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Open Access Methodology article

Detection of novel 3' untranslated region extensions with 3' expression microarrays

Lieven Thorrez12, Leon-Charles Tranchevent23, Hui Ju Chang3, Yves Moreau23 and Frans Schuit12*

Author Affiliations

1 Gene Expression Unit, Department of Molecular Cell Biology, Katholieke Universiteit Leuven, Leuven, Belgium

2 SymBioSys, K.U.Leuven Center for Computational Systems Biology, Leuven, Belgium

3 SCD-ESAT, Department of Electrical Engineering, Katholieke Universiteit Leuven, Leuven, Belgium

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BMC Genomics 2010, 11:205  doi:10.1186/1471-2164-11-205

Published: 26 March 2010

Additional files

Additional file 1:

Table S1: List of Affymetrix mouse MOE 430 2.0 probe sets detecting extended 3'UTRs, obtained by stringent automated identification. Some probe sets were already predicted by Ensembl to target the extended 3'UTR, based on the presence of a longer human 3'UTR with high homology in the extended part. These probe sets are marked with an × in the appropriate column. Probe sets which are correctly annotated in Affymetrix' NetAffx are also indicated.

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Additional file 2:

Figure S1: Examples of correlated probesets. A. Extended probe set 6 kb downstream of D10Bwg1379e gives better signals than the currently annotated probe set. Transcriptional direction is from right to left (negative strand). B. Confirmation of our extended probe sets by Ensembl gene predictions. Lpin2 is displayed with the RefSeq annotation (blue) and the Ensembl gene prediction (red). Transcriptional direction is from left to right (positive strand). C. Expression profiles of a probe set pair where the primary probe set has a low expression (log2expression <6) in all arrays. We found 44 similar cases on a genome-wide basis, but these were not retained for further downstream analysis in this study. Transcriptional direction of Slc43a2 is from left to right (positive strand).

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Additional file 3:

Figure S2: Histogram of the distances between the primary and extended probe sets. The majority of predicted 3' UTR extensions are less than 2 kb. The graph is truncated at 10 kb but 5% of extensions are in the range of 10-500 kb.

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Additional file 4:

Table S2: Occurrence of 3'UTR regulatory elements in the murine extended regions. Regions currently not annotated by Refseq to be part of the 3'UTR and < 1 kb were scanned for the occurrence of several known motifs. To classify predicted extensions as either 3' UTR or intergenic sequence, we repeated this analysis on 100 datasets of the same size randomly extracted from either known 3'UTR sequences or intergenic sequence. Each set contained 214657 bp. 95% confidence intervals are shown.

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Additional file 5:

Figure S3: Sequencing analysis of Olfml1 PCR products. Both S (short, known 3'UTR) and L (long, extended 3'UTR) products revealed 3 different alternative splicing forms, indicated as S1-S3 and L1-L3. S1 and L1 are most abundant as can be seen on Figure 3B. Note that all sequencing products start more upstream in the last but one exon of Olfml1; this region is not depicted and only the alignment with the 3'UTR is shown.

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Additional file 6:

Figure S4: Detection of extended 3' UTRs on the Affymetrix human U133 Plus 2.0 platform. A. Histogram with Pearson's correlations for human expression data. The red histogram depicts correlations between the 2126 probe set pairs before the final filtering step. The blue histogram depicts correlations between random probe sets. Similar to the mouse data, a Pearson correlation of 0.6 was chosen as a cut-off value. B. Human conservation score graphs, calculated with PhastCons based on the multiple alignment of 17 vertebrate species. Distributions in red and green represent intergenic and 3' UTR conservation respectively. Black arrow indicates the conservation score of the extended regions. C. GIS-PET track in the UCSC genome browser. GIS-PET tags are displayed in blue. Target regions for the Affymetrix U133 Plus 2.0 are indicated in black, PhastCons conservation score indicated in green.

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Additional file 7:

Table S3: Sequences of PCR primers used for validation experiment

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Additional file 8:

Table S4: List of Affymetrix human U133 Plus 2.0 probe sets detecting extended 3'UTRs, obtained by the same algorithm as applied to mouse data. Some probe sets were already predicted by Ensembl to target the extended 3'UTR and are marked with an × in the appropriate column. Probe sets which are correctly annotated in Affymetrix' NetAffx are also indicated.

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