Evaluation of the chicken transcriptome by SAGE of B cells and the DT40 cell line - PubMed (original) (raw)

doi: 10.1186/1471-2164-5-98.

Randolph B Caldwell, Andrzej M Kierzek, Hiroshi Arakawa, Eduardo Eyras, Nina Hubner, Christian Jung, Manuel Soeldenwagner, Manuela Cervelli, Yan-Dong Wang, Volkmar Liebscher, Jean-Marie Buerstedde

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Evaluation of the chicken transcriptome by SAGE of B cells and the DT40 cell line

Matthias B Wahl et al. BMC Genomics. 2004.

Abstract

Background: The understanding of whole genome sequences in higher eukaryotes depends to a large degree on the reliable definition of transcription units including exon/intron structures, translated open reading frames (ORFs) and flanking untranslated regions. The best currently available chicken transcript catalog is the Ensembl build based on the mappings of a relatively small number of full length cDNAs and ESTs to the genome as well as genome sequence derived in silico gene predictions.

Results: We use Long Serial Analysis of Gene Expression (LongSAGE) in bursal lymphocytes and the DT40 cell line to verify the quality and completeness of the annotated transcripts. 53.6% of the more than 38,000 unique SAGE tags (unitags) match to full length bursal cDNAs, the Ensembl transcript build or the genome sequence. The majority of all matching unitags show single matches to the genome, but no matches to the genome derived Ensembl transcript build. Nevertheless, most of these tags map close to the 3' boundaries of annotated Ensembl transcripts.

Conclusions: These results suggests that rather few genes are missing in the current Ensembl chicken transcript build, but that the 3' ends of many transcripts may not have been accurately predicted. The tags with no match in the transcript sequences can now be used to improve gene predictions, pinpoint the genomic location of entirely missed transcripts and optimize the accuracy of gene finder software.

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Figures

Figure 2

Figure 2

Mappings of SAGE unitags downstream of Ensembl transcripts compared to simulated genomic tags. The number of tags falling within windows of 10 bp is plotted on the y-axis whereas the distance from the 3' end of the nearest predicted Ensembl transcript is plotted on the x-axis. Sage unitags coordinates are indicated by crosses and randomly selected tag coordinates by diamonds.

Figure 3

Figure 3

Confirmation of differential gene expression using semi-quantitative PCR. Primers derived from reference genes for SAGE tags were used for the amplification of cDNA from bursal cells and DT40 employing different cycle numbers as indicated on top of the lanes. Based on the SAGE tag counts, the reference genes were classified as likely to be equally expressed (left part), higher expressed in bursal cells (middle part) or higher expressed in DT40 (right part). The size of the expected PCR product is indicated by a bar adjacent to the gel image. The numbers of tags found for the busage and dt40sage libraries as well as the calculated significance for differential expression are indicated in brackets under the gene names.

Figure 1

Figure 1

Outline of SAGE tag production and reference gene assignment.

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