Computational protocol: Comparative Transcriptome Analysis Reveal Candidate Genes Potentially Involved in Regulation of Primocane Apex Rooting in Raspberry (Rubus spp.)

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Protocol publication

[…] Sequencing reads of low quality that were adaptor-polluted or had a high content of unknown base (N) reads were filtered. De novo assembly with clean reads was carried out to obtain the final unigenes, which were annotated by querying against National Center for Biotechnology Information (NCBI) nucleotide and protein (NR) databases and Clusters of Orthologous Groups, Kyoto Encyclopedia of Genes and Genomes (KEGG), and SwissProt databases (Altschul et al., ). Blast2GO with NRwas used for Gene Ontology (GO) annotation (Conesa et al., ), and InterProScan5 was used for InterPro annotation (Quevillon et al., ). Unigenes that could not be matched to any database mentioned above were identified by ESTScan with BLAST-predicted coding DNA sequences as a model (Yu et al., ). In total, six pairs of DGE profiles of different sample libraries (SAP1 vs. SAF1, SAP2 vs. SAF2, SAP3 vs. SAF3, SAP2 vs. SAP1, SAP3 vs. SAP2, SAP3 vs. SAP1, where the former was used as control and the latter as the experimental group) were compared to assess gene expression changes during AR formation in Rubus. A strict algorithm for identifying DEGs between two samples—the false discovery rate (FDR)—was used to determine the threshold P-value in multiple tests and analyses. FDR ≤ 0.001 and |log2Ratio| ≥ 1 were used as thresholds to identify DEGs. The possible functions of DEGs were determined by searching the GO database ( Web Gene Ontology Annotation Plot (WEGO) was also used for GO classification of genes identified in each DGE library (Ye et al., ). To further characterize gene function, pathway enrichment analysis of the DGE results was performed by BLAST searches of the KEGG database ( Cluster analysis of gene expression patterns was performed using Multi Experiment Viewer ( A Q ≤ 0.05 was selected as the threshold for significant enrichment of gene sets (Cheng et al., ). […]

Pipeline specifications

Software tools Blast2GO, InterProScan, ESTScan, WEGO, TM4
Databases KEGG
Application Transcription analysis
Chemicals Nucleotides