@@ -57,22 +57,23 @@ Imputed count data were tested for differential expression using DESeq2 v1.38.3
### Sample normalization
The normalization of the count matrix was conducted using the median of ratios method (Love et al., 2014).
### Correlation analysis
The natural logarithm was determined for all normed counts before averaging triplicates. Pearson correlation coefficients were calculated for all sample pairs. Correlation coefficients varied from 0.71 to 1.0. All constant-acetate 25°C samples highly correlated with each other (Sup. Fig. X) with the lowest correlation of 0.993 between the preheat and 48h time point.
### PCA
PCA was performed on averaged triplicates using FSharp.Statsv0.4.11 on transcripts that were nonzero in at least 26 of the 30 samples (Venn et al., 2022a).
### Functional set figures
Functional descriptions were determined for each transcript (Merchant et al., 2007; Usadel et al., 2009; Venn and Muehlhaus, 2022b). Transcripts were grouped according to their functional description and time courses of their averaged normed counts were visualized as z score and log2 fold change respectively. An ANOVA was performed for each transcript at each treatment to elucidate whether a transcript underwent a relevant change during its time course (Venn et al., 2022a).
### Ontology enrichments
Ontology enrichment was performed using extended MapMan annotations (e.g. "PS.lightreaction.LHC" became "PS.Lightreaction.LHC", "PS.Lightreaction" and "PS"). The measured and filtered transcripts served as background. If a transcript showed significant differential expression in the respective comparison (FDR < 0.05), it was considered as significant for its annotations. Enrichment p values were determined using hypergeometric tests. Multiple testing correction was performed using the Benjamini-Hochberg method (Benjamini & Hochberg 1995; Venn et al., 2022a).
A second ontology enrichment was performed using ChlamyCyc annotations (v2023-01-04). This pathway ontology combines KEGG, MapMan, and JGI pathway information (May et al., 2009). The enrichment procedure was the same as described before.
### Visualization
Heatmaps, PCA, and functional sets figures were created using Plotly.NETv4.0.0 (Schneider et al., 2022).
### References:
- Benedikt Venn, Lukas Weil, Kevin Schneider, David Zimmer & Timo Mühlhaus. (2022a). fslaborg/FSharp.Stats. Zenodo. https://doi.org/10.5281/zenodo.6337056
@@ -88,3 +89,4 @@ Heatmaps, PCA, and functional sets figures were created using Plotly.NETv4.0.0 (
- Zhang, N., Mattoon, E.M., McHargue, W. et al. Systems-wide analysis revealed shared and unique responses to moderate and acute high temperatures in the green alga Chlamydomonas reinhardtii. Commun Biol 5, 460 (2022). https://doi.org/10.1038/s42003-022-03359-z
- featureCounts: Liao Y, Smyth GK, Shi W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2014 Apr 1;30(7):923-30.
- Usadel B, Poree F, Nagel A, Lohse M, Czedik-Eysenberg A, Stitt M (2009) A guide to using MapMan to visualize and compare Omics data in plants: a case study in the crop species, Maize. Plant Cell Environment, 32: 1211-1229
- May P, Christian JO, Kempa S, Walther D. ChlamyCyc: an integrative systems biology database and web-portal for Chlamydomonas reinhardtii. BMC Genomics. 2009 May 4;10:209. doi: 10.1186/1471-2164-10-209. PMID: 19409111; PMCID: PMC2688524.
