Computational protocol: A transcriptome analysis of two grapevine populations segregating for tendril phyllotaxy

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Protocol publication

[…] Raw RNA-Seq sequence data in fastq format were cleaned by removing sequence artifacts such as adapter sequences, low quality trailing and leading reads using FastQC and Trimmomatic (Illumina, San Diego, CA, USA). The cleaned sequence reads were then individually aligned to the Vitis reference genome (12× V. vinifera, Phytozome ver 12) using the recommended alignment parameters for splice-aware transcriptome mapping as outlined in the Tophat2 workflow (http://ccb.jhu.edu/software/tophat/manual.shtml). Intermediary processing of the mapped reads (sam and bam files) such as read deduplication and read group renaming was done using Samtools, Bamtools and Picardtools.Once aligned, the count data required by edgeR were obtained as a sum of overlapping reads on the annotated gene feature (gff3 file) using HTseq. Differential gene expression analysis was done in R using the Bioconductor package, edge-R. The standard normalization step that adjusts differences in initial library sizes was done using the default trimmed means of median (TMM) method. TMM is a normalization of library reads based on the scaling of the library size that minimize the log-fold change. In addition, the ad-hoc minimum expression level for downstream analyses was set at 2 (count per millions of reads aligned) CPM for all libraries. DEGs were declared significant at the FDR ⩽0.05 and a 1.5-fold change. [...] To relate the biological significance of the discovered DEGs, gene ontology (GO) enrichment was conducted using Plant MetGenMAP. The resulting list of GO terms and their P values were reduced to a representative GO terms by clustering similar terms and projected into an MDS plot, as implemented by a web-based GO enrichment engine, Revigo.Transcription factor gene families as well as gene annotations were identified by cross-referencing two online plant functional databases: Plant MetGenMAP and PlantTFD ver. 4. Regulatory cis network prediction and TF enrichment tests were facilitated using the algorithm implemented by Plant RegMap.To assess changes in co-expression topology in the mutant and wild-type background, network-based analysis was carried out using the differential dependency network (DDN) algorithm as implemented in its Cytoscape-based version, knowledge-fused differential dependency network (KDDN), where the predicted cis-regulatory connections among the DEGs determined from Plant RegMap, was used as a priori information. Cytoscape was used to visualize the resulting co-expression network. […]

Pipeline specifications

Software tools Plant MetGenMAP, REViGO, DDN, KDDN
Application Protein interaction analysis
Organisms Vitis vinifera