. However, circRNAs generated from multiple exons may contain different combinations of exons and/or introns arising from alternative splicing, while the backsplice junction sequence is the same. To be able to identify circRNA splice variants, we developed a method termed circRNA-Rolling Circle Amplification (circRNA-RCA). This method detects full-length circRNA sequences by performing reverse transcription (RT) in the absence of RNase H activity, followed by polymerase chain reaction (PCR) amplification of full-length circRNAs using a forward primer spanning the backsplice junction sequence and a reverse primer exactly upstream of the forward primer. By sequencing the PCR products, circRNA splice variants bearing the same backsplice junctions, which were otherwise only predicted computationally, could be experimentally validated. The splice variants were further predicted to associate with different subsets of target RNA-binding proteins and microRNAs, supporting the notion that different circRNA splice variants can have different biological impacts. In sum, the circRNA-RCA method allows the accurate identification of full-length circRNA sequences, offering unique insight into their individual function.

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