To verify the viruses infecting the two TBTD-symptoms tobacco field samples, the YBSh and YKMPL leaf samples were collected and then quick-frozen by liquid nitrogen and stored at − 80 °C tentatively. The two samples were sent to Biomarker Technologies (Beijing, China) for High throughput sequencing (HTS) RNA-Seq sequencing after depletion of the rRNAs with Epicentre Ribo-ZeroTM kit, which was then sequenced using the Illumina HiSeq X-ten platform with PE150 bp (Illumina, San Diego, CA, USA). Sequence data were analyzed using CLC Genomic Workbench 9.5 (QIAGEN, Hilden, Germany) as described19 (link). Reads without sequence similarity and not mapping to the reference tobacco genome were assembled de novo by Trinity program. The generated contigs were used as queries for BLAST searches; contigs that were not identified as sequences already included in the databases were sorted out as candidate genomic fragments of the novel virus.
Clc genomic workbench 9
Manufactured by Qiagen
Sourced in Germany
The CLC Genomic Workbench 9.5 is a software application designed for analyzing and visualizing genomic data. It provides a comprehensive suite of tools for tasks such as sequence alignment, assembly, annotation, and data exploration.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq x ten platform, by Illumina (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Ribo zero kit, by Illumina (1 mentions)
Pe150 bp, by Illumina (1 mentions)
Ribo zero magnetic kit, by Illumina (1 mentions)
Truseq rna sample prep kit, by Illumina (1 mentions)
Qubit 3, by Thermo Fisher Scientific (1 mentions)
Agilent 2100, by Agilent Technologies (1 mentions)
Easyspin plus complex plant rna kit, by Aidlab (1 mentions)
Ion torrent suite, by Thermo Fisher Scientific (1 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions)
Digoxigenin dig 11 dutp, by Roche (1 mentions)
Tri reagent, by Merck Group (1 mentions)
4 protocols using clc genomic workbench 9
1
Virus Detection in Tobacco Samples
2
Identification of Viral RNA Transcripts
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Total RNA was extracted using the EASY spin Plus Complex Plant RNA Kit (Aidlab, China), and then tested using the Nanodrop (Thermo Fisher Scientific, USA), Qubit 3.0 (Invitrogen, USA), and Agilent2100 (plant RNA Nano Chip, Agilent, USA) for purity, concentration, and integrity, respectively. After the removal of ribosome RNA by the Ribo-Zero Magnetic Kit (Epicenter, USA), the libraries were built using a TruSeq RNA Sample Prep Kit (Illumina, USA). An Illumina HiSeq X-ten platform (Illumina) set with length of 150-bp pair-end reads was then used for sequencing (Mega Genomics, China). Sequences of adaptor and low-quality trait were trimmed from raw reads, and the rest reads were mapped to the genome sequences of common tea (C. sinensis) (Wei et al., 2018 (link)), using the CLC Genomic Workbench 9.5 (Qiagen, USA). The reads with sequence similarities of >60% to the tea genome sequences were eliminated to reduce interference of the host background, and the remaining unique reads were de novo assembled using the Trinity program (Grabherr et al., 2013 (link)). The resulted contigs were subjected to BLASTx and BLASTn searches against viral (taxid:10239) and viroidal (taxid:2559587) sequences of local datasets retrieved from the National Center for Biotechnology Information (NCBI) databanks. These processes allowed the identification of the contigs with viral sequence attributes.
3
Ion Torrent-based Viral Identification Pipeline
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The sequence data obtained was automatically trimmed by the Ion Torrent Suite software (ThermoFisher), and the resulting BAM files were imported into CLC Genomic Workbench 9.5.1 (Qiagen, Hilden, Germany) for analysis. The pipeline for viral identification is illustrated in Supplemental Figure A1 . Low-quality reads, reads below 30 bp, and the primer sequences were removed. The sequences were mapped to a human hg19 reference genome using stringent criteria, with length fraction set at 0.5 and similarity fraction set at 0.9. The mapped reads were discarded, while unmapped reads were assembled de novo using the built-in CLC de novo assembler with a word size of 20 and a minimal contig size of 80 bp.
The contigs obtained from the de novo assembly and singleton reads longer than 50 bp were blasted against the NCBI GenBank non-redundant nucleotide database (nr/nt) using BLASTn and a cut-off with an E-value of 10−3 for significant hits. After BLAST, contigs and singletons that satisfied an E-value <10−3 and an HSP length >80 bp were selected, and viral hits among them were used for further analysis.
The contigs obtained from the de novo assembly and singleton reads longer than 50 bp were blasted against the NCBI GenBank non-redundant nucleotide database (nr/nt) using BLASTn and a cut-off with an E-value of 10−3 for significant hits. After BLAST, contigs and singletons that satisfied an E-value <10−3 and an HSP length >80 bp were selected, and viral hits among them were used for further analysis.
4
Fungal Genomic and Transcriptomic Analysis
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Fungal genomic DNA was obtained using a Genomic DNA Mini Purification kit (BioKit, Taipei, Taiwan) or a phenol/chloroform DNA extraction protocol. Fungal RNA was isolated using TRI reagent (Sigma-Aldrich, St. Louis, MO, USA) and purified further using PureLink RNA Mini Kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. DNA digestion with restriction endonucleases, electrophoresis, DNA ligation, bacterial transformation, Southern blot hybridizations, and post-hybridization washing were carried out according to standard procedures. Digoxigenin (DIG)-11-dUTP (Roche Applied Science, Indianapolis, IN, USA) was incorporated by PCR into a DNA fragment with the gene-specific primers and used for Southern blot analyses. The probe was detected by an immunological assay according to the manufacturer’s instructions (Roche Applied Science, Indianapolis, IN, USA). The protein-coding genes were predicted by GlimmerHMM [41 (link),42 (link)]. Pairwise sequence comparison was performed using CLC Genomic Workbench 9.5.1 (CLC Bio, Qiagen, Aarhus, Denmark) to calculate genetic distance using the Jukes–Cantor model and percent identity. Conserved domains were predicted with the CD-search tool [43 (link)] available in the National Center for Biotechnology Information (NCBI) and the MEME Suite server [44 (link)].
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