Computational protocol: Infection of Apple by Apple Stem Grooving Virus Leads to Extensive Alterations in Gene Expression Patterns but No Disease Symptoms

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Protocol publication

[…] Raw RNA sequences were cleaned by removal of adaptor sequences, N-containing reads with more than 10% of unknown bases, low-quality sequences containing 50% bases of quality value ≤5, and adaptor-alone sequences. The clean reads were mapped to reference sequences (Malus×domestica Whole Genome v1.0) in the Genome Database of Rosaceae (http://www.rosaceae.org/species/malus/malus_x_domestica/genome_v1.0) using SOAPaligner/soap2 allowing no more than 2 nucleotide mismatches. The reads mapped to multiple gene sequences were filtered, and the remaining reads were designated as unambiguous reads. For gene expression analysis, the number of unambiguous reads for each gene was calculated and then normalized to reads per kb per million reads (RPKM) . The gene ontology (GO, http://www.geneontology.org/) classification system and Uniprot database (http://www.uniprot.org/) were used to infer the functions of all genes. RNA-Seq data have been deposited in NCBI's Gene Expression Omnibus under accession number GSE53825 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE53825). [...] To validate the DEG results, qRT-PCR was performed with RNA samples prepared following the method for RNA-seq library construction. Ten ASGV-infected and 10 virus-free in vitro grown plantlets were pooled separately. Total RNA was extracted from each pool and subjected to DNase I treatment (TaKaRa, Dalian, China). The first-strand cDNA synthesis was performed with Oligo(dT) primer and random hexamer primer using M-MLV reverse transcriptase (Promega, Madison, Wisconsin, USA) with 2 µg of total RNA according to the manufacturer's instructions. Eight randomly selected genes with relevant expression profiles from the RNA-seq data were tested. Specific primers (Supplementary ) were designed using DNAMAN (v5.2.2) software. The SYBR Green real-time PCR assay was carried out in a total volume of 20 µL that contained 10 µL of 2×SYBR Premix Ex Taq II (Tli RNaseH Plus) (TaKaRa, Dalian, China), 0.4 µM (each) specific primers, 0.4 µL ROX Reference Dye II (50×), and 100 ng of template cDNA. The amplification program consisted of one cycle of 95°C for 30 s followed by 40 cycles of 95°C for 5 s and 60°C for 34 s. The fluorescent product was detected in the last step of each cycle. Following the amplification, melting temperatures of PCR products were analyzed to determine the specificity of the PCR products. Melting curves were obtained by slow heating at 1.6°C/s, from 60°C to 95°C, while continuously monitoring the fluorescence signal. A negative control without a cDNA template was run with each analysis to evaluate the overall specificity. Amplifications were carried out in 8 strip tubes (0.2 mL) in a ViiA7 Real Time PCR System (Applied Biosystems). All samples were run in triplicate. Amplification of an apple ACTIN gene (MDP0000774288) was used as an internal control. In total, three biological replicates (with 10 plantlets pooled for each replicate) were used for qRT-PCR analyses to obtain an average value. The average values from the three biological replicate were then used to calculate the mean and standard error. Using the 2-ΔΔCT method, the data of relative gene expression were analyzed . […]

Pipeline specifications

Software tools SOAPaligner, DNAMAN
Application RNA-seq analysis
Organisms Malus domestica, Apple stem grooving virus
Diseases Infection, Virus Diseases