Computational protocol: Transcriptomics Analysis of Apple Leaves in Response to Alternaria alternata Apple Pathotype Infection

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Protocol publication

[…] Raw reads from the image data output from the sequencing machine were generated by Base Calling and saved in FASTQ format. Clean reads were generated by removing reads with adaptors, reads where the number of unknown bases was more than 10%, and low-quality reads (the percentage of the low-quality bases with which value ≤5 was more than 50% in one read).Clean reads were then aligned to reference sequences using the software SOAPaligner/soap2 (Li et al., ) with no more than two mismatches were allowed in each alignment. The Malus × domestica Borkh. cv. “Golden Delicious” genome assemblies as well as related gene annotations were downloaded from the Phytozome V9.0 database (, ( expression levels were calculated using the “reads per kb per million reads” (RPKM) method (Mortazavi et al., ), while differential expressed genes (DEGs) were analyzed as described previously (Audic and Claverie, ). In addition, the false discovery rate (FDR) ≤ 0.001 and an absolute value of log2|Ratio| ≥ 1 were used as the thresholds to judge the significance of differences in gene expression.Gene ontology (GO) categories were assigned to all genes via a BLASTX hit using the Blast2GO software. DEGs were first mapped to GO terms using a standard database (, gene numbers for each term were calculated, and GO terms significantly enriched in DEGs compared to the background genome were determine with a hypergeometric test. All calculated P-values were then subjected to Bonferroni Correction, using a corrected P ≤ 0.05 as the threshold. GO terms that fulfilled this criterion were defined as significantly enriched in DEGs. On the basis of the Kyoto Encyclopedia of Gene and Genome (KEGG), pathway enrichment was then analyzed using the same method, while the WEGO ( software was used for functional classification of GO terms following DEG GO annotation. [...] Gene-specific primers for qRT-PCR were designed using the Beacon Designer 7.0 program (Premier Biosoft International, California, USA) and are listed in Table . Template cDNAs were synthesized using M-MLV reverse transcriptase (Promega, USA) from 1.0 μg of total RNAs following the manufacturer's instructions. 2 × SYBR-Green I RT-PCR Master Mix (Takara, Japan) was used as the labeling agent, while tubulin from M. domestica served as the internal reference gene. These reactions were performed on an Applied biosystems 7300 Real Time PCR System, with the reaction mixture (20 μL) containing 10 μL 2 × Master Mix, 10 μmol·L−1 forward and reverse primers (0.4 μL each), and 1 μL template cDNA. The PCR program in this case was 95°C for 1 min, followed by 40 cycles at 95°C for 20 s, 60°C for 20 s and 72°C for 40 s. Three independent biological replicates were performed for each sample, while the relative expression levels of the selected unigenes were calculated using the relative 2−ΔCT method (Livak and Schmittgen, ). The results represented in this case represent mean standard deviations for the three experimental replicates. […]

Pipeline specifications

Software tools SOAPaligner, BLASTX, Blast2GO, WEGO, Beacon Designer
Applications RNA-seq analysis, qPCR
Organisms Malus domestica, Alternaria alternata
Diseases Cat-Scratch Disease, Infection, Mycoses