Open Access Research article

Transcriptome profiling of the demosponge Amphimedon queenslandica reveals genome-wide events that accompany major life cycle transitions

Cecilia Conaco1, Pierre Neveu124, Hongjun Zhou1, Mary Luz Arcila1, Sandie M Degnan3, Bernard M Degnan3 and Kenneth S Kosik1*

Author Affiliations

1 Neuroscience Research Institute and Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, CA, 93106, USA

2 Kavli Institute for Theoretical Physics, University of California, Santa Barbara, CA, 93106, USA

3 Centre for Marine Science, School of Biological Sciences, University of Queensland, Brisbane, QLD, 4072, Australia

4 Present address: Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, 69117, Germany

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BMC Genomics 2012, 13:209  doi:10.1186/1471-2164-13-209

Published: 30 May 2012

Additional files

Additional file 1:

Figure S1. Read normalization and setting the transcript detection threshold. (A) Distribution of transcript read counts after global normalization for sequencing depth. (B) Comparison of normalized read counts from two sequencing runs of the same library preparation and (C) comparison of normalized read counts from sequencing runs of two independent libraries made from the same RNA sample show that determination of transcript expression is reproducible above a cutoff threshold of 64 reads (red lines).

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Additional file 2:

Figure S2. Comparison of expression trends from sequencing and quantitative RT-PCR. The expression of 50 transcripts was determined by quantitative RT-PCR (qPCR) from three separate pools of individuals at each developmental stage. The expression profile for each transcript is shown relative to the precompetent sample (sequencing, dashed blue lines; qPCR, solid red lines). The relative fold change in expression estimated by the two methods showed similar trends across development for 40 of the genes tested (Pearson r ≥ 0.70). Variance in pooled sample replicates was assessed using one-way ANOVA with Bonferroni’s post-test (***, p < 0.0001; **, p < 0.001; *, p < 0.01). Error bars indicate the standard deviation.

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Additional file 3:

Figure S3. Expression of annotated and non-annotated genes. Sponge genes were aligned to sequences in the UniProt database. Sequences with significant matches (e-value ≤ 1x10-4) were designated as ‘annotated’ and those without as ‘non-annotated.’ (A) Non-annotated genes (red line) have lower overall expression compared to annotated genes (blue line). (B) Both gene sets exhibit similar patterns of variation across development. Heatmaps show relative expression of annotated and non-annotated genes (red, high; blue, low). The number of genes in each set is indicated to the left of each heatmap.

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Additional file 6:

Table S1. Four-fold differentially expressed genes at stage transitions. List of genes that are differentially expressed (>4-fold and greater than sampling noise) at indicated stage transitions grouped by direction of change (up or downregulation). Transcript length, normalized read counts, name and accession number of best sequence match in the UniProt database, Gene Ontology (GO) annotation, PFAM domains, and PANTHER annotation is indicated for each gene.

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Additional file 7:

Table S2. Two-fold differentially expressed genes at stage transitions. List of genes that are differentially expressed (>2-fold and greater than sampling noise) at indicated stage transitions grouped by direction of change (up or downregulation). Transcript length, normalized read counts, name and accession number of best sequence match in the UniProt database, Gene Ontology (GO) annotation, PFAM domains, and PANTHER annotation is indicated for each gene.

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Additional file 8:

Table S3. Gene ontology (GO) analysis for genes exhibiting greater than two- or four-fold change in expression between successive stages. Selected functional terms enriched in the set of genes that are upregulated or downregulated at specific stage transitions are shown with the corresponding p-values. The number of genes belonging to each functional category in the genome or within each differentially expressed group is indicated.

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Additional file 9:

Table S4. PANTHER functional group enrichment analysis for genes that exhibit greater than four-fold change in expression between successive stages. Functional groups enriched in the set of genes that are upregulated or downregulated between stages are shown (enrichment p-value ≤ 0.001). The number of genes in the genome or within each differentially expressed group that belong to a category is indicated.

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Additional file 4:

Figure S4. Expression of genes in selected functional groups. Genes within each category were retrieved using the Gene Ontology (GO) annotation of their best UniProt sequence match or by the presence of PFAM domains. Transcription factors, G-protein coupled receptors, and kinase genes were obtained from previous studies [23,56,57]. Similar functional categories are shown together: (A) general cellular processes; (B) metabolic processes; (C) metazoa-associated processes; (D) regulators of gene expression; (E) transcription factors; (F) receptors and signaling mechanisms; (G) kinases. (Left) Percent of genes in each functional category that are detected by sequencing. The red and blue lines indicate the expected percent of genes to be found in at least one stage (45%) and at each stage (30%), respectively, based on the overall number of gene models detected by sequencing. The total number of predicted genes belonging to each category is shown. (Right) The percent of expressed genes in each functional category that are found within the top 25% of their expression range across the four developmental stages included in the study.

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Additional file 10:

Table S5. Genes exhibiting greater than 100-fold upregulation in relative expression level during sponge development. Maximum upregulation (max. upreg.) is the highest relative expression ratio for the gene, given as the log2 of reads for a gene in one stage minus the average of the log2 of reads in the other three stages.

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Additional file 5:

Figure S5. Relative expression heatmaps for genes belonging to developmental signaling pathways, transcription factor families, or kinase classes. (A) Genes in the Wnt, Notch, and TGF-β pathways have homologs in A. queenslandica (blue, present; gray, absent) and are expressed throughout sponge development (P, precompetent; C, competent; PL, postlarva; A, adult). Major receptors and ligands in each pathway are highlighted in red. (B) Relative expression of genes in transcription factor families. (C) Relative expression of genes in kinase classes. Heat maps show the relative expression of genes across stages (red, high; blue, low). Genes were retrieved from previous studies [10,11,23,87]. Stage enrichment of genes, based on the number that are found within the top 25% of their expression range across the four developmental stages, was estimated using Fisher’s exact test (p-values shown).

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Additional file 11:

Table S6. Quantitative RT-PCR validation cycle numbers, read counts, and primer sequences.

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