Computational protocol: Identification and genome analysis of tomato chlorotic spot virus and dsRNA viruses from coinfected vegetables in the Dominican Republic by high-throughput sequencing

Similar protocols

Protocol publication

[…] Leaf samples of symptomatic tomato (Solanum lycopersicum), potato (S. tuberosum), long beans (Vigna unguiculata) and chili pepper (Capsicum frutescens) were collected in the province of La Vega, and sweet pepper (C. annuum) in the province of Monseñor Nouel (Fig. ). Total RNA isolation was performed using the commercial kit mirVana™ (Ambion™) to improve RNA quality sent for sequencing. Aliquots of each sample were pooled to compose a single sample.Fig. 1Whole transcriptome shotgun sequencing of the RNA pool was done using an Illumina Hi Seq 2000 platform, which ended up in the production of about 53 million reads. The paired-ends reads were quality-filtered, the adapter sequences were removed, and contigs were assembled de novo using CLC Genomics Workbench version 6.0.3. Contigs covering virus-derived genomes were built by BLASTn and BLASTx searches against the virus reference database available in the National Center for Biotechnology Information (NCBI). The Geneious software was used for further characterization and BLASTx searches.Specific primers were designed to determine by RT-PCR which plants were infected with the viruses found in the deep sequencing analysis (Table ). The total RNA extracted from tomato, potato, long bean, chilli pepper and sweet pepper were used as template for cDNA synthesis. For first-strand cDNA synthesis, 2 μl of each RNA were mixed with 1 μl specific forward and reverse primer pair [10 μM], 1 μl dNTPs solution [10 mM] and 8 μl RNAse free water under 70 °C during 5 min, followed by a rapid cooling on ice. Then, it was added per reaction 2 μl [5×] First Strand® Buffer, 1 μl DTT (DL-Dithiothreitol) [0.1 M], 0.5 μl Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) (Invitrogen) [200 units./μl], and water up to a final volume of 20 μL. The reaction was incubated at 37 °C for 1 h, followed by a final denaturation at 70 °C for 15 min. For PCR, Taq Platinum® DNA polymerase (Invitrogen) was used as it follows: 1.5 μl MgCl2 [50 Mm], 2.5 μl 10X buffer, 0.5 μl primer forward [10 μM], 0.5 μl primer reverse [10 μM], 0.5 μl dNTPs solution [10 mM], 0.1 μl Taq DNA polymerase, 1 μl cDNA, and water up to 25 μL. The amplification program consisted of a primary denaturation at 94 °C for 2 min, followed by 35 cycles at 94 °C for 30 s, 55 °C for 30 s and 72 °C for 1 min, and one step final extension at 72 °C for 5 min. PCR products were sent for Sanger sequencing at Macrogen Inc. (South Korea). The sequences were then compared with those deposited in the GenBank database via BLAST.For phylogenetic analysis, multiple alignments were performed by MUSCLE implemented in Seaview v.4.5.4 and the phylogenetic trees were built by PhyML software [–]. Maximum Likelihood was used as the criterion to infer phylogenetic relationships between the isolates. The appropriate nucleotide substitution model was selected by JmodelTest program version 2.1.4 []. The visualization and edition of the phylogenetic trees were performed using FigTree v.1.4.1. […]

Pipeline specifications

Software tools CLC Genomics Workbench, BLASTN, BLASTX, Geneious, MUSCLE, SeaView, PhyML, jModelTest, FigTree
Applications Phylogenetics, Nucleotide sequence alignment
Organisms Solanum lycopersicum, Human poliovirus 1 Mahoney, Viruses, Bell pepper alphaendornavirus, Southern tomato virus, Porcine circovirus 2
Diseases Infection