Superoxide is promoted by sucrose and affects amplitude of circadian rhythms in the evening Ángela Román, Xiang Li, Dongjing Deng, John W. Davey, Sally James, Ian A. Graham, Michael J. Haydon Contact: m.haydon@unimelb.edu.au, johnomics@gmail.com This repository contains the following files and folders: adjusted.pvalues.csv - stageR p values after multiple testing correction Araport11_genes.201606.cdna.fasta.gz - reference transcript sequences Araport11_GFF3_genes_transposons.201606.gtf.gz - reference annotation Araport11_GFF3_genes_transposons.201606.salmon.geneMap.tsv - gene to transcript mapping cluster_analysis.R - R code to generate and analyse clusters clusters.14.tsv - clusters of genes used in paper from k=14 analysis dark_vs_sucrose_0.5h.wald.csv - pairwise Sleuth Wald test result for 0.5h Dark vs Sucrose comparison dark_vs_sucrose_2h.wald.csv - pairwise Sleuth Wald test result for 2h Dark vs Sucrose comparison dark_vs_sucrose_8h.wald.csv - pairwise Sleuth Wald test result for 8h Dark vs Sucrose comparison full_abundances.csv - Sleuth scaled reads per base for each sample and gene full_lrt.csv - Sleuth likelihood ratio test output for full model including all samples light_vs_dcmu_0.5h.wald.csv - pairwise Sleuth Wald test result for 0.5h Light vs DCMU comparison light_vs_dcmu_2h.wald.csv - pairwise Sleuth Wald test result for 2h Light vs DCMU comparison light_vs_dcmu_8h.wald.csv - pairwise Sleuth Wald test result for 8h Light vs DCMU comparison README.txt - this file run_sleuth.R - R code to run Sleuth differential expression analyses run_stageR.R - R code to run stageR multiple testing correction salmon - folder of Salmon output for all samples samples.tsv - Sleuth sample table with Salmon paths and Treatment, Time, Experiment and Replicate variables sugar_activated_genes.txt - list of sugar activated genes (also in Dataset 2) sugar_repressed_genes.txt - list of sugar repressed genes (also in Dataset 2) Full details of each of these files follows. REFERENCE --------- Araport 11 reference files downloaded from https://www.arabidopsis.org/download/index-auto.jsp?dir=%2Fdownload_files%2FGenes%2FAraport11_genome_release on 26 April 2017: Araport11_genes.201606.cdna.fasta.gz Araport11_GFF3_genes_transposons.201606.gtf.gz Transcript to gene map for Salmon: Araport11_GFF3_genes_transposons.201606.salmon.geneMap.tsv SALMON ------ The salmon folder contains 53 folders, one for each sample. Each folder contains Salmon transcript counts (quant.sf), Salmon gene counts (quant.genes.sf) and the complete Salmon output in Kallisto format (abundance.h5), including bootstrap values. The following samples are included: Control_0h_Exp1_Rep1 Control_0h_Exp1_Rep2 Control_0h_Exp1_Rep3 Control_0h_Exp2_Rep2 Control_0h_Exp2_Rep3 DCMU_0.5h_Exp1_Rep1 DCMU_0.5h_Exp1_Rep2 DCMU_0.5h_Exp1_Rep3 DCMU_2h_Exp1_Rep1 DCMU_2h_Exp1_Rep2 DCMU_2h_Exp1_Rep3 DCMU_8h_Exp1_Rep1 DCMU_8h_Exp1_Rep2 DCMU_8h_Exp1_Rep3 Dark_0.5h_Exp1_Rep1 Dark_0.5h_Exp1_Rep2 Dark_0.5h_Exp1_Rep3 Dark_2h_Exp1_Rep1 Dark_2h_Exp1_Rep2 Dark_2h_Exp1_Rep3 Dark_2h_Exp2_Rep1 Dark_2h_Exp2_Rep2 Dark_2h_Exp2_Rep3 Dark_8h_Exp1_Rep1 Dark_8h_Exp1_Rep2 Dark_8h_Exp1_Rep3 Dark_8h_Exp2_Rep1 Dark_8h_Exp2_Rep2 Dark_8h_Exp2_Rep3 Light_0.5h_Exp1_Rep1 Light_0.5h_Exp1_Rep2 Light_0.5h_Exp1_Rep3 Light_2h_Exp1_Rep1 Light_2h_Exp1_Rep2 Light_2h_Exp1_Rep3 Light_8h_Exp1_Rep1 Light_8h_Exp1_Rep2 Light_8h_Exp1_Rep3 Sucrose_0.5h_Exp1_Rep1 Sucrose_0.5h_Exp1_Rep2 Sucrose_0.5h_Exp1_Rep3 Sucrose_2h_Exp1_Rep1 Sucrose_2h_Exp1_Rep2 Sucrose_2h_Exp1_Rep3 Sucrose_2h_Exp2_Rep1 Sucrose_2h_Exp2_Rep2 Sucrose_2h_Exp2_Rep3 Sucrose_8h_Exp1_Rep1 Sucrose_8h_Exp1_Rep2 Sucrose_8h_Exp1_Rep3 Sucrose_8h_Exp2_Rep1 Sucrose_8h_Exp2_Rep2 Sucrose_8h_Exp2_Rep3 There are two experiments (Exp1, Exp2). Each experiment has three replicates. The first experiment covers all conditions (times 0h, 0.5h, 2h, 8h, treatments Dark, Sucrose, Light, DCMU); the second includes Control 0h, Dark 2h, Dark 8h, Sucrose 2h, Sucrose 8h. Control_0h_Exp2_Rep1 failed sequencing and is not included. SLEUTH ------ Sleuth was run using the script run_sleuth.R as follows: ./run_sleuth.R -s samples.tsv This script produced the following outputs: full_lrt.csv - Sleuth likelihood ratio test output for full model including all samples full_abundances.csv - Sleuth scaled reads per base for each sample and gene Wald test pairwise comparison output: dark_vs_sucrose_0.5h.wald.csv dark_vs_sucrose_2h.wald.csv dark_vs_sucrose_8h.wald.csv light_vs_dcmu_0.5h.wald.csv light_vs_dcmu_2h.wald.csv light_vs_dcmu_8h.wald.csv Three columns in Dataset 1 are taken from these Sleuth pairwise comparison outputs: Log2 Effect Size - b Log2 Effect Size SE - b_se Log2 Mean Abundance - mean_obs STAGER ------ Sleuth output was processed and corrected for multiple tests with StageR with run_stageR.R, which produces adjusted.pvalues.csv, a file of adjusted p values after multiple testing correction for each gene and pairwise comparison. These p values are the AdjustedP values in Dataset 1. SUGAR-REGULATED GENE SETS ------------------------- The sugar regulated genes listed in Dataset 2 are provided here as text files, as they are required for the cluster analysis (below): sugar_activated_genes.txt sugar_repressed_genes.txt CLUSTER ANALYSIS ---------------- The cluster_analysis.R script requires samples.tsv, sugar_activated_genes.txt, sugar_repressed_genes.txt, full_lrt.csv and full_abundances.csv as input. It produces the following: clusters.14.tsv - clustering output for k=14 clusters, as used in the paper elbow_plot.pdf - Elbow plot for Figure S4 trajectories.pdf - cluster trajectories plot for Figure 1D Dataset3.xlsx - raw cluster data and GO analysis for Dataset 3 enrichment_plot.pdf - GO enrichment diagram for Figure 1E / Dataset 4