Gene expression analysis

Differentially expressed genes were identified using the edgeR (Robinson, et al. 569 2010) package in R. Genes were considered differentially expressed if upregulation or downregulation was ≥ 4-fold relative to the controls with a False Discovery Rate (FDR) ≤ 0.05. Pairwise comparisons were conducted between the Present Day samples and samples subjected to the other treatments.Gene co-expression networks were reconstructed using rlog-transformed expected counts through WGCNA (Langfelder and Horvath 2008) implemented in R. Hub genes (gene significance > |0.2|; module membership > |0.8|) within a module were identified and used in downstream analyses.Pfam and GO enrichment analyses for DEGs and module members were performed using a script (github.com/fle1/canolab_scripts/blob/master/Pfam_enrichment.R) and the topGO package (Alexa and Rahnenführer 2009) implemented in R. Pfam domains and GO terms with p-value < 0.05 were considered significantly enriched.Blastp top hits for each hub gene were used as input in the KEGG pathway enrichment analysis (score > 0.400 and FDR < 0.01) in STRING v.11 (Mering, et al. 614 2003). Relative expression of sponge gene homologs in RCP 8.5 relative to the Present Day control was computed as the average sum of transcripts per million (TPM).

本研究采用R语言中的edgeR（Robinson等，2010）包鉴定差异表达基因（Differentially Expressed Genes, DEGs）：当基因表达上调或下调幅度相较于对照组≥4倍，且错误发现率（False Discovery Rate, FDR）≤0.05时，即判定为差异表达基因。以当代样本与经其他干预处理的样本开展两两比较。通过R语言中实现的加权基因共表达网络分析（Weighted Gene Co-expression Network Analysis, WGCNA，Langfelder与Horvath 2008）包，对经rlog转换的期望计数进行基因共表达网络重构。鉴定模块内的枢纽基因（hub gene），筛选标准为基因显著性＞|0.2|且模块隶属度＞|0.8|，并将其用于后续分析。针对差异表达基因与模块成员，分别通过自定义脚本（github.com/fle1/canolab_scripts/blob/master/Pfam_enrichment.R）以及R语言中实现的topGO包（Alexa与Rahnenführer 2009）开展Pfam结构域与基因本体（Gene Ontology, GO）富集分析，当Pfam结构域与GO术语的p值＜0.05时，即认定为显著富集。将每个枢纽基因的Blastp最优比对结果作为输入，利用STRING v.11数据库（Mering等，2003）开展京都基因与基因组百科全书（KEGG）通路富集分析，筛选阈值为得分＞0.400且FDR＜0.01。通过计算每百万转录本（Transcripts Per Million, TPM）的平均总和，得到RCP 8.5情境下海绵基因同源物相较于当代对照组的相对表达量。