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The head-regeneration transcriptome of the planarian Schmidtea mediterranea

Thomas Sandmann12*, Matthias C Vogg3, Suthira Owlarn3, Michael Boutros14 and Kerstin Bartscherer3*

Author Affiliations

1 Division Signaling and Functional Genomics, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany

2 CellNetworks Cluster of Excellence, Heidelberg University, Im Neuenheimer Feld 267, 69120 Heidelberg, Germany

3 Max Planck Research Group Stem Cells and Regeneration, Max Planck Institute for Molecular Biomedicine, Von-Esmarch-Str. 54, 48149 Münster, Germany

4 Department of Cell and Molecular Biology, Faculty of Medicine Mannheim, Heidelberg University, Ludolf-Krehl-Straße 13-17, 68167 Mannheim, Germany

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Genome Biology 2011, 12:R76  doi:10.1186/gb-2011-12-8-r76

Published: 16 August 2011

Additional files

Additional file 1:

Transcriptome assembly from public 454 data. (a-c) Schematic overview of 454 transcriptome assembly approaches. Publicly available 454 reads were assembled either separately (a, b) or as a combined dataset (c) using the Newbler 2.5 assembler. Colors indicate the three different assemblies: yellow, Abril et al. [17] (a); orange Blythe et al. [18] (b); red, combined (c). Quality metrics shown include the shortest and longest sequences in each assembly, as well as N50, for which 50% of all bases are contained in sequences at least as long as N50. (d) Kernel densities of the length distributions for the assembled sequences. For multi-isoform loci, only the longest isoform was considered. Colors as in (a-c). (e) Kernel densities of ortholog hit ratios obtained by comparing sequences from the different assemblies or computational prediction to the Schistosoma mansoni proteome using blastx. For multi-isoform loci, only the longest isoform was considered. Colors as in (a-c).

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Additional file 2:

Genomic supercontigs. This histogram shows the sequence length distribution of all genomic S. mediterranea supercontigs (version 3.1 [13]). The red line indicates a sequence length of 10 kb.

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Additional file 3:

Identification of supercontig-spanning transcripts. Illustration of 413 high-confidence supercontig-joining transcripts identified, with a description of the procedure.

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Additional file 4:

Experimental validation of the continuity of genomic supercontigs v31.005068 and v31.000152. (a) Agarose gel electrophoresis showing the PCR amplicon produced with forward and reverse primers annealing to supercontigs v31.005068 and v31.000152, respectively. (b) The amplicon sequence was verified by Sanger sequencing. (c) Blastn results showing the 5' end of the sequence aligning to supercontig v31.000152 and the 3' end matching supercontig v31.005068.

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Additional file 5:

Experimental validation of detected transcripts by RT-PCR. (a) Agarose gel electrophoresis of amplicons amplified using primers designed against 14 different sequences from the Illumina+ assembly and two control sequences. (b) Primer sequences used in the PCRs.

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Additional file 6:

Blastx comparison of de novo transcriptome sequences with the NCBI non-redundant protein database. (a) Distribution of similarity detected in the best blastx hit for each transcript (blastx e-value < 10-3). (b) Distribution of blastx e-values for the best blastx hit for each transcript. For multi-isoform loci, only the longest isoform was used as a blastx query.

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Additional file 7:

Illumina+ assembly annotations and expression data. A spreadsheet containing the length, annotation and differential gene expression data for all sequences of the Illumina+ assembly.

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Additional file 8:

Mapping statistics of Illumina and SOLiD raw reads. Statistics of mapping raw Illumina and SOLiD reads onto the Illumina+ assembly.

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Additional file 9:

MA plots illustrating differential gene expression from both Illumina and SOLiD data. For the longest isoform of each locus from the Illumina+ assembly, the expression fold change (log2 scale) relative to the control (0 h) is plotted against its log average abundance (MA plot). Statistically significant up- or down-regulation (adjusted P-value < 0.001 and log2 fold change > 0.7 or < -0.7 (red lines)) is indicated in yellow and blue, respectively. Genes chosen for qRT-PCR validation (Figure 5) are labeled. (a-e) MA plots show a comparison of Illumina transcriptome sequencing (RNAseq) data from 0.5 to 1 h samples (a), 2 to 3 h samples (b), 4 to 8 h samples (c), 10 to 18 h samples (d) and 24 to 72 h (e) relative to controls. (f, g) SOLiD reads from control or regeneration samples were aligned to the genomic supercontigs with Bioscope. MA plots show a comparison of SOLiD RNAseq data from 1 h sample (f) and 6 h sample (g) relative to controls.

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Additional file 10:

Gene ontology enrichment analysis for each temporal expression class. Gene ontology term enrichment and depletion of each cluster compared to the full set of annotated sequences using GOSeq.

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Additional file 11:

Conservation, knockdown efficiency, and RNAi phenotype of smed-runt-like1. (a) Alignment (ClustalX) of Runt domains of proteins from vertebrate and invertebrate species: Danio rerio (Runt-related transcription factor 3, NP_571679); Homo sapiens (Runt-related transcription factor 2, EAX04279); Hydra magnipapillata (Runx, XP_002165633); Drosophila melanogaster (Lozenge, NP_001096919); Schmidtea mediterranea (Smed-Runt-like1, JF720854). The planarian Runt domain shares at least 45% sequence identity with Runt domains from other organisms. (b) qRT-PCR analysis of smed-runt-like1 expression levels in intact and regenerating planarians 6 h after head and tail dissection. Prior to dissection, animals had been injected with three pulses of 32.2 nl of a 1.5 μg/μl dsRNA solution containing either dsRNAs against smed-runt-like1 or a control gene (gfp), for three days in a row for two consecutive weeks (day 1 to 3, 8 to 10; cut on day 11). Expression levels were normalized against those of a housekeeping gene (AY068123). Error bars represent standard deviations of the mean of three independent biological replicates of five worms each. Note that the smed-runt-like1 mRNA levels are reduced but not completely abolished upon RNAi in regenerating animals. (c) Immunofluorescence analysis (anti-Arrestin) of photoreceptor neurons in regenerating control and smed-runt-like1 RNAi animals at day 14 after decapitation. Note that smed-runt-like1 RNAi animals have severe eye patterning defects. Animals were treated as described in the Materials and methods section of the main text.

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Additional file 12:

Expression data mapped onto computational gene predictions. Differential gene expression data for all gene models predicted by MAKER.

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Additional file 13:

Custom perl script used for quality filtering of Illumina reads. Custom perl script used for quality filtering of Illumina reads.

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Additional file 14:

Illumina+ assembly sequences in fasta format. Compressed .fasta file containing all Illumina+ assembly sequences.

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Additional file 15:

Primers used for experimental validation of expression profiles by qRT-PCR. Primer sequences used for qRT-PCR analysis.

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