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A de novo assembly of the newt transcriptome combined with proteomic validation identifies new protein families expressed during tissue regeneration

Mario Looso1, Jens Preussner1, Konstantinos Sousounis3, Marc Bruckskotten1, Christian S Michel1, Ettore Lignelli1, Richard Reinhardt2, Sabrina Höffner1, Marcus Krüger1, Panagiotis A Tsonis3*, Thilo Borchardt1* and Thomas Braun1*

Author Affiliations

1 Max-Planck-Institute for Heart and Lung Research, Ludwigstrasse 43, 61231 Bad Nauheim, Germany

2 Max-Planck Genome Centre Cologne, Carl-von-Linné-Weg 10, 50829 Köln, Germany

3 Department of Biology and Center for Tissue Regeneration and Engineering at Dayton, University of Dayton, OH 45469-2320, USA

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Genome Biology 2013, 14:R16  doi:10.1186/gb-2013-14-2-r16

Published: 20 February 2013

Additional files

Additional file 1:

Performance of different assembly strategies. Performance comparison of four different assembly strategies comparing total number of transcripts, N50, transcripts >500 bp and transcripts >1,000 bp.

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Additional file 2:

Overall distribution of transcript annotation rate as a function of sequence length. Transcript length (x-axis, log scale) is plotted against the percentage of overall annotation (y-axis). E-value cut-offs from e-10 to e-200 are marked in different colors. The dashed line demarks the sequence length above which transcripts were chosen for further analysis.

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Additional file 3:

Coverage of de novo assembled newt transcript with high quality annotations on human signaling pathways. Fifty-eight members of the human p53 signaling pathway are matched by 47 proteins present in the assembled newt transcriptome. The use of high quality threshold criteria might have prevented detection of all family members.

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Additional file 4:

Coverage of de novo assembled newt transcript with high quality annotations on human signaling pathways. The transforming growth factor beta signaling pathway containing 51 members is covered by 41 newt transcripts. Candidates identified by gene symbols are marked in pink, candidates that were not identified are marked in purple. Pathway nodes including multiple candidates that are only partially represented in the newt transcriptome are marked in dark red.

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Additional file 5:

All transcripts assigned to orthologues and corresponding length distribution with respect to the subject sequences.

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Additional file 6:

All potential protein-coding transcripts that were validated by corresponding peptides. The number of identified frameshifts and the total number of identified peptides is listed. The second sheet gives the example of a single candidate where peptides and alignments identified a frameshift in the nucleotide sequence.

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Additional file 7:

All identified Pfam domains in transcripts that lacked any sequence similarity to higher organisms. The second sheet includes the domains found in candidates presented in the manuscript.

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Additional file 8:

Comparative hierarchical clustering of heart and lens expression values. Hierarchical clustering of expressions levels in regenerating hearts (columns 1 to 9), and lenses (dorsal iris, columns 10 to 12; ventral iris, columns 13 to 15) during regeneration. Only transcripts with valid array expressions for at least 13 columns are represented. The blue cluster represents a subset of transcripts that are down-regulated at at least two stages of heart and lens regeneration. The yellow cluster marks a set of transcripts that are up-regulated during late stages of heart regeneration but lacks an obvious pattern in the regenerating lens. The red cluster represents a set of transcripts that are inversely regulated at late stages of lens regeneration but lacks an obvious pattern in the regenerating heart with the exception of a smaller subfraction that was strongly up-regulated during early heart regeneration (6 hours after heart injury). The green cluster marks a set of transcripts that are uniformly up-regulated during late stages of heart and lens regeneration. The purple cluster highlights a set of transcripts that are strongly up-regulated in regenerating lens and in the regenerating heart between 1 to 4 days after damage. All heatmap members with cluster affiliation and expression values are provided in Additional file 9.

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Additional file 9:

Expression data of all microarray spots presented in the heatmap (Additional file 8).

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Additional file 10:

Microarray expression data of regenerating heart and lens tissues. All candidates described in the study and represented on microarrays are shown.

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Additional file 11:

Expression of newly identified genes in regenerating newt tissues. Real time RT-PCR analysis (n ≥ 3) of selected candidates in regenerating adult newt hearts, lenses and limbs. Values were normalized to the 0 time point and to tissues with the highest expression levels.

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Additional file 12:

Nucleotide and translated amino acid sequences of all candidate molecules described in the manuscript.

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Additional file 13:

List of all primers used.

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