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Journal articles on the topic 'Ribo-depletion'
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Alkan, Ferhat, Joana Silva, Eric Pintó Barberà, and William J. Faller. "Ribo-ODDR: oligo design pipeline for experiment-specific rRNA depletion in Ribo-seq." Bioinformatics 37, no. 17 (2021): 2659–67. http://dx.doi.org/10.1093/bioinformatics/btab171.
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Abstract Motivation Ribosome Profiling (Ribo-seq) has revolutionized the study of RNA translation by providing information on ribosome positions across all translated RNAs with nucleotide-resolution. Yet several technical limitations restrict the sequencing depth of such experiments, the most common of which is the overabundance of rRNA fragments. Various strategies can be employed to tackle this issue, including the use of commercial rRNA depletion kits. However, as they are designed for more standardized RNAseq experiments, they may perform suboptimally in Ribo-seq. In order to overcome this, it is possible to use custom biotinylated oligos complementary to the most abundant rRNA fragments, however currently no computational framework exists to aid the design of optimal oligos. Results Here, we first show that a major confounding issue is that the rRNA fragments generated via Ribo-seq vary significantly with differing experimental conditions, suggesting that a ‘one-size-fits-all’ approach may be inefficient. Therefore we developed Ribo-ODDR, an oligo design pipeline integrated with a user-friendly interface that assists in oligo selection for efficient experiment-specific rRNA depletion. Ribo-ODDR uses preliminary data to identify the most abundant rRNA fragments, and calculates the rRNA depletion efficiency of potential oligos. We experimentally show that Ribo-ODDR designed oligos outperform commercially available kits and lead to a significant increase in rRNA depletion in Ribo-seq. Availability and implementation Ribo-ODDR is freely accessible at https://github.com/fallerlab/Ribo-ODDR. Supplementary information Supplementary data are available at Bioinformatics online.
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Guo, Yan, Jie Wu, Shilin Zhao, et al. "RNA Sequencing of Formalin-Fixed, Paraffin-Embedded Specimens for Gene Expression Quantification and Data Mining." International Journal of Genomics 2016 (2016): 1–10. http://dx.doi.org/10.1155/2016/9837310.
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Background. Proper rRNA depletion is crucial for the successful utilization of FFPE specimens when studying gene expression. We performed a study to evaluate two major rRNA depletion methods: Ribo-Zero and RNase H. RNAs extracted from 4 samples were treated with the two rRNA depletion methods in duplicate and sequenced (N=16). We evaluated their reducibility, ability to detect RNA, and ability to molecularly subtype these triple negative breast cancer specimens.Results. Both rRNA depletion methods produced consistent data between the technical replicates. We found that the RNase H method produced higher quality RNAseq data as compared to the Ribo-Zero method. In addition, we evaluated the RNAseq data generated from the FFPE tissue samples for noncoding RNA, including lncRNA, enhancer/super enhancer RNA, and single nucleotide variation (SNV). We found that the RNase H is more suitable for detecting high-quality, noncoding RNAs as compared to the Ribo-Zero and provided more consistent molecular subtype identification between replicates. Unfortunately, neither method produced reliable SNV data.Conclusions. In conclusion, for FFPE specimens, the RNase H rRNA depletion method performed better than the Ribo-Zero. Neither method generates data sufficient for SNV detection.
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Thompson, Mary Kay, Maria Kiourlappou, and Ilan Davis. "Ribo-Pop: simple, cost-effective, and widely applicable ribosomal RNA depletion." RNA 26, no. 11 (2020): 1731–42. http://dx.doi.org/10.1261/rna.076562.120.
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Glaub, Alina, Christopher Huptas, Klaus Neuhaus, and Zachary Ardern. "Recommendations for bacterial ribosome profiling experiments based on bioinformatic evaluation of published data." Journal of Biological Chemistry 295, no. 27 (2020): 8999–9011. http://dx.doi.org/10.1074/jbc.ra119.012161.
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Ribosome profiling (RIBO-Seq) has improved our understanding of bacterial translation, including finding many unannotated genes. However, protocols for RIBO-Seq and corresponding data analysis are not yet standardized. Here, we analyzed 48 RIBO-Seq samples from nine studies of Escherichia coli K12 grown in lysogeny broth medium and particularly focused on the size-selection step. We show that for conventional expression analysis, a size range between 22 and 30 nucleotides is sufficient to obtain protein-coding fragments, which has the advantage of removing many unwanted rRNA and tRNA reads. More specific analyses may require longer reads and a corresponding improvement in rRNA/tRNA depletion. There is no consensus about the appropriate sequencing depth for RIBO-Seq experiments in prokaryotes, and studies vary significantly in total read number. Our analysis suggests that 20 million reads that are not mapping to rRNA/tRNA are required for global detection of translated annotated genes. We also highlight the influence of drug-induced ribosome stalling, which causes bias at translation start sites. The resulting accumulation of reads at the start site may be especially useful for detecting weakly expressed genes. As different methods suit different questions, it may not be possible to produce a “one-size-fits-all” ribosome profiling data set. Therefore, experiments should be carefully designed in light of the scientific questions of interest. We propose some basic characteristics that should be reported with any new RIBO-Seq data sets. Careful attention to the factors discussed should improve prokaryotic gene detection and the comparability of ribosome profiling data sets.
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Telzrow, Calla L., Paul J. Zwack, Shannon Esher Righi, et al. "Comparative analysis of RNA enrichment methods for preparation of Cryptococcus neoformans RNA sequencing libraries." G3 Genes|Genomes|Genetics, August 26, 2021. http://dx.doi.org/10.1093/g3journal/jkab301.
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Abstract RNA sequencing (RNA-Seq) experiments focused on gene expression involve removal of ribosomal RNA (rRNA) because it is the major RNA constituent of cells. This process, called RNA enrichment, is done primarily to reduce cost: without rRNA removal, deeper sequencing must be performed to compensate for the sequencing reads wasted on rRNA. The ideal RNA enrichment method removes all rRNA without affecting other RNA in the sample. We tested the performance of three RNA enrichment methods on RNA isolated from Cryptococcus neoformans, a fungal pathogen of humans. We find that the RNase H depletion method is more efficient in depleting rRNA and more specific in recapitulating non-rRNA levels present in unenriched controls than the commonly-used Poly(A) isolation method. The RNase H depletion method is also more effective than the Ribo-Zero depletion method as measured by rRNA depletion efficiency and recapitulation of protein-coding RNA levels present in unenriched controls, while the Ribo-Zero depletion method more closely recapitulates annotated non-coding RNA (ncRNA) levels. Finally, we leverage these data to accurately map the C. neoformans mitochondrial rRNA genes, and also demonstrate that RNA-Seq data generated with the RNase H and Ribo-Zero depletion methods can be used to explore novel C. neoformans long non-coding RNA genes.
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Engelhardt, Florian, Jürgen Tomasch, and Susanne Häussler. "Organism-specific depletion of highly abundant RNA species from bacterial total RNA." Access Microbiology 2, no. 10 (2020). http://dx.doi.org/10.1099/acmi.0.000159.
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High-throughput sequencing has become a standard tool for transcriptome analysis. The depletion of overrepresented RNA species from sequencing libraries plays a key role in establishing potent and cost-efficient RNA-seq routines. Commercially available kits are known to obtain good results for the reduction of ribosomal RNA (rRNA). However, we found that the transfer-messenger RNA (tmRNA) was frequently highly abundant in rRNA-depleted samples of Pseudomonas aeruginosa , consuming up to 25 % of the obtained reads. The tmRNA fraction was particularly high in samples taken from stationary cultures. This suggests that overrepresentation of this RNA species reduces the mRNA fraction when cells are grown under challenging conditions. Here, we present an RNase-H-based depletion protocol that targets the tmRNA in addition to ribosomal RNAs. We were able to increase the mRNA fraction to 93–99% and therefore outperform not only the commercially Ribo-off kit (Vazyme) operating by the same principle but also the formerly widely used Ribo-Zero kit (Illumina). Maximizing the read share of scientifically interesting RNA species enhances the discriminatory potential of next-generation RNA-seq experiments and, therefore, can contribute to a better understanding of the transcriptomic landscape of bacterial pathogens and their used mechanisms in host infection.
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Jang, Jin Sung, Brianna Berg, Eileen Holicky, et al. "Comparative evaluation for the globin gene depletion methods for mRNA sequencing using the whole blood-derived total RNAs." BMC Genomics 21, no. 1 (2020). http://dx.doi.org/10.1186/s12864-020-07304-4.
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Abstract Background There are challenges in generating mRNA-Seq data from whole-blood derived RNA as globin gene and rRNA are frequent contaminants. Given the abundance of erythrocytes in whole blood, globin genes comprise some 80% or more of the total RNA. Therefore, depletion of globin gene RNA and rRNA are critical steps required to have adequate coverage of reads mapping to the reference transcripts and thus reduce the total cost of sequencing. In this study, we directly compared the performance of probe hybridization (GLOBINClear Kit and Globin-Zero Gold rRNA Removal Kit) and RNAse-H enzymatic depletion (NEBNext® Globin & rRNA Depletion Kit and Ribo-Zero Plus rRNA Depletion Kit) methods from 1 μg of whole blood-derived RNA on mRNA-Seq profiling. All RNA samples were treated with DNaseI for additional cleanup before the depletion step and were processed for poly-A selection for library generation. Results Probe hybridization revealed a better overall performance than the RNAse-H enzymatic depletion method, detecting a higher number of genes and transcripts without 3′ region bias. After depletion, samples treated with probe hybridization showed globin genes at 0.5% (±0.6%) of the total mapped reads; the RNAse-H enzymatic depletion had 3.2% (±3.8%). Probe hybridization showed more junction reads and transcripts compared with RNAse-H enzymatic depletion and also had a higher correlation (R > 0.9) than RNAse-H enzymatic depletion (R > 0.85). Conclusion In this study, our results showed that 1 μg of high-quality RNA from whole blood could be routinely used for transcriptional profiling analysis studies with globin gene and rRNA depletion pre-processing. We also demonstrated that the probe hybridization depletion method is better suited to mRNA sequencing analysis with minimal effect on RNA quality during depletion procedures.
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Eshraghi, Mehdi, Pabalu P. Karunadharma, Juliana Blin, et al. "Mutant Huntingtin stalls ribosomes and represses protein synthesis in a cellular model of Huntington disease." Nature Communications 12, no. 1 (2021). http://dx.doi.org/10.1038/s41467-021-21637-y.
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AbstractThe polyglutamine expansion of huntingtin (mHTT) causes Huntington disease (HD) and neurodegeneration, but the mechanisms remain unclear. Here, we found that mHtt promotes ribosome stalling and suppresses protein synthesis in mouse HD striatal neuronal cells. Depletion of mHtt enhances protein synthesis and increases the speed of ribosomal translocation, while mHtt directly inhibits protein synthesis in vitro. Fmrp, a known regulator of ribosome stalling, is upregulated in HD, but its depletion has no discernible effect on protein synthesis or ribosome stalling in HD cells. We found interactions of ribosomal proteins and translating ribosomes with mHtt. High-resolution global ribosome footprint profiling (Ribo-Seq) and mRNA-Seq indicates a widespread shift in ribosome occupancy toward the 5′ and 3′ end and unique single-codon pauses on selected mRNA targets in HD cells, compared to controls. Thus, mHtt impedes ribosomal translocation during translation elongation, a mechanistic defect that can be exploited for HD therapeutics.
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Harrington, Christina A., Suzanne S. Fei, Jessica Minnier, et al. "RNA-Seq of human whole blood: Evaluation of globin RNA depletion on Ribo-Zero library method." Scientific Reports 10, no. 1 (2020). http://dx.doi.org/10.1038/s41598-020-62801-6.
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Newton, Yulia, Andrew J. Sedgewick, Luis Cisneros, et al. "Large scale, robust, and accurate whole transcriptome profiling from clinical formalin-fixed paraffin-embedded samples." Scientific Reports 10, no. 1 (2020). http://dx.doi.org/10.1038/s41598-020-74483-1.
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Abstract Transcriptome profiling can provide information of great value in clinical decision-making, yet RNA from readily available formalin-fixed paraffin-embedded (FFPE) tissue is often too degraded for quality sequencing. To assess the clinical utility of FFPE-derived RNA, we performed ribo-deplete RNA extractions on > 3200 FFPE slide samples; 25 of these had direct FFPE vs. fresh frozen (FF) replicates, 57 were sequenced in 2 different labs, 87 underwent multiple library analyses, and 16 had direct microdissected vs. macrodissected replicates. Poly-A versus ribo-depletion RNA extraction methods were compared using transcriptomes of TCGA cohort and 3116 FFPE samples. Compared to FF, FFPE transcripts coding for nuclear/cytoplasmic proteins involved in DNA packaging, replication, and protein synthesis were detected at lower rates and zinc finger family transcripts were of poorer quality. The greatest difference in extraction methods was in histone transcripts which typically lack poly-A tails. Encouragingly, the overall sequencing success rate was 81%. Exome coverage was highly concordant in direct FFPE and FF replicates, with 98% agreement in coding exon coverage and a median correlation of whole transcriptome profiles of 0.95. We provide strong rationale for clinical use of FFPE-derived RNA based on the robustness, reproducibility, and consistency of whole transcriptome profiling.
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Culviner, Peter H., Chantal K. Guegler, and Michael T. Laub. "A Simple, Cost-Effective, and Robust Method for rRNA Depletion in RNA-Sequencing Studies." mBio 11, no. 2 (2020). http://dx.doi.org/10.1128/mbio.00010-20.
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ABSTRACT The profiling of gene expression by RNA sequencing (RNA-seq) has enabled powerful studies of global transcriptional patterns in all organisms, including bacteria. Because the vast majority of RNA in bacteria is rRNA, it is standard practice to deplete the rRNA from a total RNA sample such that the reads in an RNA-seq experiment derive predominantly from mRNA. One of the most commonly used commercial kits for rRNA depletion, the Ribo-Zero kit from Illumina, was recently discontinued abruptly and for an extended period of time. Here, we report the development of a simple, cost-effective, and robust method for depleting rRNA that can be easily implemented by any lab or facility. We first developed an algorithm for designing biotinylated oligonucleotides that will hybridize tightly and specifically to the 23S, 16S, and 5S rRNAs from any species of interest. Precipitation of these oligonucleotides bound to rRNA by magnetic streptavidin-coated beads then depletes rRNA from a complex, total RNA sample such that ∼75 to 80% of reads in a typical RNA-seq experiment derive from mRNA. Importantly, we demonstrate a high correlation of RNA abundance or fold change measurements in RNA-seq experiments between our method and the Ribo-Zero kit. Complete details on the methodology are provided, including open-source software for designing oligonucleotides optimized for any bacterial species or community of interest. IMPORTANCE The ability to examine global patterns of gene expression in microbes through RNA sequencing has fundamentally transformed microbiology. However, RNA-seq depends critically on the removal of rRNA from total RNA samples. Otherwise, rRNA would comprise upward of 90% of the reads in a typical RNA-seq experiment, limiting the reads coming from mRNA or requiring high total read depth. A commonly used kit for rRNA subtraction from Illumina was recently unavailable for an extended period of time, disrupting routine rRNA depletion. Here, we report the development of a “do-it-yourself” kit for rapid, cost-effective, and robust depletion of rRNA from total RNA. We present an algorithm for designing biotinylated oligonucleotides that will hybridize to the rRNAs from a target set of species. We then demonstrate that the designed oligonucleotides enable sufficient rRNA depletion to produce RNA-seq data with 75 to 80% of reads coming from mRNA. The methodology presented should enable RNA-seq studies on any species or metagenomic sample of interest.
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Garcia, Anastacia M., Kathryn C. Chatfield, Genevieve C. Sparagna, et al. "Abstract 17010: Metabolic Gene Expression and Mitochondrial Function Are Altered in the Failing Single Ventricle Myocardium." Circulation 138, Suppl_1 (2018). http://dx.doi.org/10.1161/circ.138.suppl_1.17010.
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Introduction: Despite current standard of care, heart failure (HF) remains a leading cause of death and indication for transplant in the single ventricle congenital heart disease (SV) population. However, little is known regarding the molecular mechanisms underlying remodeling and eventual HF in SV patients. The purpose of this study was to characterize the transcriptional profile of SV myocardium in both failing (SVHF) and non-failing (SVNF) SV patients compared to biventricular NF controls (BVNF). Furthermore, we conducted high resolution respirometry to assess mitochondrial function in each of these populations. Methods: Library prep was performed using the TruSeq Ribo Zero rRNA depletion kit, and 2x150 total RNAseq (Illumina HiSEQ 4000) was performed on age-matched explanted RV myocardium from BVNF (n=4 biventricular donors), SVNF (n=8 SV primary transplants, normal function), and SVHF (n=9 SV systolic HF transplants). Samples were aligned to hg19 and were normalized and annotated using the edgeR pipeline. Significant changes in gene expression were calculated using an FDR adjusted p-value (q<0.1; p<0.025). Respiration of myocardial mitochondria was measured using a stepwise protocol to evaluate respiratory capacity in an Oroboros Oxygraph system; n=6 SVHF, n=6 SVNF, n=18 BVNF. Results: RNAseq identified 1,007 differentially expressed genes in SVNF and 2,109 in SVHF myocardium relative to BVNF controls. Transcriptome pathway analysis demonstrated multiple pathways that are similarly dysregulated in SVNF and SVHF, while pathways involved in mitochondrial metabolism and function were significantly dysregulated specifically in the SVHF population. Moreover, mitochondrial oxygen flux was significantly decreased, particularly through complexes I and II, in SVHF relative to BVNF controls. Conclusions: Our results provide new insights into SVHF by identifying unique gene expression changes, including those related to metabolism, and impaired mitochondrial function. Together these data suggest dysregulated metabolic gene expression and mitochondrial dysfunction are phenotypes associated with the failing single ventricle and may serve as potential therapeutic targets for the treatment or prevention of HF in the SV population.
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Liu, Dongcheng, Yan Wang, and Yan Yuan. "Kaposi's Sarcoma-Associated Herpesvirus K8 Is an RNA Binding Protein That Regulates Viral DNA Replication in Coordination with a Noncoding RNA." Journal of Virology 92, no. 7 (2018). http://dx.doi.org/10.1128/jvi.02177-17.
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ABSTRACTKaposi's sarcoma-associated herpesvirus (KSHV) lytic replication and constant primary infection of fresh cells are crucial for viral tumorigenicity. The virus-encoded bZIP family protein K8 plays an important role in viral DNA replication in both viral reactivation andde novoinfection. The mechanism underlying the functional role of K8 in the viral life cycle is elusive. Here, we report that K8 is an RNA binding protein that also associates with many other proteins, including other RNA binding proteins. Many protein-protein interactions involving K8 are mediated by RNA. Using a UVcross-linking andimmunoprecipitation (CLIP) procedure combined with high-throughput sequencing, RNAs that are associated with K8 in BCBL-1 cells were identified, including both viral (PAN, T1.4, T0.7, etc.) and cellular (MALAT-1, MRP, 7SK, etc.) RNAs. An RNA binding motif in K8 was defined, and mutation of the motif abolished the ability of K8 to bind to many noncoding RNAs, as well as viral DNA replication duringde novoinfection, suggesting that the K8 functions in viral replication are carried out through RNA association. The functions of K8 and associated T1.4 RNA were investigated in detail, and the results showed that T1.4 mediates the binding of K8 to ori-Lyt DNA. The T1.4-K8 complex physically bound to KSHV ori-Lyt DNA and recruited other proteins and cofactors to assemble a replication complex. Depletion of T1.4 abolished DNA replication in primary infection. These findings provide mechanistic insights into the role of K8 in coordination with T1.4 RNA in regulating KSHV DNA replication duringde novoinfection.IMPORTANCEGenomewide analyses of the mammalian transcriptome revealed that a large proportion of sequence previously annotated as noncoding regions is actually transcribed and gives rise to stable RNAs. The emergence of a large number of noncoding RNAs suggests that functional RNA-protein complexes, e.g., ribosomes or spliceosomes, are not ancient relics of the last ribo-organism but would be well adapted to a regulatory role in biology. K8 has been puzzling because of its unique characteristics, such as multiple regulatory roles in gene expression and DNA replication without DNA binding capability. This study reveals the mechanism underlying its regulatory role by demonstrating that K8 is an RNA binding protein that binds to DNA and initiates DNA replication in coordination with a noncoding RNA. It is suggested that many K8 functions, if not all, are carried out through its associated RNAs.
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Qin, YanHong, Suxia Gao, Yuxia Liu, et al. "First Report of Dasheen Mosaic Virus Infecting Typhonium giganteum Engl. (Baifuzi) in Henan Province of China." Plant Disease, August 18, 2021. http://dx.doi.org/10.1094/pdis-03-21-0589-pdn.
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Typhonium giganteum Engl. (Baifuzi ) is a perennial plant of the family Araceae. In China, its root is commonly used as an antispasmodic for stroke and cancer treatment (Chi et al. 2010; Gao et al. 2014; Khalivulla et al. 2019). Yuzhou city in Henan Province is the main producing area of T. giganteum Engl., and in July 2020, a survey of viral disease infecting T. giganteum Engl. was conducted in the city. In the surveyed fields (n =5), over 60% of plants displayed varying levels of virus-like symptoms, including mosaic, chlorotic and leaf distortion (Supplementary Figure S1) . To identify possible viral pathogens associated with the disease symptoms afflicting T. giganteum Engl., one leaf each from 25 symptomatic plants was collected and analyzed by high-throughput sequencing (HTS) as well as PCR. For HTS analysis, total RNA was extracted from one pooled sample containing a portion of all abovementioned leaves using RNAprep Pure Plant Plus Kit (TIANGEN Biotech, Beijing, China). After removing ribosomal RNA with Ribo-off rRNA depletion kit (Vazyme Biotech, Nanjing, China), a sequencing library was generated using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA) and sequenced on an Illumina Novaseq6000 sequencing system at Berry Genomics Corporation (Beijing, China). A total of 6,899,143 high-quality clean reads were obtained after trimming and used for contig assembly. BLASTn and BLASTx analyses on the contigs (n = 128,400) showed that one contig (9,245 bp in length) exhibited a sequence identity of 84.0% with the reference sequence of dasheen mosaic virus (DsMV, NCBI reference seq. NC_003537, genus Potyvirus, family Potyviridae) , suggesting infection of the plants by DsMV. No other viral sequences were detected in the sample. To confirm these results, a near full-length genomic sequence of DsMV was obtained from one sample (sample no. 39) by reverse transcription polymerase chain reaction (RT-PCR) of three overlapping fragments with the following primer pairs: DsMV-1F (5′-AAATTAAAACATCTCAACAAAACCTACA-3′) /DsMV-4130R (5′-TTCATGGTCCTCGTGGAGTATA-3′), DsMV-3870F (5′-GAGGACGTGAGAATTCAAAGTCT-3′)/DsMV-8250R (5′-GTCCAACCTTGCTTGATGCATGC-3′), DsMV-7690F (5′-GGAGCGACTCCTCTTCCAAAGTTGTG-3′)/DsMV-10100R (5′-TGAACACCGTGCACGAAGCATCTC-3′). The PCR products were cloned into pMD19-T vector (TAKARA Biotech, Dalian, China) and sequenced. The near full-length genomic sequence of the isolate (DsMV-BF39) was 9,737 nt in length and deposited into GenBank under the accession no. MZ043618. BLASTn analysis of this sequence demonstrated that it shared an identity ranging from 78.6% (MG602234) to 85.6% (MG602227) with various DsMV isolates. To determine whether DsMV was closely associated with the symptoms observed in T. giganteum Engl., leaf tissues from 30 symptomatic plants and 22 asymptomatic plants were analyzed by RT-PCR using primer pairs DsMV-CPF (5′-TGTTCTGTGAACATGATGAAGTTG-3′, sense) and DsMV-CPR (5′-GTAACTGTGGCCTGTTTACCAG-3′, antisense) targeting a 916 bp fragment of the CP gene of DsMV. Amplicons with the expected size were detected from the 30 symptomatic plants but not from the 22 asymptomatic plants, suggesting a close association between DsMV infection and the observed symptoms. To our knowledge, this is the first report of DsMV infecting T. giganteum Engl.. Further study is needed to identify the specific symptoms induced by this virus in T. giganteum Engl. and to understand the biological characteristics, epidemiology, prevalence of this virus in China.
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He, Chengyong, Xiaoli Zhao, Lingjiao Fan, Shifang Li, and Hongqing Wang. "Strawberry, a new natural host of Brassica yellows virus in China." Plant Disease, September 21, 2021. http://dx.doi.org/10.1094/pdis-08-21-1617-pdn.
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Brassica yellows virus (BrYV; genus Polerovirus, family Solemoviridae) has an icosahedral spherical virion with a positive-sense single-stranded RNA genome and it is distinguished from turnip yellows virus (TuYV) based on differences in ORF0 and ORF5 (Xiang et al., 2011). To investigate the occurrence and distribution of viruses infecting strawberry (Fragaria ananassa) in the main production areas in China, a survey of nine greenhouses (667 m2 each) was conducted in the cities of Yantai and Beijing, China in August 2020. About 1% of strawberry plants in each greenhouse showed virus-like symptoms of chlorotic spots; 89 symptomatic leaf samples were randomly collected for virus testing. Total RNA was extracted from a pool of eight samples of four different cultivars (Hokowase: 2, Mibao: 2, Sagahonoka: 2, Monterey: 2) from Yantai using RNAprep Pure Plant Plus Kit (TianGen, China). A cDNA library was constructed by NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA) after ribosomal RNA-depletion using an Epicentre Ribo-Zero™ rRNA Removal Kit (Epicentre, USA). High-throughput sequencing was done on Illumina Hiseq 4000, generating 70,931,850 high-quality 150 bp paired-end reads. Clean reads were de novo assembled by Trinity (v2.2.0) and the resulting contigs were screened by BLASTn and BLASTx against GenBank database as described previously (Grabherr et al., 2013). A total of 1,432,164 high-quality reads unmapped to the strawberry genome were obtained and assembled into 93 contigs (ranging from 33 to 8,031 nt). Seven of these contigs (277 to 1,254 nt) shared 98.2 to 100% nt identities with BrYV-A (accession no. HQ388348) and covered 89.5% of the genome of BrYV-A. Subsequent analyses indicated the presence of Strawberry pallidosis-associated virus and Strawberry mottle virus in the analyzed sample, both have been reported in strawberry in China (Shi et al., 2018; Fan et al., 2021). To confirm BrYV infection, total RNA was isolated from the eight samples used for HTS and reverse transcription polymerase chain reaction (RT-PCR) was conducted with two pairs of specific primers (CP and rtp, Supplementary Table 1) designed based on the assembled contigs. PCR products with expected sizes (587 and 609 bp) were observed in one sample (cv. Mibao). BLASTn analysis indicated that the amplicons (accession no. MW548437 and MW548438) shared 98.6% and 99.3% nt identity with BrYV-A, respectively. To obtain the complete sequence of the putative BrYV isolate, the gaps were bridged and the terminal sequences were determined using 5ʹ and 3ʹ RACE kits (Clontech, China) based on the assembled contigs. The complete genome sequence of the putative BrYV isolate has a length of 5,666 nt (accession no. MZ666129) and shares more than 94.3% nt identities with other BrYV isolates. Phylogenetic analysis indicated that the isolate grouped closely with BrYV and further from TuYV (Figure S1). In addition, 11 samples (cv. Benihoppe) of the remaining 81 symptomatic strawberry samples tested positive for BrYV by RT-PCR with the two pairs of primers mentioned above. The sequences (accession no. MZ407232 and MZ407233) revealed 99.5% and 99.3% nt identities with MW548437 and MW548438. To the best of our knowledge, this is the first report of natural infection of BrYV in strawberry plants. Our findings expand the host range of BrYV, but disease association is difficult to establish due to presence of mixed infection and non-fulfillment of Koch's postulates.