Computational protocol: Infection by a Giant Virus (AaV) Induces Widespread Physiological Reprogramming in Aureococcus anophagefferens CCMP1984 – A Harmful Bloom Algae

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Protocol publication

[…] Sequencing reads were initially trimmed in CLC Genomics Workbench 9.0 (Qiagen, Hilden, Germany). Reads with a quality score cut-off of ≤0.03, or with ambiguous bases (“N”s), were discarded. Reads passing quality control were mapped to the Aureococcus (NCBI Accession No. ACJI00000000) and AaV genome sequence (NCBI Accession No. NC_024697) with stringent mapping criteria (95% similarity, 70% length matching). Differential expression of genes in the virus-treated samples compared to the controls was determined at each time point using edgeR () program implemented in the CLC Genomics Workbench 9.0. P-values were adjusted for false discovery rate (FDR) using Benjamini–Hochberg (BH) procedure (). Heatmaps representing data from these analyses (Figures , and Supplementary Figures , , ) were constructed using statistical computing environment R (). The number of reads mapped to each AaV gene was rarefied by library size. Values from biological replicates at each time point were averaged prior to hierarchical clustering of the viral gene expression. Figure summarizing this data was constructed using BRIG software (). The putative early promoter motif of AaV was detected using MEME () in discriminative mode. The E-values associated with the discovered motifs are a conservative estimate of the number of motifs having equal or higher log-likelihood ratio if the target sequences were randomly generated () given a background model.Functional enrichment within the framework of Gene Ontology (GO) terms (positive or negative fold changes) was determined using BiNGO (). GO enrichment for differentially expressed genes is complicated by at an arbitrary fold-change cut-off imposed prior to the enrichment analysis, which excludes the genes with fold-change values even marginally similar to that cut-off. To partially alleviate this problem, we ran the enrichment analysis on gene sets selected using two absolute fold-change cut-offs: >1.5 and >1.3. Using both these cut-offs recovered mostly same GO processes; however, some of the processes were missed by each of the individual approaches. Since our analysis is largely exploratory, results obtained from both cut-off were investigated for interesting GO processes. We report all the GO terms recovered by this approach in Supplementary Dataset . The up- or down-regulation of KEGG pathway-related gene expression was determined using z-test as implemented in “GAGE” R package (). This analysis employed input from all the genes, irrespective of fold-change level or statistical significance, and looked for coordinated expression changes within a particular pathway. The resulting P-values for both the analyses were corrected for FDR using BH procedure (). We considered a FDR-corrected P-value ≤ 0.1 to be significant for both GO and KEGG pathway enrichment. Heatmaps showing the presence or absence of significantly enriched GO processes (Figure and Supplementary Figure ) were constructed in statistical computing environment R (). nMDS and clustering analysis of the virus and host gene expression data and associated figure construction were done using the statistical program PRIMER v6.0 (). […]

Pipeline specifications

Software tools CLC Genomics Workbench, edgeR, BRIG, MEME, BiNGO, GAGE
Databases KEGG PATHWAY
Application Genome data visualization
Organisms Viruses, Human poliovirus 1 Mahoney, Adeno-associated virus, Aureococcus anophagefferens, Aureococcus anophagefferens virus